EUROPEAN FEDERATION FOR MEDICINAL CHEMISTRY AND CHEMICAL BIOLOGY BOOK OF ABSTRACTS 9 th BBBB International Conference on Pharmaceutical Sciences Pharma Sciences of Tomorrow Ljubljana, Slovenia, 15th-17th September, 2022 https://bbb1 b2022.sfd.si/ Impressum 9th BBBB International Conference on Pharmaceutical Sciences Pharma Sciences of Tomorrow: Book of Abstracts Editors: Aleš Obreza, Rok Dreu, Alenka Zvonar Pobirk, Barbara Sterle Zorec Reviewers: Mirjana Gašperlin, Janez Ilaš, et al. Authors: Stanislav Gobec, Hatice Yeşim Karasulu et al. Prepress design: Barbara Sterle Zorec, Alenka Zvonar Pobirk Design: Sebastjan Jenko Publisher: Slovensko farmacevtsko društvo in Univerza v Ljubljani, Fakulteta za farmacijo URL address: The book of abstracts will be pdf format, available at: https://bbbb2022.sfd.si/programme/scientific-programme/ Ljubljana, 2022 Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v Ljubljani COBISS.SI-ID 120548355 ISBN 978-961-94230-4-2 (Slovensko farmacevtsko društvo, PDF) 2 Welcome letter Dear colleagues, We warmly welcome you to the 9th BBBB Conference in Ljubljana, where we have decided to continue the tradition of organizing international BBBB conferences after a break due to the Covid-19 pandemic. Unfortunately, the situation did not allow us to hold the meeting in 2021, when we celebrated three anniversaries, the 100th anniversary of the University of Ljubljana, the 70th anniversary of the Slovenian Pharmaceutical Society and the 60th anniversary of continuous pharmacy studies at the University of Ljubljana. The theme of this year's symposium is "Pharma sciences of tomorrow". The program consists of plenary and keynote lectures from different areas of pharmaceutical sciences, coming from all BBBB partners and broader scientific community. There will also be plenty of opportunity for younger researchers to present their results in the form of oral and poster presentations in an international environment. The conference will offer opportunities for exchange of scientific ideas between young and established scientists and professionals, as well as between people from academia, industry and regulatory authorities. At the conference, we invite you to also visit the capital of Slovenia, which was designated as the European Best Destination 2022 for 2022. Conference Chair Prof Dr Aleš Obreza Chair of the Scientific Committee Prof Dr Rok Dreu General Secretary or the Conferece: Assoc Prof Dr Alenka Zvonar Pobirk 3 CONTENT Committees 7 Conference Floor Plane 9 Scientific Programme 11 Plenary Lectures 18 Keynote Lectures 25 Oral Presentations 59 Poster Presentations 140 Sponsors 296 (Photo: Zmajski most / The Dragon bridge; Luka Esenko, Ljubljana Tourism photo library) 4 OUR R KNOWLEDG GE FOR YOUR TH R HEAL . We c contribute to effe f g ctive treatment through our hrough our own development, in novative procedu ures and state e-of-the-art produ ucts. 10% o of total sales revenues in nvested in research and development Advan nced pharmaceutical dos sage forms and technolo ogies Patent t-protected innovations protected innovations .krka www a.biz 12:15 COMMITTEES Scientific committee Organizing Committee • Rok Dreu (president), Slovenia • Zrinka Abramovič • Nevin Çelebi, Turkey • Marko Anderluh • Mirjana Gašperlin, Slovenia • Tomaž Bratkovič • Stanislav Gobec, Slovenia • Nataša Karas Kuželički • Iztok Grabnar, Slovenia • Petra Kocbek • Judit Hohmann, Hungary • Janez Ilaš • Julijana Kristl, Slovenia • Odon Planinšek • Ivo Laidmäe, Estonia • Robert Roškar • Jasmina Lovrić, Croatia • Tomaž Vovk • Panos Macheras, Greece • Barbara Sterle Zorec • Janja Marc, Slovenia • Natalija Škrbina Zajc • Irena Mlinarič Raščan, Slovenia • Jurij Trontelj • Urve Paaver, Estonia • Anže Zidar • Jelena Parojčić, Serbia • Leena Peltonen, Finland President of the Conference • Luka Peternel, Slovenia Aleš Obreza • Jarkko Rautio, Finland • Selma Şahin, Turkey General Secretary • Zvone Simončič, Slovenia of the Conference • Dieter Steinhilber, Germany Alenka Zvonar Pobirk • Gabriele Stocco, Italy • Tamás Tábi, Hungary Secretary of the Conference • Gordana Wozniak-Knopp, Austria Andrijana Tivadar The BBBB International Conferences have been initiated under the auspices of the EUFEPS at the joint proposal of four founding associations: the Estonian Academic Society of Pharmacy and the Finnish Pharmaceutical Society (Baltic), the Hungarian Society of Pharmaceutical Sciences (Balaton), the Slovenian Pharmaceutical Society (Bled), the Turkish Pharmaceutical Technology Scientists' Association (Bosphorus), and later the Finnish Pharmaceutical Society (Baltic). The founding board consisted of Prof Dominique Duchene (APGI, France), Prof Istvan Hermecz (HSPS, Hungary), Prof Atilla Hincal (TÜFTAD, Turkey), Prof Hans Linden (EUFEPS), Prof Aleš Mrhar (SPS, Slovenia), Prof Christian R. Noe (EUFEPS) and Prof Peep Veski (EPS, Estonia). The aim of the BBBB conferences is to support young, promising scientists from these regions and to create a strong and stable background for an ongoing dialog between pharmacists and other scientists that goes beyond the traditional European links in the fields of Pharmaceutical Sciences, Research and Drug Development. The first BBBB conference was held in Siófok, Hungary, in 2005, followed by Talinn-Tartu, Estonia (2007); Antalya, Turkey (2009); Bled, Slovenia (2011); Athens, Greece (2013); Helsinki, Finland (2015), Balatonfüred, Hungary (2017); and most recently Izmir, Turkey (2019). 7 www.brinox.eu Brinox is a company with almost forty years of experience offering complete turnkey solutions and equipment for the biopharmaceutical, pharmaceutical, and food industries. The company’s performance and growth are based on quality, innovation, flexibility and reliability. Brinox guides the customer along the entire path from a complex process challenge to an optimal solution, tailor-made to meet the customer’s needs. With the aim of producing first-rate products and systems, we carry out all the steps necessary for success, from research and development, process engineering, design, manufacturing, automation, testing and qualification, installation as well as all after-sales services. Here at Brinox, our focus on customer satisfaction, adaptability, as well as our wealth of experience and innovative technical solutions, are the traits that attract and maintain our client base and lead the company to the realization of its vision – to stay the leading provider of unique technological process solutions for the biopharmaceutical and food industry in Europe by 2025. Brinox d.o.o. Sora 21 | SI-1215 Medvode | Slovenija T: +386 1 361 97 30 | info@brinox.eu Brinox Deutschland GmbH Maria-und-Georg-Dietrich Str. 2 | 77652 Offenburg Deutschland | T: +49 (0) 781 970 678 10 | info@brinox.de Brinox Innovations AG Poststrasse 14 | CH-6300 Zug | Switzerland T: +41 41 727 81 50 | info@brinox-innovations.ch Technology that delivers CONFERENCE FLOOR PLAN 15 14 Violet lecture hall 85 - 86 1 - 15 74 - 84 56 - 73 Lunch Poster exhibition 16 - 36 wc 37 - 55 9 Red lecture hall 3 4 4 1 2 5 6 7 8 9 10 11 12 13 wc Exhibitors: 1. Krka 2. Lek/Novartis 3. Pfizer 4. Merck 5. Brinox 6. ASAHI KASEI 7. Harke Pharma 8. Melt Prep 9. Bioinicia 10. Munit 11. ABL&E Group / Teledyne Hanson 12. Chemass 13. Labtim 14. Simplivia 15. Mettler Toledo 10 DETAILED SCIENTIFIC PROGRAMME 9th BBBB International Conference on Pharmaceutical Sciences PHARMA SCIENCES OF TOMORROW Thursday, September 15, 2022, MORNING SESSION Red Lecture Hall Chairpersons: Mirjana Gašperlin, Jelena Parojčić 8.45–9.15 Conference Opening [PL1] Stanislav Gobec, University of Ljubljana, Faculty of Pharmacy, Slovenia 9.15–10.00 Development and translation of lead compounds for the treatment of neurodegenerative diseases Session 1 - NEW TRENDS IN INDUSTRIAL PHARMACY Chairpersons: Mirjana Gašperlin, Jelena Parojčić [KL1] Hatice Yeşim Karasulu, Ege University, Turkey 10.10–10.40 Scientific and regulatory approaches in formulation development with poorly soluble drugs [KL2] Biljana Janković, Lek, Sandoz & University of Ljubljana, Slovenia 10.40–11.10 The power of data science in pharmaceutical industry 11.10–11.45 Coffee Break & Poster session Leo Ohrem, Merck 11.45–12.00 Continuous manufacturing in solid dose – how to leverage opportunities of this new technology [OP1] Klemen Kreft, University of Ljubljana, Slovenia 12.00–12.15 Influence of the binder jetting process parameters and binder liquid composition on the relevant attributes of 3D printed tablets [OP2] Aljoša Gradišek, Krka, Slovenia 12.15–12.30 Optimization of radial extrusion and pellet coating processes using PAT approaches [OP3] Gábor Vasvári, University of Debrecen, Hungary 12.30–12.45 From batch technology to continouos manufacturing: formulation of gastroretentive dosage form based on melt foaming technique [OP4] Mila Kovačević, University of Ljubljana, Slovenia 12.45–13.00 The influence of polymeric binder type and concentration on flow and dissolution properties of Syloid® 244FP-based SMEDDS granules [OP5] Mahwash Mukhtar, University of Szeged, Hungary 13.00–13.15 Pulmonary dry powder for inhalation for targeted drug delivery to macrophages in tuberculosis 13.15–14.15 Luncheon Thursday, September 15, 2022, MORNING SESSION Violet Lecture Hall Session 2 - NEW MOLECULES FOR TREATING NEVRODEGENERATIVE DISEASES Chairpersons: Tamás Tábi, Stanko Gobec [KL3] István Szatmári, University of Szeged, Hungary 10.10–10.40 Synthesis and transformations of kynurenic acid derivatives with potential neuroprotective activity Plenary lecture (PL), Keynote lecture (KL), Oral Presentations (OP) 11 [KL4] Urban Košak, University of Ljubljana, Slovenia 10.40–11.10 Simple syntheses of synthons for potential anti-Alzheimer and antiviral drugs 11.10–11.45 Coffee Break & Poster session [KL5] Aleš Obreza, University of Ljubljana, Slovenia 11.45–12.15 Design, synthesis and biochemical evaluation of low-molecular-weight human neutrophil elastase inhibitors [OP6] Damijan Knez, University of Ljubljana, Slovenia 12.15–12.30 Fluorescent probes for detection of misfolded proteins in biological samples [OP7] Urša Pečar Fonović, University of Ljubljana, Slovenia 12.30–12.45 Targeting cathepsin X in neurodegenerative diseases with novel triazole-benzodioxine inhibitors [OP8] Anže Meden, University of Ljubljana, Slovenia 12.45–13.00 Chalcogen carbamates as covalent cholinesterase inhibitors – surveying group 16 (VI) of the periodic table [OP9] Selin Parmaksız, Hacettepe University, Turkey 13.00–13.15 In vivo efficiency of a new liposomal adjuvant system based on porins 13.15–14.15 Luncheon Thursday, September 15, 2022, AFTERNOON SESSION Red Lecture Hall Chairpersons: Petra Kocbek, Karin Kogermann [PL2] Gerhard Winter, Ludwig Maximilian University of Munich, Germany 14.15–15.00 30 years in formulation of protein pharmaceuticals: What has been achieved – where do we stand today – what is next? Session 3 - FORMULATION AND DELIVERY CHALLENGES OF SMALL MOLECULES AND BIOTECH DRUGS Chairpersons: Petra Kocbek, Karin Kogermann [KL6] Kairi Lorenz, University of Tartu, Estonia 15.10–15.40 Electrospun antimicrobial peptides – novel approach for improved wound infection treatment [KL7] Slavko Kralj, J. Stefan Institute, Slovenia 15.40–16.10 Bioinspired anisotropic magnetic nanoparticles: design, synthesis and biomedical applications 15.10–16.45 Coffee break & Poster session [KL8] Tapani Viitala, University of Helsinki & Åbo Akedemi University, Finland 16.45–17.15 Real-time label-free sensing platforms for nanoparticle and extracellular vesicle analysis Peter Balogh, Merck 17.15–17.30 The viscosity reduction platform enabling subcutaneous delivery [OP10] Hayrettin Tonbul, Inonu University, Turkey 17.30–17.45 In vivo evaluation of doxorubicin and elacridar co-loaded PLGA/silica hybrid nanoparticles against multidrug resistant breast cancer [OP11] Nina K. Grilc, University of Ljubljana, Slovenia 17.45–18.00 Nanofibers for local delivery of two bacillus strains with antibacterial activity Plenary lecture (PL), Keynote lecture (KL), Oral Presentations (OP) 12 [OP12] Fabiola Guareschi, University of Parma, Italy 18.00–18.15 Development of cyclosporine A – loaded micelles exibiting a promising antiviral activity against SARS-CoV-2 18.15 Welcome reception Thursday, September 15, 2022, AFTERNOON SESSION Violet Lecture Hall Session 4 - NEW MOLECULES FOR TREATING BACTERIAL AND VIRAL INFECTIONS Chairpersons: Anikó Borbás, Janez Ilaš [KL9] Anikó Borbás, University of Debrecen, Hungary 15.10–15.40 Semisynthetic glycopeptide antibiotics – one weapon against two deadly enemies [KL10] Lucija Peterlin Mašič, University of Ljubljana, Slovenia 15.40–16.10 Rational design of balanced dual-targeting antibacterial compounds with limited resistance 15.10–16.45 Coffee break & Poster session [KL11] Marko Anderluh, University of Ljubljana, Slovenia 16.45–17.15 Potent DNA gyrase inhibitors bind to their target through symmetrical bifurcated halogen bonding. [OP13] Margherita Brindisi, University of Naples Federico II, Italy 17.15–17.30 HDAC6 inhibition in cystic fibrosis: in vivo proof-of-concept study anti-inflammatory profile, effects on bacterial load, formulation and biodistribution studies [OP14] Sveva Pelliccia, University of Naples Federico II, Italy 17.30–17.45 New covalent reversible inhibitors of SARS-CoV-2 main protease [OP15] Ivana Perković, University of Zagreb, Croatia 17.45–18.00 Synthesis and biological evaluation of quinoline and anthranilic acid derivatives as potential quorum sensing inhibitors [OP16] Peter Peršolja, University of Ljubljana, Slovenia 18.00–18.15 Expanding the chemical space of n-phenylpyrrolamides as DNA gyrase B inhibitors 18.15 Welcome reception Friday, September 16, 2022, MORNING SESSION Red Lecture Hall Chairpersons: Ildikó Bácskay, Rok Dreu [PL3] Daniel Markl, University of Strathclyde, United Kingdom 9.00–9.45 Mechanistic Understanding of long-term performance of oral medicines [PL4] Niklas Sandler, Nanoform, Finland (on-line presentation) 9.45–10.30 Adoption of 3D printing technologies in pharmacies and hospital pharmacies Session 5 - HOT-MELT PROCESSING AND PRINTING TECHNOLOGIES IN PHARMACEUTICAL MANUFACTURING Chairpersons: Ildikó Bácskay, Rok Dreu [KL12] Amrit Paudel, Graz University of Technology, Austria Long-acting drug delivery to female health via polymeric intravaginal ring: Innovative 10.40–11.10 and rational principles underlying formulation design and hot-melt extrusion processing 11.10–11.45 Coffee Break & Poster session Plenary lecture (PL), Keynote lecture (KL), Oral Presentations (OP) 13 [KL13] Gregor Lorbek, Krka, d.d., Slovenia 11.45–12.15 Application of hot-melt technologies in designing formulations with poorly water-soluble active ingredient [OP17] Ognen Jakasanovski, Lek.d.d., Slovenia 12.15–12.30 Rational development and process optimization of an amorphous solid dispersion generic drug product prepared by hot-melt extrusion (a case study) [OP18] Serena Bertoni, University of Bologna, Italy 12.30–12.45 Use of additives to control the polymorphism of solid lipid formulations: a simple way to face a complex problem [OP19] Diren Sarısaltık Yaşın, Dicle University, Turkey 12.45–13.00 Fabrication of 3DP colon-targeted tablets including fluticasone with enhanced solubility by γ-cyclodextrin 13.00–14.00 Luncheon Friday, September 16, 2022, MORNING SESSION Violet Lecture Hall Session 6 - EMERGING CONTAMINANTS IN EVNIRONMENTAL SAMPLES AND PHARMACEUTICAL PRODUCTS Chairpersons: Robert Roškar, Jurij Trontelj [KL14] Žiga Hodnik, Krka, d, d,, Slovenia 10.40–11.10 Mass spectrometry as a powerful tool for analysing degradation pathways: 1,4-benzodiazepine case study 11.10–11.45 Coffee Break & Poster session [KL15] Mira Petrović, ICRA Catalan Institute for Water Research, Spain 11.45–12.15 Pharmaceutical residues in the aquatic environment – Challenges and opportunities of using advanced analytical methods for their monitoring [OP20] Andrej Grobin, University of Ljubljana, Slovenia 12.15–12.30 A useful method for routine monitoring of endocrine disruptors in surface waters by SPE-LC-MS/MS [OP21] Nejc Golob, Lek d.d., Slovenia 12.30–12.45 Study of different sources of nitrosamine traces in pharmaceuticals by highly sensitive hyphenated mass spectrometry techniques [OP22] Nika Osel, University of Ljubljana, Slovenia 12.45–13.00 Selective determination of whey proteins by a chromatographic analytical approach 13.00–14.00 Luncheon Friday, September 16, 2022, AFTERNOON SESSION Red Lecture Hall Chairpersons: Odon Planinšek, Martin Brandl [PL5] Sulev Reisberg. University of Tartu, Estonia 14.00–14.45 Personalised medicine in Estonia – moving from research to practice Session 7 - CREATING USER FRIENDLY ORAL DOSAGE FORMS Chairpersons: Odon Planinšek, Martin Brandl [KL16] Cansel Köse Özkan, University of Health Sciences, Turkey 14.55–15.25 Personalized medicine: Drug development and usage Plenary lecture (PL), Keynote lecture (KL), Oral Presentations (OP) 14 [KL17] István Antal, Semmelweis University, Hungary 15.25–15.55 Microparticles and multi-unit systems for advanced drug delivery 15.55–16.55 Coffee break & Main Poster session (16.15–16.55) [OP23] Krisztián Pamlényi, University of Szeged, Hungary 16.55–17.10 Preparation of buccal films in Parkinson’s disease [OP24] Genada Sinani, Altinbas University, Turkey 17.10–17.25 Design and evaluation of composite film formulations using quality by design for paediatric use [OP25] Gülbeyaz Yıldız Türkyılmaz, Ege University, Turkey 17.25–17.40 Development of a pediatric orally disintegrating tablet dosage form by masking the bitter taste of ornidazol with Eudragit E PO [OP26] Katarina Bolko Seljak, University of Ljubljana, Slovenia 17.40–17.55 Nanocellulose-based orodispersible films as a potential drug delivery system [OP27] Pawel Balcerzak, Lubrizol Advanced Materials, Inc., USA 17.55–18.10 Excipient’s role in formulating user friendly oral dosage forms [OP28] Ece Türkmen, Hacettepe University, Turkey 18.10–18.25 A chitosan based bioadhesive gel for delivery of combined antimicrobials with enhanced activity in local treatment of skin infections in humans and animals 19.30 Conference Dinner Friday, September 16, 2022, AFTERNOON SESSION Violet Lecture Hall Session 8 - THERAPEUTIC DRUG MONITORING & PHARMACOGENOMICS AS A TOOL TO IMPROVE DRUG EFFICIENCY AND SAFETY IN PERSONALISED MEDICINE Chairpersons: Nataša Karas Kuželički, Tomaž Vovk [KL18] Gabriele Stocco, University of Trieste, Italy 14.55–15.25 Therapy personalization of anti-TNF drugs in pediatric patients with immune mediated diseases [KL19] Dunja Urbančič, University of Ljubljana, Slovenia 15.25–15.55 Pharmacogenomic and biological implications of thiopurine S-methyltransferase 15.55–16.55 Coffee break & Main Poster session (16.15–16.55) [KL20] Eva Germovšek, Boehringer Ingelheim, Germany 16.55–17.20 PK-PD modelling to optimise therapy and facilitate therapeutic drug monitoring of antimicrobials in neonates [KL21] Jurij Aguiar Zdovc, University of Ljubljana, Slovenia 17.20–17.45 Model-informed precision dosing of ustekinumab in Crohn's disease [KL22] Eva del Amo Páez, University of Eastern Finland, Finland 17.45–18.10 Crossing ocular barriers: pharmacokinetic models to support drug development in retinal diseases [OP29] Alenka Šmid, University of Ljubljana, Slovenia 18.10–18.25 Transcriptome analysis reveals involvement of thiopurine s-methyltransferase in oxidation-reduction processes 19.30 Conference Dinner Plenary lecture (PL), Keynote lecture (KL), Oral Presentations (OP) 15 Saturday, September 17, 2022, MORNING SESSION Red Lecture Hall Chairpersons: Julijana Kristl, Stane Srčič [PL6] Helder Santos, University of Groningen, The Netherlands 9.00–9.45 Microfluidics manufacturing of nanoparticle based formulations for pharmaceutical and biomedical applications Session 9 - CONTEMPORARY CHALLENGES IN PHARMACEUTICS Chairpersons: Julijana Kristl, Stane Srčič [KL23] Panos Macheras, National and Kapodistrian University of Athens, Greece 10.00–10.30 Physiologically based finite time pharmacokinetic (PBFTPK) models: the end of the beginning in oral drug absorption [OP30] Martin Brandl, University of Southern Denmark, Denmark Microdialysis and nanofiltration allow to distinguish molecularly dissolved from colloid-10.30–10.45 associated drug concentrations during biomimetic dissolution testing of supersaturating formulations [OP31] Gizem Tezel, Hacettepe University & Süleyman Demirel University, Turkey 10.45–11.00 Memantine loaded PLGA nanoparticles for Alzheimer’s disease [OP32] Tamás Sovány, University of Szeged, Hungary 11.00–11.15 An assessments of titanate nanotubes performance as protein carriers [OP33] Anže Zidar, University of Ljubljana, Slovenia 11.15–11.30 Immunomodulatory properties of simvastatin embedded in liposomes and electrospun nanofibers for wound healing 11.30–12.00 Coffee Break [OP34] Jolanta Pyteraf, Jagiellonian University Medical College, Poland 12.00–12.15 The effect of the structure – the evaluation of the disintegration and the dissolution processes of 3D printed orodispersible tablets [OP35] Maja Bjelošević, University of Ljubljana, Slovenia 12.15–12.30 Exploring the potential of new bulking agents for lyophilised biopharmaceutical formulations intended for subcutaneous administration [OP36] Ágnes Rusznyák, University of Debrecen, Hungary 12.30–12.45 Cyclodextrin polymer-based siRNA delivery systems [OP37] Arle Kõrkjas, University of Tartu, Estonia 12.45–13.00 Open liquid-surface ultrasound-enhanced electrospinning for generating multilayered nanofiber structures 13.00 Closing remarks Saturday, September 17, 2022, MORNING SESSION Violet Lecture Hall Session 10 - PEPTIDES AS THERAPEUTICS AND BIORECOGNITION ELEMENTS, M-RNA VACCINES Chairpersons: Tomaž Bratkovič, Taavi Lehto [KL24] Taavi Lehto, University of Tartu, Estonia 10.00–10.30 Peptides-based drug delivery systems for RNA therapeutics [KL25] Mojca Lunder, University of Ljubljana, Slovenia 10.30–11.00 Short peptides and their role in research and immunotherapy of allergies Plenary lecture (PL), Keynote lecture (KL), Oral Presentations (OP) 16 [KL26] Gordana Wozniak Knopp, University of Natural Resources and Life Sciences, Austria 11.00–11.30 CD81 large extracellular loop as a novel alternative scaffold: laminin binding CD81 enhances specific uptake of extracellular vesicles 11.30–12.00 Coffee Break Aleš Štrancar, BIA Separations, Slovenia 12.00–12.15 Process related impurities in mRNA manufacturing [OP38] Tomaž Bratkovič, University of Ljubljana, Slovenia 12.15–12.30 Development of peptides as affinity ligands for human antibody purification [OP39] Abida Zahirović, Jožef Stefan Institute, Slovenia 12.30–12.45 Engineering IL-6-binding lactic acid bacteria for alleviation of inflammatory bowel disease [OP40] Pasquale Russomanno, University of Naples Federico II, Italy 12.45–13.00 Expression, purification, and NMR investigation of human KHSRP DNA binding protein using E. coli system 13.00 Closing remarks Saturday, September 17, 2022, MORNING SESSION UL-FFA WORKSHOPS FOR YOUNG SCIENTISTS Introduction to electrospinning / Introduction to flow cytometry 10.30–12.30 Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7 13.00 Closing remarks Plenary lecture (PL), Keynote lecture (KL), Oral Presentations (OP) 17 Plenary Lectures 18 PL2 30 YEARS IN FORMULATION OF PROTEIN PHARMACEUTICALS What has been achieved- where do we stand today- what is next? Gerhard Winter 1 Pharmaceutical Technology and Biopharmaceutics, Dept. Pharmacy, LMU München, Germany This lecture reviews the last, most relevant With the high dose antibody products other decades in protein formulation research. previously unknown complications evolved, the Since recombinant protein drugs have entered need to deal with high product viscosities, the market in the early 1990ies this class of making fil and finish operations and therapeutics has changed the scene of modern application via injection needles quite therapy dramatical y. Since the turn of the problematic. It became clear, that only limited century antibodies have outnumbered al other resolution of the problem can be achieved by types of therapeutic proteins and the focus of classical formulation efforts, and a workaround formulation research efforts has focused in that via patch pumps, using higher volume or even direction. the design of new protein molecules by protein engineering could help. The lat er il ustrates the The overarching goal of al formulation work most remarkable change that has happened in has been and stil is optimal storage stability at the last 30 years of protein galenics, i.e. the shift reasonable temperatures. Over the years, certain from starting formulation work late in the problems could be resolved, others came up overal development process to a modern new. For example, some of the first generation integrative development cycle, where stability, proteins were particularly low dose products viscosity and other aspects are considered and use of surfactants had focused on reducing equal y important as e.g. binding affinities losses due to surface adsorption. With high dose when designing new antibodies. At that point, antibodies, such losses became mostly molecular model ing/MD simulations are part irrelevant, but surfactants remained necessary of the toolbox to optimize proteins and their ingredients useful to minimize aggregation. formulation features. Considered to be inert excipients first, they later became notorious to eventual y degrade and Final y, the vision to select the most ( long term) contaminate formulations with their stable formulation from a few experiments, degradation products. With this background it carried out in a few weeks or less, stimulated a has become necessary to also monitor their lot of work in the past years. A combination of concentration in a formulation over time, low volume-high sensitivity analytics, robotics, adding significantly to the overal workload to MD-simulations and bet er knowledge on qualify a marketable formulation. aggregation pathways has brought us much closer, but we are not yet there. At least, for Related to the issue of aggregate formation and normal antibodies, platform approaches have particle contamination is the evolution of more replaced “from scratch” formulation programs sophisticated analytical methods to quantify and quite successful y. characterize such contaminations. The community stil has to find the balance between Now, new “formats” have received more high acclamations for new methods, al owing to interest and with viral vectors comprising measure submicron particle contaminations and physical y spoken self-assembled protein the problem of the extremely smal sample sizes envelopes a continuation of previous work with and difficult interpretation of such data. a bit higher complexity has become a recent chal enge. Some of our own work in that field shal be presented to conclude the review circle. 19 PL5 PERSONALISED MEDICINE IN ESTONIA - MOVING FROM RESEARCH TO PRACTICE Sulev Reisberg Research fellow of health informatics Institute of Computer Science, University of Tartu balance between cost, technical options, 1. INTRODUCTION outcome, good healthcare service, data Estonia is a global leader in the digitalisation of protection, legal and ethical aspects? public services. Today, the central government and al municipalities provide services online 5. CONCLUSION through various e-government systems. One can’t optimise everything before putting Currently, Estonia is building a national IT first services into live - novel approaches infrastructure for bringing personalised require brave decisions to prevent losing time medicine into common clinical practice. What and human energy in an endless optimisation have been the lessons learned so far? loop. 2. METHODS We outline the most important factors in Estonian policymaking and scientific works that have brought us to the current state of personalised medicine. We highlight the chal enges that are stil there to be solved. 3. RESULTS When Estonia redeclared its independence in 1992 after the Soviet occupation, it had to build al organisations and processes from scratch. At the same time, the role of IT exploded global y, and Estonians were lucky to integrate IT into their processes from the start. In 2000, Estonia launched a national population-based biobank, which now covers 20% of the adult population and is one of the global research flagships on personalised medicine. After providing personal feedback for 3000 biobank participants in 2017 which included disease risks and pharmacogenetic information, it became clear that a special national IT infrastructure is needed for scaling personalised medicine services for the whole population. Since 2019, such a project has been ongoing. During this period, we have been facing several chal enges. 4. DISCUSSION Genetic data differs from other health data as it also reveals information about the others - how to deal with this? What is correct for most of the research cohort may be stil incorrect for a single patient. Stricter regulations (for minimising risks) make personalised medicine more expensive - how to find an optimal 20 PL6 MICROFLUIDICS MANUFACTURING OF NANOPARTICLE BASED FORMULATIONS FOR PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS Hélder A. Santos1,2,3,4 1 Department of Biomedical Engineering, University Medical Center Groningen / University of Groningen, 9713 AV Groningen, The Netherlands 2 W.J. Kolff Institute for Biomedical Engineering and Materials Science, University Medical Center Groningen / University of Groningen, 9713 AV Groningen, The Netherlands 3 Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland 4 Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Ruijin Second Road, 200025 Shanghai, PR China alternative technique to the conventional Drug carriers are gaining increased at ention methods for the preparation of nanoparticles. In during recent years, due to their advantages. comparison to the conventional methods, the This includes increased drug stability, increased particle preparation process is miniaturized in protection of drug against enzymatic the microfluidic device, thereby leading to a metabolism, possibility of control ed drug reduction in the consumption of regents. release, high drug loading capacity, Furthermore, microfluidic approaches enable biocompatibility, less variability in release the continuous online synthesis of particles, mechanisms and their kinetics, potential for which could reduce the batch-to-batch increased permeability due to lipid and variations in the physicochemical properties of surfactant contents, and hence, enhanced the obtained nanoparticles. bioavailability, and ligand mediated or passive targeting due to their smal size through oral, parenteral, dermal, nasal, ocular, and pulmonary routes of administration. Scale-up of nano/micro-particles fabrication process using batch techniques typical y results in a reduction of control over the synthesis process, leading to wide particle size distributions and, in some cases, to uncontrol ed particle aggregation. Current methods of particle synthesis rely largely on batch stirred Figure 1. Figure legend. Please delete the square homogenizers. However, major chal enges above and insert your figure in high resolution. persist in these systems with regard to process control ability and reproducibility, owing to the rapidity of the involved processes of mixing, In this plenary talk, several examples on how nucleation, growth and agglomeration and their different microparticles and nanoparticles can complex interactions when they take place be prepared and scaled-up using microfluidics, concurrently. as wel as how they can be used to enhance the drug’s targetability, intracellular drug delivery Microfluidics has been defined as the for both cancer chemo- and immune-therapy manipulation of fluids in channels with applications as wel as other applications, wil dimension of tens of micrometer. Microfluidics be highlighted and discussed. has been extensively applied in the fabrication of materials with precisely control ed Overal , our results suggest that microfluidics is physicochemical features [1–16]. As a result of a versatile technique to prepare advanced drug its excel ent ability to manipulate nanoliters delivery systems for different pharmaceutical flows, microfluidics has emerged as an and biomedical applications 21 PL6 15. P. Zhang, C. Li, T. Huang, Y. Bai, P. Quan, W. 1. H. Zhang, W. Cui, X. Qu, H. Wu, L. Qu, X. Li, Z. Zhang, F. Zhang, Z. Liu, B. Wan, A. Zhang, E. Mäkilä, J. Salonen, Y.-Q. Zhu, Z. Correia, J. Zhang, X. Wu, J.T. Hirvonen, H.A. Yang, D. Chen, H. A. Santos*, M. Hai*, D. A. Santos, J. Fan, T. Cai, D. Liu*, Nano Lett. 2021, Weitz*, 21(22), 9458–9467. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 7744. 16. W. Li, J. Chen, S. Zhao, T. Huang, H. Ying, C. 2. Z. Liu*, Y. Li, W. Li, C. Xiao, D. Liu, C. Dong, Trujil o, G. Molinaro, Z. Zhou, T. Jiang, W. Liu, M. Zhang, E. Mäkilä, M. Kemel , J. Salonen, J. L. Li, Y. Bai, P. Quan, Y. Ding, J. Hirvonen, G. T. Hirvonen, H. Zhang, D. Zhou, X. Deng, H. Yin*, H.A. Santos*, J. Fan*, D. Liu*, Nature Commun. A. Santos*, 2022, 13, 1262. Adv. Mater. 2018, 30, 1703393. 3. F. Fontana*, M.-A. Shahbazi, D. Liu, H. Zhang, E. Mäkilä, J. Salonen, J. T. Hirvonen, H. A. Santos*, Adv. Mater. 2017, 29, 1603239. ACKNOWLEDGMENT 4. T. Yong, X. Zhang, N. Bie, H. Zhang, X. Zhang, Sigrid Jusélius Foundation, the Academy of F. Li, A. Hakeem, J. Hu, L. Gan, H. A. Santos*, Finland (Grant No. 331151), China Scholarship X. Yang*, Nature Commun. 2019, 10, 3838. 5. M. Fusciel o, F. Fontana, S. Tähtinen, C. Council and UMCG Research Funds. Capasso, S. Feola, B. Martins, J. Chiaro, K. Peltonen, L. Ylösmäki, E. Ylösmäki, F. Hamdan, O. K. Kari, J. Ndika, H. Alenius, A. Urt i, J. T. Hirvonen, H. A. Santos*, V. Cerullo*, Nature Commun. 2019, 10, 5747. 6. J. Zhang, Cheng Ji, Hongbo Zhang, Hui Shi, Fei Mao, Hui Qian, Wenrong Xu, Dongqing Wang, Jianming Pan, Xinjian Fang*, Hélder A. Santos*, Xu Zhang*, Sci. Adv. 2022, 8, eabj8207. 7. Z. Liu, W. Lian, Q. Long, R. Cheng, G. Torrieri, B. Zhang, A. Koivuniemi, M. Mahmoudzadeh, A. Bunker, H. Gao, H. He, Y. Chen, J. Hirvonen, R. Zhou, Q. Zhao*, X. Ye*, X. Deng*, H.A. Santos*, Adv. Funct. Mater. 2022. 8. Z. Wei, S.Wang, J. Hirvonen, H.A. Santos*, W. Li*, Adv. Healthcare Mater. 2022. 9. I. Arduino, Z. Liu, R.M. Iacobazzi, A.A. Lopedota, A. Lopalco, A. Cutrignel i, V. Laquintana, L. Porcel i, A. Azzariti, M. Franco, H.A. Santos*, N. Denora*, Int. J. Pharm. 2021, 610, 121246. 10. I. Arduino, Z. Liu, A. Rahikkala, P. Figueiredo, A. Correia, A. Cutrignel i, N. Denora*, H.A. Santos, Acta Biomater. 2021, 121, 566–578. 11. C. Costa, Z. Liu, S. Simões, A. Correia, A. Rahikkala, J. Seitsonen, J. Ruokolainen, A. Aguiar-Ricardo*, H.A. Santos, M. Luísa Corvo*, Colloids Surf. B: Biointerfaces 2021, 199, 111556. 12. C. Costa, Z. Liu, J.P. Martins, A. Correia, P. Figueiredo, A. Rahikkala, W. Li, J. Seitsonen, J. Ruokolainen, S.-P. Hirvonen, A. Aguiar- Ricardo, L. Corvo*, H.A. Santos*, Biomater. Sci. 2020, 8, 3270–3277. 13. S. Wang, S. Wannasarit, P. Figueiredo, J. Li, A. Correia, B. Xia, R. Wiwat anapatapee, J. Hirvonen, D. Liu, W. Li*, H. A. Santos*, Mater. Horiz. 2020, 7, 1573–1580. 14. P. Zhang, C. Du, T. Huang, S. Hu, Y. Bai, Cong Li, G. Feng, Y. Gao, Z. Li, B. Wang, J.T. Hirvonen, J. Fan, H.A. Santos*, D. Liu*, Small 2022, 18(15), 2200449. 22 EUFEPS was eestablished on 21.09.1991 in Strasbourg as an indeependent scientifi c European b ody promotin g the interests of t h e pharmaaceutical sciences. EUFFEPS is representing 15 societies fro m differen t cou untries wit h more t han 15 5.0 00 pharmacist s. Th e Mission of EUFEPS is to promot e excellence in th e pharmaceutical sciences and innovative dru g rresearc h in Euro Eu pe . EUFEPS is a Matrix x organization b ased on re cr atin g a platform fo r the productive interrelations an d join t initiatives between member societies an d nd in ividuals resulting EUFEPS Networks. Upcoming events : WK*%+ ,0HHWLQ J (8)(36$$36* /2%$ / % LR( TXLYDOHQFH+ $5021,6$ $7 7,2 1 ,1,7 $ ,$7 7,9( $PVWHUGDP7 KH1 HWKHUODQGV _ 6HSWHPEHU , 2022 KWWSVJEKLHXIHSVRUJ (8)(36$ QQXDO0HHWLQ J ,QWHUQDWLRQD O0HHWLQ J WRJHWKH U Z LWKWK H 3 RUWXJXHVH3KDUPDFHXWLFDO6 RFLHW\ /LVERQ3RUWXJDO_0 D \ ± -XQH , 202 KWWSVHXIHSVRUJHXIHSVDQQXDOPHHWLQJFRQIHUHQFHVHULHVKWP O S m n ca e Cont act u s: Follo w u s: WEB: www.eufeps.or g Mail: secretariat@eufeps.org 23 24 Keynote Lectures 25 KL1 SCIENTIFIC AND REGULATORY APPROACHES IN FORMULATION DEVELOPMENT WITH POORLY SOLUBLE DRUGS H.Yesim KARASULU Department of Pharmaceutical Technology, Faculty of Pharmacy, Ege University, Bornova Izmir, Turkiye 1. INTRODUCTION This study includes development studies for In recent years, studies have at ributed more many water-insoluble drugs for the oral than 40 percent of failures in new drug administration, il ustrated by formulation development to poor biopharmaceutical studies and regulations [4,5]. The purpose of properties, particularly poor water solubility. these studies includes researchers and Issues surrounding low solubility of the active regulations in addressing formulation problems ingredient, in particular can delay or completely related to water-insoluble drugs [1-3]. Due to hinder new drug development. New approaches the essential influence of solubility on drug in the formulation of water-insoluble drugs bioavailability, numerous strategies have been have provided significant benefits in developed to improve the solubility, and reformulating many of the products currently on consequently absorption and bioavailability of the market. More recently, it has been reported poorly water-soluble drugs [1-5]. that 90 percent of new chemical substances in discovery and 75 percent for compounds under 3. RESULTS AND DISCUSSION development show low solubility. For the The formulation of drugs is carried out with the poorly soluble and low bioavailability drugs, principal objective of enhancing their among the most promising approaches are bioavailability. One of the most promising developed lipid formulations. Lipid-based drug approaches is to also formulate as lipid-based delivery systems (LBDDS) are one of the formulations. Encapsulating or solubilizing the future-promising technologies designed to drug in lipid excipients can lead to increased address such chal enges [1,2]. A potential solubilization and absorption, resulting in advantage of LBDDS is that the drug is enhanced bioavailability drugs [1-3]. In this administered to the gastrointestinal tract in context, we would like to discuss the results of solution and eliminates the need for dissolution. our recent work with these systems. Firstly, in The drug remains in solution by digestion and our last study, different lipid-based drug occurs micel ar dissolution caused by its own delivery systems for valsartan were developed emulsification. The most important step in and then compared to a commercial tablet in developing the formulation is to distinguish vitro and in vivo. The new F2-SEDD which excipient (s) and their proportions wil formulation has been proposed as an ideal drug resolve the drug dose. Lipid-based systems delivery system for valsartan with low increase drug absorption from the solubility and bioavailability. As valsartan is a gastrointestinal tract by accelerating the substance which solubility increases with dissolution process, facilitating the formation of increasing pH, the important thing is to increase the dissolved phase by reducing the particle size its solubility at pH 1.2. At the end of the 24th to molecular levels, changing the drug intake, hour at pH 1.2, the commercial formulation increasing the transportation of the drug to the dissolved 63%, powder valsartan 9.4%, F2- systemic circulation through the intestinal SEDD 90.7%. Dissolution studies were also lymphatic system by the enterocyte-based performed in fed and fasted media made with transport [3]. valsartan powder, F2-SEDD and commercial formulation (Fig. 1). The F2-SEDD formulation 2. MATERIALS AND METHODS was released in gastric juice at a rate of 44.6%, 2.1. Materials and Methods while the commercial formulation was only released at a rate of 34.4%. This result indicates 26 KL1 that the F2-SEDD formulation may be more Pharmacokinetic RCa- Commercial bioavailable than the commercial formulation Parameters SNEDDs Tablet due to the increased solubility of valsartan in an AUC0՜24 12867.9± 28.3± acidic environment. In vitro release results (h*ng/mL) 5688.7 7.1 obtained with both the F2-SEDD formulation AUC0՜∞ 12883.9± 34.3± and the commercial formulation support the in (h*ng/mL) 5695.8 16.1 vivo results. Under fasting (FaSSIF) and feding Cmax (ng/mL) 30.3±14 4.7±1.4 (FeSSIF) conditions, F2-SEDD and the Tmax (ng/mL) 3±0.3 5.3±1.6 commercial formulation show similar release rates in the intestinal fluid due to the high 4. CONCLUSION solubility of valsartan at pH 6.8 [4]. In this article, it was aimed to develop different lipid-based formulations containing poorly soluble drugs and to compare these formulations among themselves with in vitro such as legal regulations of these systems and in vivo characterization studies. Furthermore, by comparing the proposed formulation with the commercial formulation in terms of pharmacokinetics and pharmacodynamics, it offered higher possibility to propose a new lipid-based formulation. Consequently, in this Figure 1. Dissolution graph of the valsartan study poorly soluble drug’s new LBDDs may be formulation in different simulated fluids (FaSSGF, suggested with increased oral bioavailability as FaSSIF, FeSSIF) an alternative to classical dosage forms. Secondly, in our study, to improve oral bioavailability, new self-nanoemulsifying 5. REFERENCES formulation (SNEDDS) of Rosuvastatin (RCa) 1. Kleberg, K. et al., Characterising the behaviour was developed and characterized. RCa is a drug of poorly water soluble drugs in the intestine : with low aqueous solubility and 20% oral application of biorelevant media for solubility, bioavailability. The self-nanoemulsifying dissolution and transport studies. Journal of system was formulated with oil (Oleic acid), Pharmacy and Pharmacology, 2010.,2: 1656- surfactant (Labrasol/Labrafil M 1944) and 1668. cosurfactant. According to the in vivo studies, 2. Savla, R., et al., Review and analysis of FDA RCa-SNEDDS has shown higher approved drugs using lipid-based formulations, pharmacokinetic parameters than commercial Drug Development and Industrial Pharmacy, RCa tablet. In addition, the RCa-SNEDDS 2017. 43(11): 1743–1758. unveiled safe pharmacodynamics effects for 3. Liu, R. (Ed), Water - Insoluble Drug Formulation, Second Edition, CRC Press, 2008. RCa in Yorkshire pig. Accordingly, the dosing 4. Gulmezoglu , E., et al., Evaluation of valsartan’s amount for RCa may be reduced using self-emulsifying drug delivery systems with in SNEDDS formulation. Table 1 lists the vitro lipolysis and in vivo and in vivo animal pharmacokinetic parameters of RCa which were models, 8th BBBB International Conference on evaluated by a noncompartmental method using Pharmaceutical Sciences, 2019. P-53. oral WinNonLin 5.3 program [5]. After RCa- 5. Karasulu, H.Y., et al., Enhancing Solubility and SNEDDS administration, the AUC Bioavailability of Rosuvastatin into Self 0→24 of RCa was significantly higher than the commercial Nanoemulsifying Drug Delivery System, RCa tablet ( P<0.05). In the case of oral Current Drug Delivery, 2018. 15 (7), 1072-administration, half-life of RCa in RCa- 1082. SNEDDS was significantly decreased (1.75- fold) when compared with commercial RCa ACKNOWLEDGMENT tablet ( P<0.05). This study was supported by The Scientific and Table 1. Pharmacokinetic parameters of Technological Council of Turkey (TUBİTAK SNEDDs - RCa and commercial tablet-RCa (p Project No: 117S821; 112S637) and was also 0.05) supported by Ege University Scientific Research Project (14/ECZ/005). 27 KL4 SIMPLE SYNTHESES OF SYNTHONS FOR POTENTIAL ANTI-ALZHEIMER AND ANTIVIRAL DRUGS Urban Košak1, Stanislav Gobec1 1Faculty of pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION There is a desperate need for new drugs for previously described synthesis of the treating neurodegenerative diseases and viral orthogonal y protected aminocyclopentitols, we infections. A simple way to develop new drugs developed a more convenient way of making is to synthesize analogues of approved drugs. these important compounds [3]. This makes convenient methods for preparing key intermediates in the synthesis of these analogues highly desirable. 2. RESULTS AND DISCUSSION As part of our development of analogues of the Orthogonal y protected piperidines were anti-Alzheimer drug, donepezil, we needed prepared from commercial y available orthogonal y protected piperidines. Because nipecotamide, isonipecotamide, nipecotic acid procedures to prepare these compounds were and isonipecotic acid in 48-78% overal yields. not wel documented outside of the patent Purification of the intermediates using this literature, we developed a straightforward procedure is not necessary, and the final method to produce these building blocks in high compounds were purified by simple flash overal yields (Fig. 1A) [1]. column chromatography [1]. To develop analogues of the experimental anti- 3- And 4-((phenylcarbamoyl)oxy)benzoic acids Alzheimer drug, phenserine, we needed 3- and were prepared from commercial y available 3- 4-((phenylcarbamoyl)oxy)benzoic acids. The and 4-hydroxybenzoic acids in 76-90% overal problem was that these compounds are not yields. The main advantages of our method are commercial y available and procedures for their the simplicity, as no purification of preparation have also not been reported yet. We intermediates or final acids is required, and solved this problem by developing a simple effectiveness [2]. procedure to produce these acids in excel ent Orthogonal y protected aminocyclopentitols overal yields (Fig. 1B) [2]. were prepared from commercial y available D- Aminocyclopentitols are structural motifs that (−)-ribose, L-(+)-ribose and and D-(−)-lyxose in are present in a number of pharmacological y 11-22% overal yields. The absolute important natural products and drugs including configurations of the synthesized final antiviral drug, peramivir. Because we identified compounds were determined using NOESY a number of potential problems in the spectroscopy [3]. 28 KL4 Figure 1. Simple syntheses to ease the development of new anti-Alzheimer drugs (A and B) and antiviral drugs (C). 29 KL4 3.CONCLUSION We have developed convenient procedures for the synthesis of orthogonal y protected piperidines, (phenylcarbamoyl)oxybenzoic acids and orthogonal y protected aminocyclopentitols. These compounds are important building blocks which can ease the development of new anti-Alzheimer and antiviral drugs. 4. REFERENCES 1. Košak, U., et al., Straightforward synthesis of orthogonally protected piperidin-3- ylmethanamine and piperidin-4-ylmethanamine derivatives. Tetrahedron Let ers, 2014. 55(12): 2037-2039. 2. Košak, U., et al., A simple and effective synthesis of 3- and 4- ((phenylcarbamoyl)oxy)benzoic acids. Acta Chimica Slovenica, 2020. 67(3): 940-948. 3. Košak U., et al., Convenient syntheses of orthogonally protected aminocyclopentitols from aldopentoses. Tetrahedron Let ers, 2015. 56(3): 529-531. ACKNOWLEDGMENTS We thank the Ministry of Higher Education, Science and Technology of the Republic of Slovenia, the Slovenian Research Agency ARRS (grant No. Z1-9195 and core funding P1- 0208) and Lek Pharmaceuticals d.d. for financial support. 30 KL5 DESIGN, SYNTHESIS AND BIOCHEMICAL EVALUATION OF LOW- MOLECULAR-WEIGHT HUMAN NEUTROPHIL ELASTASE INHIBITORS Aleš Obreza1, Nina Porovne Černe1, Sara Koščak1, Špela Skala1, Damjan Avsec1, Maša Kandušer1, Irena Mlinarič Raščan1 1 University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia Correspondence: ales.obreza@ffa.uni-lj.si 1. INTRODUCTION Serine proteases account for approximately one 2. RESULTS AND DISCUSSION third of al known proteolytic enzymes. Their The aim of current study was the synthesis of importance for organisms is shown by the fact several related groups of N-benzylpiperazine-1- that they are found in al kingdoms of living carbohydrazide derivatives, thereby optimizing beings. In humans, they are involved in a variety the physicochemical and pharmacological of processes, such as blood clot ing, properties of previously reported compounds. intracel ular signaling, apoptosis, immune In particular, we tried to improve the potency response, and other processes. They are and selectivity of compounds and increase their at ractive targets in medicinal chemistry since water solubility, which in turn may affect their the catalytic mechanisms of this class of bioavailability as wel as facilitate the enzymes have been extensively investigated biochemical evaluation of the compounds in over the past few decades and the three- vitro. dimensional structure of numerous enzymes has . been published1. Human neutrophil elastase (HNE), a highly basic glycoprotein, is an important serine protease found in the azurophilic granules of the neutrophils. The potential substrates of an enzyme include almost al components of the extracel ular matrix, as wel as diverse proteins that include components of complement, immunoglobulins, clot ing factors and cytokines.2 It has been identified as a validated therapeutic target in several neurodegenerative and inflammatory diseases, its activity is upregulated in various cancer types. Many types of synthetic inhibitors have been reported in the literature and the inhibition of HNE has been Figure 1. Structure of compound 1 with fragments reviewed.3 that were replaced during the optimisation in the In the past years our research group has current study. designed and synthesized a large number of serine protease inhibitors, which were screened Compound 1 was optimized in three positions, for their activity against various enzymes. marked in Figure 1. Instead of amidoxime we Compound 1 has been published recently and introduced nitrile, carboxylic, aminomethyl and presented a useful lead compound in the design amino groups at positions 3 and 4 of aromatic of new potent and selective derivatives.4 ring, piperidine in the side chain was replaced with other cyclic secondary amines, especial y derivatives of piperazine. In the end we tried to 31 KL5 introduce more drug-like fragments instead of highly lipophilic and planar naphthalene ring. For this reason, we used sterical y smal er, substituted benzene rings and alkyl groups of different length. 3. CONCLUSION We prepared a medium-sized library of compounds that were in vitro biochemical y evaluated against HNE, trypsin, chymotrypsin, cathepsin G and proteinase 3. The results show promising inhibition of HNE and selectivity against other enzymes of some derivatives, when tested in low micromolar concentrations. 4. REFERENCES 1. Di Cera, E. Critical Review: Serine Proteases. Life, 2009. 61(5): 510-515. 2. Korkmaz, B., et al. Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases. Pharmacological reviews 2010, 62(4): 726-759. 3. Groutas, WC., et al. Neutrophil elastase inhibitors. Expert opinion on therapeutic patents 2011, 21(3): 339-354. 4. Wang X, et al. A Putative Serine Protease is Required to Initiate the RIPK3-MLKL— Mediated Necroptotic Death Pathway in Neutrophils. Frontiers in pharmacology 2021, doi: 10.3389/fphar.2020.614928 ACKNOWLEDGMENT The authors acknowledge the financial support from the Slovenian Research Agency. 32 KL6 ELECTROSPUN ANTIMICROBIAL PEPTIDES - NOVEL APPROACH FOR IMPROVED WOUND INFECTION TREATMENT Kairi Lorenz1, Celia Ramos1,2, Marta Putrinš1,3, Andres Meos1, Tanel Tenson3, James Mason2, Karin Kogermann1 1Institute of Pharmacy, University of Tartu, Estonia, Country 2Institute of Pharmaceutical Science, Kingś College London, United Kingdom 3Institute of Technology, University of Tartu, Estonia 1. INTRODUCTION Non-healing wounds are a huge problem for tested out. For efficacy studies, different society due to increased healthcare costs and pathogenic wound bacteria were used, such as significant burden to patients. It is known that A. baumannii, P. aeruginosa, S. aureus, E. coli. biofilm formation is one of the main problems associated with chronic wounds. [1] That is why 2.2. ES fiber matrix development novel management strategies have been Ten different peptide loaded ES matrices were proposed such as topical antimicrobials in created. Polymer solutions were made 24h prior advanced wound dressings which can be used to ES. For matrix preparation an ESR200RD eliminate the microbes. In addition, these robotized ES system (NanoNC, Seoul, Republic advanced dressings can interact with the wound of Korea) was used. Different ES methods and and support normal wound healing. conditions were tested. One of the methods that enables to produce such 2.3. ES fiber matrix characterisation wound dressings is electrospinning (ES). [2] ES Morphology of the ES matrices was nanofiber mats, as wound matrices, can be investigated using scanning electron functionalized using antimicrobial peptides microscopy, SEM (Zeiss EVO 15 MA, (AMPs). [3] AMPs have many advantages Germany). Fiber diameter was measured in compared to traditional antibiotics and topical Image J (N=100). AMP preparations are expected to have bet er Drug content and distribution across the matrix treatment outcomes local y in the wound than was analysed using high performance liquid systemic AMPs due to their antimicrobial, anti- chromatography (HPLC) (Shimadzu Europa inflammatory and/or immune modulating GmbH, Germany). Three random 1 cm2 or 4 properties. [4] cm2 pieces were used for this. The aim of the study was to understand the Drug release was studied from the fiber matrix behaviour of AMPs during ES for preparing to a 5 mL buffer solution (1xPBS, pH 7.4) at 37 functionalised AMP-loaded wound matrices C. Samples were col ected at fixed timepoints and characterise their physicochemical and analysed by using HPLC. properties and antimicrobial efficacy relevant for the treatment of infected wounds. Hence, we Bacterial studies such as modified minimum formulated different AMP-loaded fiber inhibitory concentration (MIC) evaluation of matrices and investigated their antimicrobial ES matrices in liquid broth and on agar plate properties in various antimicrobial assays. were carried out using pathogenic bacteria. 2. MATERIALS AND METHOD 2.1. Materials 3. RESULTS AND DISCUSSION Different AMPs (Pleurocidin, its analogue D- 3.1. Electrospinning (ES) and fiber Pleurocidin-KR, D-Pleurocidin and Temporin morphology L) were purified and used (Cambridge Research Successful ES was performed with al ten Biomedicals, UK). For developing ES different formulations in terms of stable and formulations, the selection of solvents and continuous ES process. SEM micrographs biodegradable high-quality polymers were revealed smooth fibers with uniform diameter 33 KL6 confirming suitable ES formulations and 3.3. Antimicrobial activity of peptide loaded conditions (Fig.1). fiber matrix Traditional disc diffusion test on agar plate was unsuccessful for our formulations. On the other A hand, formulations that successful y released the peptide from the matrix showed a drug loading dependent bacteriostatic or bactericidal effects in modified MIC test in liquid broth. 4. CONCLUSIONS To conclude, our study showed that ES of antimicrobial peptides (AMP) can be affected by many parameters. It is of importance to find suitable solvents, polymers, and ES methods for B the peptide to have a desired final fiber matrix with sufficient drug load and release from the fibers. Only then successful antimicrobial effects can be obtained. Further studies wil investigate these matrices in our developed in vitro and ex vivo wound infection models. 5. REFERENCES 1. Lindholm C, Searle R. 2016. Wound management for the 21st century: combining effectiveness and efficiency. Int Wound J 13:5– Figure 1. The morphology of AMP-loaded 15. fiber matrices. SEM micrographs of two 2. Preem L, Kogermann K. 2019. Electrospun electrospun Pleurocidin-loaded fiber matrices, Antimicrobial Wound Dressings: Novel A. with a hydrophobic polymer, and B. with a Strategies to Fight Against Wound Infections, p. hydrophilic polymer are shown as examples. 1–41. In Recent Clinical Techniques, Results, Magnification 10 000x. and Research in Wounds. Springer, Cham. 3. Felgueiras HP, Amorim MTP. 2017. 3.2. Peptide content in the fiber matrix and Functionalization of electrospun polymeric release into buffer solution wound dressings with antimicrobial peptides. AMP content in fiber matrix varied largely Col oids Surfaces B Biointerfaces 156:133– between different formulations. Results showed 148. that some solvents such as acetic acid degraded 4. Gera S, Kankuri E, Kogermann K. 2022. Antimicrobial peptides – Unleashing their the peptide prior and during ES, whereas in therapeutic potential using nanotechnology. other solvents the peptide was stable. Also, ES Pharmacology & Therapeutics 232, 107990 process affected the peptide stability, resulting in much higher peptide content when core-shel structured fibers were obtained. The release of ACKNOWLEDGMENT AMP from the fibers was dependent on the chosen polymer. Our results showed that using This work was funded by the Estonian Research hydrophobic polymers inhibited the drug Council grant PRG 1507. This presentation is release from the fiber matrix, but with supported by Doctoral School of Clinical hydrophilic polymer more than 50% of AMP Medicine, financed by the European Union was released into the buffer solution after 24 h. Development Foundation (University of Tartu ASTRA Project PER ASPERA). 34 KL9 SEMISYNTHETIC GLYCOPEPTIDE ANTIBIOTICS - ONE WEAPON AGAINST TWO DEADLY ENEMIES Anikó Borbás Department of Pharmaceutical Chemistry, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary Antibiotic resistance is a very serious global against RNA viruses such as influenza and healthcare issue, which we have to face human coronaviruses 229E and SARS- every day. While Gram-negative bacteria CoV-2.1-4 Moreover some of these are at the top of the list in causing severe, derivatives also exerted high antibacterial often incurable infections, antibiotic effect against resistant Gram positive strains.5 Modification of the peptide core of resistant Gram-positive bacteria also teicoplanin with the superbasic guanidino- represent a major threat. Both MRSA and group resulted in high antibacterial effect enterococci, namely Enterococcus faecalis against resistant enterococcus strains,6 and E. faecium are often resistants to while introduction of or multiple cationic traditional drugs such as vancomycin and aminoethyl moieties into the vancomycin other glycopeptide antibiotics. The aglycone led to synergistic activity against Gram-negative bacteria. COVID-19 pandemic has drawn at ention to the limited scope of antiviral drugs, In this lecture, our recent results on which, together with the increasing problem synthesis, antibacterial and antiviral evaluations and SAR analysis of our of antiviral drug resistance, also poses a semisynthetic glycopeptide derivatives wil serious global health threat. be presented. Glycopeptides are a class of cel wal biosynthesis inhibitor antibiotics used to treat drug-resistant Gram-positive bacterial REFERENCES: infections. In addition to the wel -known 1. Z. Szűcs, V. Kelemen, S. L. Thai, M. Csávás, E. antibacterial effect, glycopeptide antibiotics Rőth, G. Bat a, A. Stevaert, E. Vanderlinden, L. have exhibited potential for use against Naesens, P. Herczegh, A. Borbás, Structure- activity relationship studies of lipophilic various enveloped viruses. teicoplanin pseudoaglycon derivatives as new Over the past decade, our group has anti-influenza virus agents, Eur. J. Med. Chem. 2018 systematical y studied modifications of , 157, 1017-1030. 2. Z.Szűcs, Li. Naesens, A, Stevaert, E, Ostorházi, glycopeptide antibiotics, vancomycin, G, Bat a, P, Herczegh, A, Borbás, teicoplanin, and ristocetin to produce Reprogramming of the antibacterial drug compounds with high activity against vancomycin results in potent antiviral agents glycopeptide resistant bacteria, and to devoid of antibacterial activity, Parmaceuticals, 2020 design and prepare new derivatives that , 13, 139; 3. I. Bereczki, M. Csávás, Z. Szűcs, E. Rőth, G. posess strong antiviral activity against Bat a, E. Ostorházi, L. Naesens, A. Borbás, P. deadly pathogens such as influenza viruses Herczegh Synthesis of antiviral perfluoroalkyl and SARS-CoV-2. derivatives of teicoplanin and vancomycin – ChemMedChem 2020, 15, 1661–1671. Among these derivatives, teicoplanin, 4. I. Bereczki, H. Papp, … P. Herczegh, A. Borbás, ristocetin and vancomycin aglycones Natural apocarotenoids and their synthetic equipped with various lipophilic tags glycopeptide conjugates inhibit SARS-CoV-2 including natural apocarotenoids, or a replication, Pharmaceuticals, 2021, 14, 1111 5. Z. Szűcs, E. Ostorházi, M. Kicsák, L. Nagy, A. dual y lipo- and hydrophobic perfluoroalkyl Borbás, P. Herczegh, New semisynthetic chain displayed either promising activity teicoplanin derivatives have comparable in vitro 35 KL9 activity to that of oritavancin against clinical isolates of VRE, J. Antibiotics, 2019, 72, 524-534. 6. Z. Szűcs, I. Bereczki, E. Rőth, M. Márton, E. Ostorházi, G. Bat a, L. Nagy, Z. Dombrádi, A. Borbás, P. Herczegh, N-Terminal guanidine derivatives of teicoplanin antibiotics strongly active against glycopeptide resistant Enterococcus faecium. J. Antibiotics 2020, 73, 603–614 36 KL10 RATIONAL DESIGN OF BALANCED DUAL-TARGETING ANTIBACTERIAL COMPOUNDS WITH LIMITED RESISTANCE Lucija Peterlin Mašič1, Martina Durcik1, Tihomir Tomašič1, Žan Toplak1, Janez Ilaš1, Petra Szili2, Csaba Pal2 1 University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana 2 Synthetic and Systems Biology Unit, Biological Research Center, Szeged, Hungary 1. INTRODUCTION 2. MATERIALS AND METHODS There is an urgent need for new therapies and new antibiotics to treat deadly infections caused 2.1. Materials by so-cal ed ESKAPE pathogens bacteria that Material and methods include hit-to-lead are often resistant to available antibiotics. Not optimisation of new DNA gyrase B and only infections caused by these pathogens are topoisomerase IV (Fig. 1) inhibitors with difficult to treat, but discovering new therapies integrated methodology to obtain high quality to overcome Gram-negative resistance is leads with antibacterial activity against particularly chal enging. The World Health methicil in-resistant (MRSA), vancomycin- Organization (WHO) classifies these pathogens intermediate (VISA) Staphylococcus aureus as a critical threat because they are resistant to and Acinetobacter baumannii isolates that are al or nearly al of the antibiotics available less prone to resistance development. Integrated today. Over 95 percent of the antibiotics in methodology includes structure-based drug development today are being researched by design, organic synthesis, PADMET profiling, smal companies and academia, not the big antibacterial testing and in vivo antibacterial pharmaceutical companies that once dominated efficacy testing [2, 3]. the field [1]. To ensure that the supply of new antibiotics keeps pace with these evolving pathogens, a robust pipeline of new antibiotics and innovative solutions is needed. One promising strategy to address this rapid evolution of resistance is the design of antimicrobial compounds that equipotently inhibit multiple bacterial targets. The rationale for this approach is that the development of resistance to multitargeting antibiotics would require the simultaneous occurrence of multiple specific mutations at al targets, which is extremely rare. Figure 1. Targeted proteins: ATP-binding sites of Therefore, multitargeting antibiotics (MTA) DNA gyrase B and topoisomerase IV (ParE). should be less susceptible to resistance compared to monotargeting antibiotics. However, designing antibiotic compounds that 3. RESULTS AND DISCUSSION equipotently inhibit two or more bacterial 3.1. Design of ULD1 and ULD2 compounds targets while exhibiting adequate antibacterial ULD1 and ULD2 compounds were designed efficacy, safety, and physicochemical using structure-based design based on several properties remains a chal enge. Although a cocrystal structures [2]. major focus of companies, only a handful of such antibiotics show balanced inhibition at multiple targets. 37 KL10 clinical antibiotic against systemic MRSA infections, but resistance against this drug is emerging rapidly [2]. Figure 2. Structures of ULD1 and ULD2. 3.2. Balanced dual enzyme inhibition Fig. 3 presents low nanomolar balanced dual inhibitions of ULD1 and ULD2 on DNA gyrase B and topoisomerase IV from several bacterial species [2]. Figure 5. Antibacterial activities of ULD1 and ULD2 against a panel of 95 S. aureus clinical isolates. 4. CONCLUSION We have discovered new multitargeting antibacterial compounds, namely ULD1 and ULD2, which belong to a new chemical class. Figure 3. Dual inhibition of compounds ULD1 and They inhibit almost equipotent in the low ULD 2. nanomolar range bacterial DNA gyrase and topoisomerase complexes IV from S. aureus, A. 3.3. Antibacterial potencies of ULD1 and baumannii and E. coli and interact with several ULD2 evolutionarily conserved amino acids in the Antibacterial potencies against Gram-negative ATP binding pockets of their target proteins. and Gram-positive bacteria are presented in the Resistance mutations to these compounds are Fig. 4 [2]. rare, have limited effects on compound sensitivity, and significantly reduce bacterial growth. Based on their antibacterial potency and lack of toxicity demonstrated in mice in the VISA infection model, these compounds could lead to new therapies against multidrug- resistant bacterial infections. 5. REFERENCES 1. Brown, ED et al. Nature, 2016, 529, 336. 2. NYERGES, A. TOMAŠIČ, T., DURCIK, M. ET AL., PLOS BIOL 18(10): E3000819. 3. TOMAŠIČ, T, ZIDAR, N, DURCIK, M, ILAŠ, Janez, ZEGA, A, DURANTE CRUZ, C, Figure 4. Antibacterial potencies of ULD1 and TAMMELA, P, PÁL, C, NYERGES, Á, ULD2. KIKELJ, D, PETERLIN-MAŠIČ, L. New class of DNA gyrase and/or topoisomerase IV 3.4. Antibacterial activities against 95 S. inhibitors with activity against gram-positive aureus MRSA and vancomycin-resistant and gram-negative bacteria : Patent application isolates in vitro publication US 2021/0323957 A1, 2021-10-21. 100% of the strains were inhibited at 1 μg/mL ACKNOWLEDGMENT concentration of ULD1 and ULD2 (Fig. 5) [2]. This research was funded by Slovenian Research 3.5. Efficacy of ULD1 (iv) in a neutropenic Agency (ARRS) grant numbers J1-9192, N1-0098, mouse infection of Staphylococcus aureus P1-0245, J1-3030 and IMI ENABLE project: VISA ATCC 700699 European Gram-negative Antibacterial Engine. The antibacterial activity of ULD1 was comparable to that of linezolid, a widely used 38 KL13 APPLICATION OF HOT-MELT TECHNOLOGIES IN DESIGNING FORMULATIONS WITH POORLY WATER-SOLUBLE ACTIVE INGREDIENTS Gregor Lorbek1, Klemen Korasa1 1 Krka, d. d., Novo mesto, Slovenia 1. INTRODUCTION 2.1. Materials The pharmaceutical industry encounters many We used hot-melt granulation to prepare a film- chal enges, e.g. poor active ingredient coated tablet containing a poorly water-soluble solubility, rising manufacturing costs, active ingredient, a low-melting point tightening environmental policies and extensive hydrophilic binder poloxamer 188 (BASF, patent protection. Newer approaches, such as USA), and other suitable commercial y hot-melt granulation, extrusion or coating are available excipients. Hot-melt extrusion process becoming recognized for their speed, cost- was investigated with polymers polyvinyl effectiveness and smal environmental alcohol (PVA) (Merck, Germany), copovidone footprint. Their versatility can be used to (Ashland, USA), and Soluplus® (BASF, USA). increase active ingredient solubility and Extrudates of solid dispersions were then bioavailability, taste masking, and for formulated into film-coated tablets with other modifying or control ing active ingredient excipients. release. For instance, the dissolution rate of 2.2. Hot-melt granulation in a high-shear poorly water-soluble active ingredients can be mixer significantly improved with melt granulation by GEA Col et e Gral 10 high-shear mixer (GEA, using hydrophilic binders that improve Germany) was used for hot-melt granulation. wet ability through intimate contact with the Temperature of the heated jacket ranged active ingredient [1]. Also, hot-melt extrusion between 70–80 °C, and end-point granulate has become a method of choice for improving temperature between 58–68 °C. A 32 ful dissolution rate through amorphous solid factorial design of experiments (Design Expert dispersions, where a systematic developmental v12, Stat-Ease, USA) was employed to assess approach of polymer screening, multivariate the influence of the two temperatures on assay statistics and Quality-by-Design is highly and content uniformity. recommended [2]. Nevertheless, several potential disadvantages should be considered, 2.3. Hot-melt extrusion with twin-screw extruder e.g. stability of thermolabile active ingredients Coperion ZSK MI18 twin-screw extruder due to high processing temperatures or potential (Coperion, Germany) was used for hot-melt recrystal ization of amorphous solid extrusion. Barrel temperature in the range dispersions. To a certain extent we stil lack between 190–230 °C, feed rate from knowledge about predictability and model ing 10–30 g/min, different active ingredient particle of these processes. We intend to show possible sizes and various active ingredient-to-polymer applications of hot-melt granulation and ratios were assessed for their effects on extrusion technologies used in development and physicochemical properties of solid dispersions. production of formulations containing poorly water-soluble active ingredients. 3. RESULTS AND DISCUSSION 2. MATERIALS AND METHODS 39 KL13 3.1. Optimization of hot-melt granulation produce amorphous solid dispersions with al process three polymers at a smal scale. Soluplus® was According to literature, solubility of a model later discarded because the dissolution rate was poorly water-soluble active ingredient can be too low compared with the other two polymers. increased by a hydrophilization process in a PVA formed a very hard extrudate that was fluid-bed granulator. Our approach was to use difficult to reduce to finer particles by mil ing, hot-melt in-situ granulation using hydrophilic which led to problems with content uniformity. binders [1] that led to a low-cost water-free In the next phase, we therefore optimized the formulation with desired active ingredient process only with copovidone in order to solubility. The assay and content uniformity achieve continuous production of physical y chal enges were investigated by varying stable amorphous solid dispersion with no temperatures of the heated jacket and end-point detectable crystal ine drug particles that could granulate temperatures using a 32 ful factorial trigger recrystal ization. The extrusion experimental design. Shorter granulation times temperature, active ingredient-to-polymer ratio, resulted in lower assay, and longer times in dwell time that was control ed by feed rate, and higher assay, whereas no effect was observed on drug particle size had an important effect on the content uniformity, which remained high [Fig. level of residual crystal inity in the amorphous 1]. This was solved with particle size reduction solid dispersion. By determining optimal values of poloxamer 188, since more nucleation points of these critical processing parameters, we were for granule growth were introduced to the able to produce physical y stable, completely granulation mixture this way, shifting from amorphous solid dispersion on the pilot scale bimodal to more uniform particle size with the desired dissolution rate. distribution of the granulate, which reduced the relative standard deviation of content three- 4. CONCLUSION fold. We were able to produce physical y stable formulations with improved active ingredient solubility, the desired dissolution rate, and good technological processability by employing hot- melt granulation and extrusion techniques. During the development, we obtained good understanding of the processes that wil hopeful y further advance the use of hot-melt Figure 1. Contour plots of the heated jacket and end-technologies in the pharmaceutical industry. point granulate temperature showing higher assay at 5. REFERENCES longer processing times (i.e. at lower jacket and higher end-point granulate temperatures), and lower assay at 1. Kukec S., Dreu R., Vrbanec T., Srčič S. and shorter processing times (i.e. higher jacket and lower Vrečer F., Characterization of agglomerated end-point granulate temperatures). No significant carvedilol by hot-melt process in a fluid bed and effect on content uniformity was observed. high shear granulator. International Journal of 3.2. Designing a hot-melt extrusion Pharmaceutics, 2012. 430(1-2): 74-85. 2. Simões M.F., Pinto R. M. A., Simões S., Hot- formulation melt extrusion in the pharmaceutical industry: In the second example, hot-melt extrusion was toward filing a new drug application. Drug applied to design a solid dosage form containing Discovery Today, 2019. 24(9):1749-1768. poorly water-soluble active ingredient. An initial screening of high-degradation ACKNOWLEDGMENT temperature polymers copovidone, Soluplus®, and PVA by DSC showed good mixing of high- This research was supported by Krka, d. d., melting point active ingredient only with Novo mesto. copovidone. Nevertheless, we were able to 40 KL14 MASS SPECTROMETRY AS A POWERFUL TOOL FOR ANALYSING DEGRADATION PATHWAYS: 1,4-BENZODIAZEPINE CASE STUDY Žiga Hodnik1, Jure Bezenšek2, Aleš Judež1 1 Pharmaceutical R&D, Analytics Development, Krka, d. d., Slovenia 2 API R&D, Chemistry, Krka, d. d., Slovenia 1. INTRODUCTION According to our experience and reference data Control of active pharmaceutical ingredient [3], aminobenzophenone, is a predominant (API) degradation products is essential for degradation product in 1,4-benzodipazepine safety of pharmaceutical products. Therefore, parenteral products, while the aforesaid batch understanding API degradation pathways works showed a significant degradation to a so far as the basis for analytical and pharmaceutical unknown impurity in our development formulation scientists when they are developing formulation. new products or improving product life cycles [1]. High-resolution mass spectrometry coupled with high- or ultra-performance liquid chromatography (LC-HRMS) is one of the most established techniques for analysing the Figure 1. Common, acid-catalysed degradation structure of degradation products, since it does pathway of 1,4-benzodiazepine versus metal- catalysed degradation pathway. not require their isolation, which is demanding due to their low presence in pharmaceutical We therefore performed a LC-HRMS analysis products [1]. On the other hand, inductively of the batch sample. After overcoming different coupled plasma mass spectrometry (ICP-MS) ionization behaviour of the degradation product represents one of the most powerful tools for compared to the parent API, the analysis detection and quantification of elemental revealed the same exact mass, together with impurities in pharmaceutical products due to comparable MS/MS spectrum. Furthermore, ICP-MS detection limits in ppm or sub-ppm hydrogen-deuterium exchange experiment ranges [2]. revealed the presence of two additional exchangeable hydrogen atoms in the Herein, we present a less common degradation degradation product, which indicated possible pathway of 1,4-benzodiazepine depressant in phenylquinoline-2-one structure. The LC- parenteral solutions, which was discovered HRMS results were later confirmed using a using aforesaid techniques. 1,4- suitable reference substance. Benzodiazepines, which act as enhancers of inhibitory neurotransmit er GABA, most often A statistical analysis of manufacturing process degrade to aminobenzophenone impurity in an and routine analyses data for al development acidic aqueous medium, while we discovered a batches was performed. The statistical analysis metal catalysed redirection of degradation did not detect any trends or outliers. While pH pathway into phenylquinoline-2-one impurity and temperature, as most common factors for (Fig. 1). directing degradation pathways in parenteral formulations, did not stand out, we decided to 2. RESULTS AND DISCUSSION perform an ICP-MS analysis of elemental While performing routine high-performance impurities. liquid chromatography (HPLC), we detected an ICP-MS revealed only a minor difference in a unusual trend in the content of related single metal ion content in the outlier batch substances of 1,4-benzodiazepine in one of the compared to other development batches. Even development batches of parenteral formulation. though the difference was present in the very 41 KL14 low-ppm range, we performed additional ICP- the stability of pharmaceutical products. The MS analyses, which revealed that glass rigorous control of contaminants in ampoules were a source of the low-ppm range pharmaceutical industry therefore represents an content of pathway-directing metal. important element for the quality and safety of Final y, the spiking of a suitable metal salt in the pharmaceutical products. otherwise stable batch of parenteral solution confirmed its pathway-directing impact. 4. REFERENCES 3. CONCLUSION 1. Baertschi, S W et al., Pharmaceutical Stress To summarize, the capacity of the state-of-the-Testing: Predicting Drug Degradation. Informa art LC-HRMS instruments to provide for a Healthcare, 2011. 2nd Edition. detailed insight into chemical structures of API 2. Barin, S J et al., Determination of Elemental degradation products together with ICP-MS Impurities in Pharmaceutical Products and high sensitivity for the presence of trace Related Matrices by ICP-Based Methods: a elemental impurities is a powerful tool for an Review. Analytical and Bioanalytical Chemistry, 2016. 408(17): 4547-4566. analytical chemist involved in the development 3. Loftsson, T et al., 1,4-Benzodiazepines: of pharmaceutical products. Chemical Stability and Cyclodextrin Despite the negligible presence of contaminants Solubilisation. Journal of Drug Delivery in low-ppm range, such contaminants can act as Science and Technology, 2021. 66: 102936. powerful catalysts, which significantly affect 42 KL16 PERSONALIZED MEDICINE: DRUG DEVELOPMENT AND USAGE Cansel KOSE OZKAN Gulhane Faculty of Pharmacy, Department of Pharmaceutical Technology, University of Health Sciences, Ankara, TURKIYE select the specific administration form that a 1. INTRODUCTION patient needs. Personalized medicine, also cal ed precision The effective delivery of tailored medications medicine in some studies, is a medical model created through pharmacy compounding to the that divides people into different groups by body's il ness locations is one ongoing study tailoring medical decisions, practices, field. For instance, by combining real-time interventions and/or products to each patient imaging and examining the pharmacodynamics according to the predicted response or risk of of the drug delivery, researchers are at empting disease [1]. The terms personalized medicine, to create nanocarriers that can precisely target precision medicine, layered medicine, and P4 the specific location. medicine are often used interchangeably to describe this concept, although the terms are Although alternative routes such as mucosa, sometimes used separately to indicate specific skin, and lung have become popular in recent points [2]. This concept is summarized as years taking advantage of advances in drug therapy in the right patient, with the right drug, delivery technology, cost is significant and at the right dose. many oral formulations can be produced at lower costs, al owing for a wide variety of The main goal of the design and development alternatives. process is to create a product that can be used safely and effectively by the patient group. The oral route remains the most important pil ar From a pharmaceutical industry perspective, of drug dosing methods due to cost, ICH Q8 guidance leads to the definition of a convenience, and patient compliance. Quality Targeted Product Profile (QTPP) for a The likelihood of patient non-adherence particular drug product and patient population. increases with the complexity of the drug It is recognized that designing drugs suitable for regimen and is of particular concern in patients paediatrics, adults and elderly patients wil with complex health conditions combined with result in multiple presentations that can lead to multiple drug experience from various smal er production batch sizes. Dosage indications. Complex dosing regimens often flexibility, easy-to-swal ow formulations and benefit from oral user-friendly dosage forms more accessible packaging are al factors to due to their reduced side effects and simplified consider. Dose flexibility can be achieved with regimens in studies of the treatment of patients. a variety of dosage forms such as oral liquids, mini-tablets, or multiple particles [3] Good taste sensation and taste masking techniques in patients who refuse or cannot Pharmaceutical compounding is one of the swal ow tablets, such as paediatric and geriatric applications of personalised medicine. Though patients, may change the drug perception, not always utilizing genetic data, the specialized especial y in paediatric patients, such as the manufacturing of a medication with a range of resistance of the bit er pil . Products that qualities (e.g. dose, ingredient selection, route disintegrate rapidly in the mouth dissolve in the of administration, etc.) are selected and crafted mouth within seconds and in such cases may for an individual patient is accepted as an area increase the clinical effect of the drug due to of personalised medicine. pre-gastric absorption in the mouth, pharynx, Pharmaceutical compounding offers a variety of and oesophagus, avoiding first pass hepatic benefits, like gives a prescriber the option to metabolism. the drug can significantly increase compared to conventional tablets. 43 KL16 In some indications, such as migraine, the faster 2. CONCLUSION onset of action associated with oral y disintegrating drug forms may increase patient In conclusion, patient comfort is a decisive satisfaction and adherence to treatment. While factor in drug compliance and bet er outcomes. advanced formulations may be more expensive The benefit of a drug can be achieved primarily than conventional dosage forms, user-friendly by designing and developing appropriate dosage forms often have more favourable formulation options tailored to the needs of pharmacological profiles and therefore have the specific groups of patients. Oral delivery potential to reduce overal treatment cost by systems save costs because they can be reducing the burden on healthcare. produced in large quantities within short production times while maintaining consistent Another aspect that is critical for certain patient quality. It should be noted that the healthcare populations is the need to take multiple system benefits financial y from user-friendly medications, especial y for the elderly who are dosage forms. However, much more research is bat ling multiple diseases. The development of needed to balance both patient dosage fixed-dose combination products may result in preferences and the needs of the caregiver, fewer prescriptions and a bet er cost-benefit prescriber, and payer. ratio than prescribing drugs individual y. The most important chal enges to be overcome 3. REFERENCES in the development of oral user-friendly 1. Academy of Medical Sciences. Stratified, formulations are physiological y impaired personalised or P4 medicine: a new direction for swal owing function in specific patient groups, placing the patient at the centre of healthcare the widespread need for taste masking in oral and health education. 2015. solutions or orodispersible tablets, the need to 2. Movafagh, A. Personalised medicine in modern develop more analytical technologies, with the era. Asian Pacific Journal of Cancer Biology, 2016, 1.(2): 31-32. addition of more excipients to make a more 3. Page, S, et al., An industrial perspective on the user-friendly product, which techniques are design and development of medicines for older more complex and more interactive thereby patients. Int J Pharm. 2016 Oct 30;512(2):352- increasing the possibility of much more 354. formulation instability. Minimum dosing 4. Thomas, F. Making the Ideal Dosage Form. frequency is particularly important in the case Pharmaceutical Technology, Supp.: APIs, of adherence, and there have been numerous Excipients, and Manufacturing, 2020. 4: 17–18. studies investigating the role the dosing 5. Tovey, G. D. (ed.). Pharmaceutical schedule can play in medication adherence. Formulation: The Science and Technology of There are significant physiological and Dosage Forms. Royal Society of Chemistry, 2018. metabolic differences between children and adults in the design of paediatric drugs, and there are significant differences in drug dose, especial y in cases where chronic disease is evident. Understanding the properties and requirements of an active ingredient in terms of formulation is critical to proper product design and performance. Before an API formulated into a dosage form, its chemical and physical properties need to be characterized. Certain physicochemical properties may make oral administration of a substance impossible; this is general y accurate for proteins. The required solubility also requires specific formulation approaches [4,5]. 44 KL19 PHARMACOGENOMIC AND BIOLOGICAL IMPLICATIONS OF THIOPURINE S-METHYLTRANSFERASE Dunja Urbančič1, Alenka Šmid1, Anita Kotar2, Janez Plavec2, Nataša Karas Kuželički1, Irena Mlinarič-Raščan1 1 Faculty of Pharmacy, University of Ljubljana, Slovenia 2 Slovenian NMR Centre, National Institute of Chemistry, Slovenia 1. INTRODUCTION represent a useful indirect pharmacogenomic Despite major therapeutic advances in the field marker for future thiopurine treatment. of biologic agents, smal molecules remain an important therapeutic pil ar in many diseases. Among them, thiopurines - cytostatics and immunosuppressants - are a regularly prescribed agents for the treatment of childhood leukemia and certain autoimmune diseases. One of the most important factors affecting thier efficacy and toxicity is the activity of an enzyme thiopurine S-methyltransferase (TPMT). Genetic polymorphisms in its gene, metabolic factors that stabilize TPMT, or drug interactions Figure 1. The evolution of motives ABnC in VNTR associated with thiopurine treatment often lead region of TPMT and their linkage with TPMT*3 to interindividual differences in response to genetic polymorphisms [2]. thiopurines. Our studies thus aim to identify novel genetic determinants of response to thiopurines and to define molecular 3. INFLUENCE OF DRUGS AND CO- mechanisms of drug interactions during FACTOR ON TPMT ACTIVITY thiopurine therapy. Since the biological role of TPMT is stil unclear, we further try to In addition to genetic variants, other factors, understand its endogenous function and its such as the cofactor S-adenosylmethionine involvement in physiological processes. (SAM), substrates, and inhibitory drugs, may modulate TPMT activity and thus influence the 2. TPMT AS FARMACOGENOMIC outcomes of thiopurine therapy. The role of BIOMARKER SAM in stabilising the TPMT structure and Individuals differ with respect to their TPMT consequently maintaining its activity is wel activity. TPMT activity depends mainly on documented [3]. On the other hand, the genetic variants, with TPMT*3C and TPMT*3A mechanisms of interaction between thiopurines being the most common, coding for lower and other drugs that are often administered enzyme activity [1]. However, in the search for concomitantly with them remain to be new pharmacogenomic markers, we realised elucidated. In this context, we specifical y that the exact DNA sequence in the TPMT focused on the interaction of TPMT with promoter, which contains a variable number of sulfasalazine, a derivative of 5-aminosalicylic tandem repeats (VNTR), is also important. The acid, which is used as a first-line treatment for ABnC motifs are in complete linkage inflammatory bowel disease. By measuring the disequilibrium with the TPMT*3C and fluorescence change of tryptophan residues in TPMT*3A genetic polymorphisms (Fig. 1) [2]. human recombinant TPMT, we confirmed the Since TPMT*3 al eles are clinical y established binding of sulfasalazine to TPMT, and by in thiopurine dosage adjustment, ABnC motifs 45 KL19 measuring TPMT activity, we determined lines (LCLs) from individuals with different mixed inhibition of the enzyme. TPMT genotypes, TPMT knock-out Hap1 cel s, 4. THE BIOLOGY OF TPMT and TPMT-transfected cel s. When we evaluated the sensitivity of the cel s to selenium Despite the importance of TPMT in clinical compounds, we found that cel s with high practice, it appears that decreased TPMT TPMT activity were less sensitive to selenium activity does not cause changes in the compounds than cel s with low or undetectable development and function of the (human) body. TPMT activity. In the absence of thioupurine exposure, individuals with inactive TPMT are 6. CONCLUSION phenotypical y indistinguishable from Our studies focus on deciphering TPMT from individuals whose TPMT has normal activity. A pharmacological and biochemical perspectives. number of orthologous TPMT proteins are Expanding the spectrum of genetic markers for known. The relatively high homology between susceptibility to thiopurines may contribute to TPMT sequences from biological species that future improvements in thiopurine treatment are very distant in evolutionary development outcomes. The demonstration of methylation of indicates that TPMT has been present for a long selenium compounds by TPMT was a good time and suggests the importance of the enzyme starting point for further elucidation of the role in the survival of individual species. In humans, of TPMT in pathological processes through the TPMT enzyme is expressed in most tissues selenium metabolism. and organs, most abundantly in the thyroid gland, fol owed by the kidneys, various parts of 7. REFERENCES the intestine, and the liver. 1. Tamm, R., et al., Polymorphic Variation in TPMT Is the Principal Determinant of TPMT 5. INVOLVEMENT OF TPMT IN Phenotype: A Meta-Analysis of Three Genome- SELENIUM METABOLISM Wide Association Studies. Clinical Since certain selenium-containing organic pharmacology and therapeutics, 2017. 10 (5): compounds have chemical properties similar to 684-695. those of thiopurines, we have investigated the 2. Urbancic, D., et al., Novel motif of variable number of tandem repeats in TPMT promoter role of this enzyme in selenium metabolism. region and evolutionary association of variable Using STD NMR spectroscopy and tryptophan number of tandem repeats with TPMT*3 alleles. fluorescence measurements, we demonstrated Pharmacogenomics, 2018. 19 (17): 1311-1322. the binding of an organic selenium compound, 3. Karas Kuzelicki N., et al., From selenocysteine (Sec), to human recombinant pharmacogenetics to pharmacometabolomics: TPMT. The incubation of Sec with TPMT and SAM modulates TPMT activity SAM, resulted in a formation of a methylated Pharmacogenomics. 2014. 15(11): 1437-1449. product, Se-methlyselenocysteine, revealing 4. Urbancic, D., et al. Methylation of Sec to be the first known biological compound selenocysteine catalysed by thiopurine S- to act as a substrate for TPMT [4]. methyltransferase. Biochimica et Biophysica Acta - General Subjects, 2019. 1863(1): 182-190. ACKNOWLEDGMENT This work was supported by Slovenian Research Agency [P1-0208], and by the Ministry of Education, Science, and Sport (MIZŠ) and the European Regional Development Fund OP20.05187 RI-SI- EATRIS. Figure 2. Selenocysteine methylation reaction catalysed by TPMT [4]. To investigate the biological relevance of this finding, we performed experiments using different in vitro systems: lymphoblastoid cel 46 KL21 MODEL-INFORMED PRECISION DOSING OF USTEKINUMAB IN CROHN'S DISEASE Jurij Aguiar Zdovc1, Jurij Hanžel2,3, Erwin Dreesen4,5, Debby Thomas4, Barbara Ostanek1, Tomaž Vovk1, David Drobne2,3, Iztok Grabnar1 1 University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia 2 Department of Gastroenterology, University Medical Centre Ljubljana, Ljubljana, Slovenia 3 University of Ljubljana, Medical Faculty, Ljubljana, Slovenia 4 Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium 5 Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California, USA 1. INTRODUCTION fit ed to the unbound serum concentrations [12, Crohn’s disease (CD) is a chronic relapsing- 13]. Stepwise covariate modeling was used for remit ing inflammatory bowel disease. The covariate testing and sequential PD modeling decreased fecal calprotectin (FC) concentration was performed to describe the relationship indicates a decreased biochemical disease between ustekinumab, latent target and FC. A activity [1, 2]. Ustekinumab is a monoclonal population of 10000 virtual patients and the antibody used in moderate-to-severe CD [3], final PK-PD model were used for simulation of but there is an unmet need for the optimization various treatment regimens. To compare the of the standard treatment based on the reported efficacy, the proportion of patients achieving proportions of patients achieving remission [4– biochemical remission (FC < 100 mg/kg) at 11]. However, the optimal dosing strategy and weeks 8, 16, 24 and 32 was calculated. the identification of patients with a benefit from 3. RESULTS AND DISCUSSION dose optimization remain unresolved dilemmas in clinical practice. To compare the efficacy of Ustekinumab exposure, as wel as the several clinical y relevant treatment regimens of probability for biochemical remission, ustekinumab in CD, we developed a population increased with increasing serum albumin, pharmacokinetic-pharmacodynamic (PK-PD) decreasing fat-free mass, decreasing C-reactive model of ustekinumab and FC. protein, previous non-exposure to biologics and FCGR3A-158 V/V variant. Model-based 2. MATERIALS AND METHODS simulation demonstrated that proportions of We performed a prospective, observational, patients in remission at week 32 receiving single-center cohort clinical study including 57 standard treatment, treatment with initial CD patients, aged 18 years or older who started induction and subsequent maintenance dosing treatment with ustekinumab between October every 4 weeks or treatment with induction 2017 and June 2019. Al patients received a dosing every 8 weeks were 41.9%, 52.2% and standard ustekinumab treatment and were 56.0%, respectively. fol owed for 32 weeks after the first dose. We 4. CONCLUSION prospectively measured unbound serum ustekinumab concentration and FC Our model could be used to guide future concentration, as wel as recorded other at empts to stratify treatment with ustekinumab patients’ characteristics, clinical data and in CD. biochemical markers. Polymorphisms in IL12B 5. REFERENCES (rs3212227, rs3213094, rs6887695), FCGR2A (rs1801274), and 1. Ng SC, Shi HY, Hamidi N, et al (2017) FCGR3A (rs396991) were Worldwide incidence and prevalence of genotyped. Target mediated drug disposition inflammatory bowel disease in the 21st (TMDD) model and its approximations were century: a systematic review of population- 47 KL21 based studies. Lancet 390:2769–2778. 8. Verstockt B, Dreesen E, Noman M, et al 2. Peyrin-Biroulet L, Sandborn W, Sands BE, (2019) Ustekinumab Exposure-outcome et al (2015) Selecting Therapeutic Targets in Analysis in Crohn’s Disease Only in Part Inflammatory Bowel Disease (STRIDE): Explains Limited Endoscopic Remission Determining Therapeutic Goals for Treat-to- Rates. J Crohn’s Colitis 1–9. Target. Am J Gastroenterol 110:1324–1338 9. Hanžel J, Zdovc J, Kurent T, et al (2021) . Peak Concentrations of Ustekinumab After 3. Feagan BG, Sandborn WJ, Gasink C, et al Intravenous Induction Therapy Identify (2016) Ustekinumab as Induction and Patients With Crohn’s Disease Likely to Maintenance Therapy for Crohn’s Disease. Achieve Endoscopic and Biochemical N Engl J Med 375:1946–1960. Remission. Clin Gastroenterol Hepatol 19:111-118.e10. 4. Iborra M, Beltrán B, Fernández-Clotet A, et al (2019) Real-world short-term 10. Zdovc JA, Hanžel J, Kurent T, et al (2021) effectiveness of ustekinumab in 305 patients Ustekinumab dosing individualization in with Crohn’s disease: results from the crohn’s disease guided by a population ENEIDA registry. Aliment Pharmacol Ther pharmacokinetic–Pharmacodynamic model. 50:278–288. Pharmaceutics 13:1–16. 5. Biemans VBC, van der Meulen - de Jong 11. Wang Z, Verstockt B, Sabino J, et al (2021) AE, van der Woude CJ, et al (2019) Population pharmacokinetic- Ustekinumab for Crohn’s Disease: Results pharmacodynamic model-based exploration of the ICC Registry, a Nationwide of alternative ustekinumab dosage regimens Prospective Observational Cohort Study. J for patients with Crohn’s disease. Br J Clin Crohn’s Colitis jjz119. Pharmacol 1–13. 6. Bat at R, Kopylov U, Bessissow T, et al 12. Gibiansky L, Gibiansky E (2009) Target- (2017) Association Between Ustekinumab mediated drug disposition model: Trough Concentrations and Clinical, approximations, identifiability of model Biomarker, and Endoscopic Outcomes in parameters and applications to the Patients With Crohn’s Disease. Clin population pharmacokinetic– Gastroenterol Hepatol 15:1427-1434.e2. pharmacodynamic modeling of biologics. Expert Opin Drug Metab Toxicol 5:803– 7. Ma C, Fedorak RN, Kaplan GG, et al (2017) 812. Clinical, endoscopic and radiographic outcomes with ustekinumab in medical y- 13. Ternant D, Monjanel H, Venel Y, et al refractory Crohn’s disease: real world (2019) Nonlinear pharmacokinetics of experience from a multicentre cohort. rituximab in non-Hodgkin lymphomas: A Aliment Pharmacol Ther 45:1232–1243. pilot study. Br J Clin Pharmacol 85:2002– 2010. 48 KL22 CROSSING OCULAR BARRIERS: PHARMACOKINETIC MODELS TO SUPPORT DRUG DEVELOPMENT IN RETINAL DISEASES Eva Maria del Amo1 1 University of Eastern Finland, School of Pharmacy, Biopharmaceutics, Yliopistonranta 1, 70210 Kuopio, Finland 1. INTRODUCTION in-house using LC-MS analytical method) after The eye is an amazing and complex organ that intravitreal injection (1-4). al ows us to interact with the surrounding world. ●In silico calculation of molecular descriptors Ocular protective mechanisms, flows, and of low-MW drugs with ACDlabs software (v 12, barriers limit the access of therapeutics drugs Advanced Chemistry Development, Inc., into the eye, and the drug delivery to the Canada), regression Quantitative Structure- posterior segment (i.e. retina) is especial y Property Relationships (QSPR) modeling with difficult. The route of administration that can Simca+v.10.5 Umetrics, Umeå, Sweden and ensure retinal drug delivery is the intravitreal pharmacokinetics analysis using compartmental injection (Fig. 1), and the pharmacokinetics is & non-compartmental analysis with Phoenix described in this presentation. A comprehensive WinNonlin (build 8.3.1, Certara L.P.) and quantitative pharmacokinetic data and analysis simulations with Stella professional, Modelling of low- and high-molecular-weight (MW) drugs & Simulation software (v1.5.1, isee systems, are presented, with the key pharmacokinetic Inc., USA) (1-5). parameters, the contributing mechanisms, and the link to the ocular physiology. 3. RESULTS AND DISCUSSION Pharmacokinetic models to predict ocular drug ● Comprehensive data collections and the concentration profiles are presented, which are intravitreal primary pharmacokinetic extremely useful when designing new parameters for low- and high-MW drugs were formulations or deciding new dosing regimens. obtained (1-3): 1) Volume of distribution (Vss,ivt) of the investigated drugs was fairly similar, 2) Clearance (CLivt) had broad variance, and predictive CLivt QSPR model was built for low- MW drugs where logD7.4 and hydrogen bonding were the key descriptors. In general, metabolism do not impact clearance, and the relevant routes are the posterior and anterior routes (for low- and high-MW drugs, respectively). ● Human and rabbit intravitreal Fig. 1. Intravitreal injection to treat diseases of the pharmacokinetic correlate wel . The scaling posterior segment of the eye. factors generated al ows reliable translation from rabbit-to-human (4). 2. MATERIALS AND METHODS ● In silico tools integrating the CLivt QSPR In vivo, in silico, and literature data and related model and Vss,ivt typical values in methods were utilized: pharmacokinetic simulation models al ow reliable calculations of drug ocular ●In vivo pharmacokinetics studies in humans concentration profiles. Intravitreal delivery (from literature) and rabbits (from literature or systems can be incorporated into the 49 KL22 simulations to estimate dose-requirement as a 2. del Amo EM, et al. Intravitreal clearance and function of the constant release of the system, volume of distribution of compounds in rabbits: or the dosing interval. This provides the In silico prediction and pharmacokinetic appropriate framework of various scenarios in simulations for drug development. Eur J Pharm designing drug delivery systems (2-5). Biopharm. 2015; 95: 215-26. doi.org/10.1016/j.ejpb.2015.01.003 3. del Amo EM, et al. Pharmacokinetic aspects of 4. CONCLUSION retinal drug delivery. Prog Retinal Eye Res. 2017;57:134-85. The generated knowledge and pharmacokinetic doi.org/10.1016/j.preteyeres.2016.12.001 models can be relevant tools to advance 4. del Amo EM et al. Rabbit as an animal model ophthalmic drug development and guide the for intravitreal pharmacokinetics: Clinical design of drug delivery systems to treat retinal predictability and quality of the published data. diseases. Exp Eye Res. 2015;137: 111-24. doi.org/10.1016/j.exer.2015.05.003 5. Subrizi A, del Amo EM et al. Design principles of ocular drug delivery systems: importance of 5. REFERENCES drug payload, release rate, and material properties. 1. del Amo EM, et al. Drug Discov Today. 2019; 24:1446- Ocular metabolism and 1457. distribution of drugs in the rabbit eye: doi.org/10.1016/j.drudis.2019.02.001 quantitative assessment after intracameral and intravitreal administrations, Int J Pharm 2022, ACKNOWLEDGMENTS 613: 121361. doi.org/10.1016/j.ijpharm.2021.121361 Al the co-authors and the corresponding funding sources of the cited publications. 50 KL23 PHYSIOLOGICALLY BASED FINITE TIME PHARMACOKINETIC (PBFTPK) MODELS: THE END OF THE BEGINNING IN ORAL DRUG ABSORPTION Panos Macheras1, Athanasios Tsekouras2 1 Department of Pharmacy, National and Kapodistrian University of Athens, Greece 2 Department of Chemistry, National and Kapodistrian University of Athens, Greece 1. INTRODUCTION standard optimization with least squares Drugs administered oral y are absorbed in the methods, while monitoring parameter blood while they are in the gastrointestinal (GI) uncertainties and correlations to establish the tract. This fact poses a time limit in the drug validity of the models [4]. The quality of each absorption duration. This observation is not fit for a given set of data determined which appreciated in most pharmacokinetic models. model was the most relevant for the data The Finite Absorption Time (FAT) concept [1] revealing basic physical characteristics of the poses constraints, leads to modifications in data absorption and disposition process of the analyses and can, eventual y, rewrite sampling corresponding formulation. schemes and regulatory requirements. 3. RESULTS AND DISCUSSION 2. MATERIALS AND METHODS 3.1. Models and equations 2.1. Materials The PBFTPK models implemented involved 1, Starting material used in this work was derived 2 or 3 consecutive input stages of finite duration from published pharmacokinetic data. in one- or two-compartment systems [4]. In the lat er case expressions needed to be derived for 2.2. Methods the peripheral compartment, although actual The presence of drugs in the GI tract and, concentrations cannot be monitored there. consequently, their absorption is limited by the 3.2. Simulations duration of gastrointestinal transit times. Given the sink conditions arising due to the high flow Concentration-time curves were generated rate in the vena cava [2], zero-order kinetics using the model equations developed in [4]. sufficiently describes the absorption process. Figure 1 shows a set of simulated data. In al Different input rates may operate at various cases, the simulated data resemble real life data. parts of the GI tract for finite duration in each part [1,3,4]. The absorption sequences can be F 1 D/V d = 100, τ 1 = 3 h described by appropriate sets of differential 60 F 2 D/V d = 30, τ 2 = 4 h equations depending on whether one or two 40 k C 21 = 0.5 h-1 compartments need to be taken into account [4]. k 12 = 0.2 h-1, α = 0.73 h-1 We coined the term Physiological y Based 20 k 10 = 0.1 h-1, β = 0.068 h-1 t Finite Time Pharmacokinetic (PBFTPK) max = 3 h 0 models for the models derived. These have been 0 5 10 15 20 25 30 solved analytical y al owing their use both for simulations and fits to experimental t (hour) pharmacokinetic data. For simulations a diverse Figure 1. Simulated drug concentration in the blood set of conditions are tried to explore a variety of in a two-compartment model with two successive situations. Fits have been performed using constant (zero-order) absorption stages. The black 51 KL23 triangle marks the end of al absorption processes Absorption Time Concepts. Pharmaceutical [4]. Research, 2020. 37: 187 https://doi.org/10.1007/s11095-020-02894-w 3.3. Data fits (cover page of the issue). Erratum. A large number of pharmacokinetic data were Pharmaceutical Research, 2020. 37: 206 https://doi.org/10.1007/s11095-020-02935-4 analysed using the PBFTPK models. In al cases, nice fit ing results were obtained; the 2. Iranpour, P., et al. Altered Doppler flow pat erns drug input rates were estimated along with the in cirrhosis patients: an overview. duration, τ, of each absorption stage. Ultrasonography, 2016. 35: 3–12. https://doi.org/10.14366/usg.15020. Representative examples are shown in Figure 2. 3. Chryssafidis, P., et al. Revising Pharmacokinetics of Oral Drug Absorption: II 0 Bioavailability-Bioequivalence Considerations. -1000 F 1D/Vd = 17.2 ± 0.9 μg/mL τ Pharmaceutical Research, 2021. 38: 1345– 1 = 0.90 ± 0.06 h L) 12 F 2D/Vd = 7.7 ± 1.8 μg/mL 1356 10.1007/s11095-021-03078-w τ 2 = 1.4 ± 0.2 h ng/m 8 k el = 0.34 ± 0.03 h-1 3 χ 2 = 4.7644e+06, R 2 = 0.9968 4. Chryssafidis, P., et al. Re-writing oral pharmacokinetics using physiological y based (x10 4 C finite time pharmacokinetic (PBFTPK) models. 0 Pharmaceutical Research, 2022. 39: 691-701. 0 2 4 6 8 10 12 https://doi.org/10.1007/s11095-022-03230-0. t (hours) 400 -40 F 1200 1 D/V d = 704±146 ng/mL, τ 1 = 1.36±0.3 h F 2 D/V d = 802±157 ng/mL, τ 2 = 2.06±0.3 h k L) 21 = 0.083 ± 0.02 h-1 800 k 12 = 0.070 ± 0.017 h-1, α = 0.170 h-1 k 10 = 0.0343 ± 0.0026 h-1, β = 0.0167 h-1 (ng/m C 400 χ 2 = 7523.2, R 2 = 0.9988 0 0 100 200 300 400 500 t (hours) Figure 2. Ibuprofen (top panel) and niraparib (bot om panel) pharmacokinetic data were used to obtain optimum parameter values (shown in the inset) that correspond to a one- or two-compartment model, respectively, with two stages of constant absorption rate (each of duration τ 1 and τ 2). 4. CONCLUSION The finite absorption time (FAT) of drugs is a physiological y sound concept and the relevant estimate for τ derived from the analysis of oral data using an immediate release formulation is a characteristic drug property. The application of PBFTPK models to the analysis of oral drug absorption data wil enhance our understanding of oral drug absorption phenomena. 5. REFERENCES 1. Macheras, P., et al. Revising Pharmacokinetics of Oral Drug Absorption: I Models Based on Biopharmaceutical/Physiological and Finite 52 KL24 PEPTIDE-MEDIATED DELIVERY OF RNA THERAPEUTICS Taavi Lehto 1,2,3 1 Institute of Technology, University of Tartu, Estonia 2 Department of Pharmacy, University of Tartu, Estonia 3 Department of Laboratory Medicine, Karolinska Institutet, Sweden 1. INTRODUCTION conformation and are further orthogonal y RNA Therapeutics, such as short modified with short hydrcocarbon/lipidic oligonucleotides, modified mRNA or modifications. We have recently shown that we CRISPR/Cas9 gene editing modalities, hold can deliver short oligonucleotide in enormous therapeutic potential for the treatment nanoparticles with hPep peptides in vitro and in of human disease1. Due to their high specificity, vivo6. Our recent studies have further any gene or gene product can be targeted with demonstrated that hPep peptides display these technologies. meaning that theoretical y excel ent delivery capacity for the transporting any disease is amenable for treatment. chemical y modified mRNAs both in vitro and However, these biomacromolecules typical y in vivo set ings. In contrast to existing leading display poor bioavailability in vivo due to non-viral delivery modalities, ie LNPs, our inefficient uptake into tissues and cel s. peptides primarily localize to lungs and spleen Therefore, a wide variety of delivery vectors and to lesser extent to liver in mice. have been devised and developed over the years Furthermore, we have also extended hPep either of biological nature, such as viruses and peptides for the delivery of CRISPR/Cas9 RNP extracel ular vesicles, or synthetic, e.g. lipid and demonstrate their potential for gene editing nanoparticles (LNPs) or cel -penetrating application in the cel culture models. peptides (CPPs, peptides)2. In particular CPPs 3. CONCLUSION is an interesting class of delivery vectors since they are rapidly taken up in entire cel Taken together, hPep peptides comprise populations and are able to transport various excel ent potential y for the delivery of various cargos ranging from impermeable smal RNA Therapeutics and has the potential to molecules to large plasmid DNA to their target chal enge the lipid-mediated delivery vectors cel s3. for extra-hepatic delivery and translation of new RNA therapeutics in the future. 2. RESULTS 4. REFERENCES We have previously developed a series of fat y acid modified CPP-based delivery vectors 1. Crooke, S. T. et al. Cell Metab. 27, 714–739 cal ed PepFect peptides4,5. These peptides (2018). demonstrated high delivery capacity for a 2. Bost, J. P. et al. ACS Nano 15, 13993–14021 variety of nucleic acid-based molecules both (2021). cel culture modes in vitro and also in local and 3. Lehto, T., et al. Adv. Drug Deliv. Rev. 106, systemic delivery in vivo. Based on our 172–182 (2016). previous experience, we have recently 4. Lehto, T. et al. Mol. Ther. 19, 1457–1467 developed a novel peptide platform, cal ed hPep (2011). peptides, with improved activity/safety profile 5. Andaloussi, S. E. et al. Nucleic Acids Res 39, and highly universality for the delivery of 3972–87 (2011). various novel classes of RNA therapeutics6. 6. Bazaz, S. et al. Biomedicines 9, 1046 (2021). hPep peptides are designed to adopt ɑ-helical 53 KL25 SHORT PEPTIDES AND THEIR ROLE IN RESEARCH AND IMMUNOTHERAPY OF ALLERGIES Mojca Lunder1, Abida Zahirović2, Ana Koren3, Jerneja Debeljak3, Peter Korošec3 1 Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, Slovenia 2 Department of Biotechnology, Jožef Stefan Institute, Slovenia 3 University Clinic Golnik, Golnik, Slovenia 1. INTRODUCTION immunodetection methods (ELISA or Peptide Al ergic diseases are affecting up to 20-30% of Microarrays; JPT Peptide Technologies). the world population. Al ergen-specific Synthetic epitope-like peptides were purchased immunotherapy emerged as an effective from commercial providers. Reactivity of treatment for some al ergic conditions, in peptides with patients’ IgE was tested by particular stinging insect hypersensitivity [1]. It immunodetection methods (western and dot typical y involves subcutaneous administration blot). Computational mapping (PepSurf of gradual y increasing quantities of al ergen algorithm) of al ergen 3D structure was extracts until immunotolerance is induced. performed to identify location of dominant However, this approach is not available for al epitopes. Selected peptides were coupled to an al ergies (e.g. peanuts), it requires repetitive immunogenic carrier. Minor coat protein pIII administrations over 3-5 years and it is often from M13 bacteriophage was used for associated with adverse events [2]. Novel mimotopes of Api m 1 and L. lactis, for therapeutic strategies with reduced side effects mimotopes of Ara h 2. Al ergenicity of and decreased treatment time are thus required. mimotopes was tested by ex vivo activation of Identification of IgE epitope-like peptides basophils from al ergic patients. Marker of enables research of immunodominant epitopes basophil activation and degranulation CD63 of relevant al ergens and their role in al ergy was monitored by flow cytometry. Peptides occurrence as wel as enables development of displayed on carrier were also tested for their next-generation al ergy vaccines containing ability to influence al ergen specific T cel s of solely the necessary epitope-like peptides or al ergic patients, by measuring cytokine epitope mimetics (mimotopes), thus eliminating response (Cytometric Bead Array; BD undesirable side effects induced by irrelevant Biosciences) on FACSCalibur flow cytometer determinants. We have focused on epitope- (BD Biosciences). Sera from al ergic patients based immunotherapeutics for life-threatening were obtained at University Clinic Golnik. al ergies, such as al ergies to insect venoms and food al ergies, particularly peanut al ergy. 3. RESULTS AND DISCUSSION We have focused on Ara h 2 (major peanut 2. MATERIALS AND METHODS al ergen), Api m1 (major bee venom al ergen) Peptide mimotopes of selected major al ergens and Ves v 5 (major wasp al ergen). By were obtained by screening biological peptide screening phage libraries displaying linear or libraries (PhD-12, PhD-7 and PhD-C7C; New cyclic peptides of various lengths against anti- England Biolabs) against anti-al ergen al ergen antibodies we obtained sequences antibodies (Indoor Biotechnologies). For binding to these paratopes and thereby al ergen Ves v 5 phage display was combined mimicking epitopes on al ergens. Sequences of with next generation sequencing (MiSeq, peptides in combination with computer Il umina) and bioinformatics processing of the algorithms enabled mapping and identification obtained sequences (PuLSE software). Binding of dominant epitopes on the selected al ergens. characteristics of peptides were evaluated by 3.1. Ara h 2 54 KL25 Selected peptides were aligned to the first part of the loop between helices 2 and 3 containing 4. CONCLUSION amino acid residues DPYSPSQD. Due to the flexibility of the loop only the first part is visible Identification of Api m 1 and Ves v 5 IgE in the crystal structure. Within the loop the epitopes is a very important step for bet er motif DPYSP(S) is repeated three times and it understanding of pathogenesis of bee or wasp has been shown previously that this loop region sting anaphylaxis and can improve diagnosis contains important epitopes. Synthetic peptides and prognosis of Hymenoptera venom al ergy. showed decreased basophil activation Furthermore, their respective mimotopes compared to Ara h 2 or peanut extract. The represent novel candidates for development of mimotope with the highest sequence similarity more precise and safer Hymenoptera venom with Ara h 2 showed some activation of immunotherapy. We have identified Ara h 2 IgE basophils suggesting its al ergenic activity is epitope-like peptides that successful y prevent partial y preserved. After stimulation with Ara biding of causative al ergen, and thereby h 2 higher production of cytokines specific for prevent degranulation and secretion of al ergic type 2 T cel response was observed. Peptides inflammation mediators from effector cel s. fused to carrier (either on L. lactis or fused to L. Such peptides represent new preventive lactis anchoring protein) yielded significantly treatment option in accidental exposures to lower concentrations of cytokines compared to al ergens or intentional exposures during the al ergen. Except production of IFN-γ was immunotherapy. Our efforts are focused higher, particularly with one IgE like peptide towards further optimization of these peptides which may suggest shifting the T cel immune for therapeutic use. response to non-al ergic type 1. Additional y, the peptides markedly reduced al ergenic activity of Ara h 2 and were able to prevent 5. REFERENCES degranulation of patients’ effector cells [4]. 1. James, C., et al., Allergen immunotherapy: an updated review of safety. Current Opinion in 3.2. Api m1 Al ergy and Clinical Immunology. 2017. Peptide sequences were arranged into two 17(1):55-59. groups according to the consensus motifs. 2. Zahirović, A., et al., B ee Venom Conformational mapping overlapped with Immunotherapy: Current Status and Future Directions. Clinical Reviews in Al ergy and primary sequence alignment, indicating that the Immunology. 2020. 58, 326–341. regions 17-24 and 119-124 represent the 3. Lunder, M., et al., IgE epitope-like peptides and epitopes of Api m 1. Both defined epitopes are uses thereof. Publication number US located on the solvent-exposed loops protruding 2021/0115097 A1. Alexandria, VA: US Patent from the al ergen surface and do not include and Trademark Office, 2021. glycosyl moiety. Corresponding peptide 4. Zahirović A, et.al., Identification of bee venom mimotopes showed no al ergenic activity in Api m 1 IgE epitopes and characterization of basophil activation test. In contrast to bee corresponding mimotopes. The Journal of venom and Api m 1, which stimulated al ergy and clinical immunology. 2019. predominantly type 2 cytokine response, 143(2):791-794.e5. mimotopes induced only T cel secretion of type 1 cytokines, suggesting a shift from TH2 to TH1 ACKNOWLEDGMENT immune response [5]. We would like thank former lab members Peter 3.3. Ves v 5 Molek, PhD and Jernej Luzar, PhD for help with From screening phage libraries fol owed by screening phage libraries. This work was NGS we obtained three groups of peptide motifs supported by the Slovenian Research Agency and align them to three major epitopes of Ves v (program P4-0127). 5. Using peptide microarrays variations of the three peptide motifs were profiled using al ergic patients and control sera. The profiling revealed peptides with strongest binding to IgE from wasp venom al ergic patients. 55 KL26 CD81 LARGE EXTRACELLULAR LOOP AS A NOVEL ALTERNATIVE SCAFFOLD: LAMININ BINDING CD81 ENHANCES SPECIFIC UPTAKE OF EXTRACELLULAR VESICLES Gordana Wozniak Knopp1,2, Stefan Vogt1,3, Gerhard Stadlmayr1,2, Katharina Stadlbauer2, Madhusudhan Reddy Bobbili1,4, Jørgen Kjems5, Florian Rüker1, Johannes Grillari1,4 1 Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria 2 Christian Doppler Laboratory for Innovative Immunotherapeutics, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria 3 acib GmbH (Austrian Centre of Industrial Biotechnology), Austria 4 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology in the AUVA Research Center, Austria 5 Centre for Cellular Signal Patterns (CellPat), Interdisciplinary Nanoscience Centre (iNANO), Department of Molecular Biology and Genetics, Aarhus University, Denmark 1.INTRODUCTION binding molecules recognizing an antigen of In recent years, extracel ular vesicles (EVs) choice. The selection platforms using LEL as an have entered the limelight as multifaceted drug independent folding unit were constructed using delivery vehicles [1]. These about 100 nm large yeast display as a validated method of membrane enveloped structures are important molecular evolution in vitro. The chosen cel -to-cel messengers, as their contents, antigen was human placental laminin, important composed of a cocktail of proteins, miRNAs in muscular and neural regeneration processes. and other non-coding RNAs, originating from the producing cel , can efficiently be transferred to a recipient cel upon fusion with its cel ular 2. MATERIALS AND METHODS membrane. EVs mediate intercel ular 2.1. Molecular design communication and thus sustain homeostasis in Solvent-exposed residues of CD81 LEL were tissues and organs, as wel as further support identified that could upon randomization form a processes such as regeneration after injury and novel antigen binding site of about 600 Å2 in limitation of tumorigenesis and malignant size (Fig. 1). The first scaffold molecule was a transformation. On the other hand, targeted CD81 LEL mutant with two novel disulfide delivery of EVs has long been recognized as a bonds with the midpoint of thermal transition feature that could crucial y improve their (TM) of 109.4°C, 43°C above that of the wild- biological effect. Directed delivery was type CD81 LEL. The second library design was enhanced when their surface has been modified based on the CD81 LEL mutant with the ability to enable the specific recognition of the target to reversibly refold when heated up to 110 °C, cel s. For this purpose, we have modified the unlike the wild-type CD81 LEL. innately overrepresented EV surface protein CD81, which is at the same time regarded as 2.2. Yeast display and binder validation their hal mark surface marker, into an antigen- Yeast display libraries with about 109 members recognition scaffold. This protein is a member were processed with MACS and FACS-based of the tetraspanin family with four antigen selection until visible enrichment. transmembrane segments (TM1–4) linked by a Unique binders were displayed on the surface of smal intracel ular loop, a smal extracel ular mammalian cel s to confirm specific antigen loop (SEL) and a large extracel ular loop (LEL). binding. Previously, we have designed highly stabilized 2.3. EV preparation and characterization mutants of CD81 LEL via introduction of de EVs were isolated from the supernatant of HeLa novo disulfide bonds [2], and 2 were chosen for cel s stably expressing CD81-eGFP variants randomization to produce libraries of potential using ultracentrifugation. Nanoparticle 56 KL26 tracking analysis, Western blot ing and Figure 1. Models of CD81 library designs (PDB: transmission electron microscopy (TEM) were 1G8Q) with novel disulphide bonds in yel ow and the used for particle characterization. Specificity of randomized residues surfaced in cyan. laminin binding was determined with a competition experiment. 4. CONCLUSION 2.4. Uptake of targeting EVs Two laminin-secreting cel lines, Huh-7 and We have established a novel selectable antigen NCI-N87 were exposed to recombinant EV binding platform based on the CD81 LEL preparations and flow cytometry was used to scaffold [3]. The recombinant laminin-binding measure the uptake levels of eGFP-positive EVs shown a higher level of internalization into EVs. laminin-secreting cel s. This method can rapidly deliver EV binding to any antigen of 2.5. Transfer of cel-miR-39 by targeting EVs choice, simply by “clicking” recombinant LEL Huh-7 cel line was treated with EVs loaded onto the ful -length CD81. with cel-miR-39 and the levels of transfer were determined after RNA isolation, cDNA synthesis and qPCR in real time. 5. REFERENCES 1. Yamamoto, T., et al., Latest advances in 3. RESULTS AND DISCUSSION extracellular vesicles: from bench to bedside. Science and Technology of Advanced 3.1. Isolation of laminin binders Materials, 2019. 20(1): 746-757. Laminin-binding clones were enriched after 3-4 2. Vogt, S., et al., Stabilization of the CD81 large selection rounds and 9 unique sequences were extracellular loop with De Novo disulfide bonds identified. improves its amenability for peptide grafting. Pharmaceutics, 2018. 10(3):138-151. 3.2. Recombinant EVs 3. Vogt S, et al., An engineered CD81-based Recombinant EVs were prepared at a high yield combinatorial library for selecting recombinant with the median size between 100 and 125 nm. binders to cell surface proteins: Laminin At least 25-60% of the particles were eGFP- binding CD81 enhances cellular uptake of fluorescent. EV marker proteins syntenin, Alix extracellular vesicles. Journal of Extracel ular and TSG101 were shown to be enriched while Vesicles, 2021. 10(11): e12139. calnexin, as a non-EV protein, was absent. TEM analysis confirmed the presence of the typical cup-shaped structures. ACKNOWLEDGMENT This project has been supported by the Austrian 3.3. Laminin-binding EVs mediate enhanced FFG-COMET-Funding Program and Evercyte cellular uptake GmbH. SV was a fel ow of the international PhD The enhanced laminin-binding EV programme “BioToP” (FWF grant W1224). This internalization was observed in both in Huh-7 project was also supported by EQ-BOKU VIBT and NCI-N87 cel s with up to 8-fold increase in GmbH and the BOKU Core Facilities for fluorescence compared to wild-type EVs. Biomolecular and Cellular Analysis and Multiscale Remarkably, cel-miR-39 was detected in Imaging recipient Huh-7 cel s, which was found to be significantly increased in Huh-7 cel s exposed to laminin-binding variant EVs compared to wild-type CD81 EVs. 57 58 Oral Presentations 59 CONTINUOUS MANUFACTURING IN SOLID DOSE - HOW TO LEVERAGE OPPORTUNITIES OF THIS NEW TECHNOLOGY Leo Ohrem 1Merck There is a constant pressure from both authorities to develop robust and safe drugs as wel from the industry to reduce cost. Continuous manufacturing can be a solution. The presentation shows up the specific chal enges of this new technology and how to tackle these by the choice of excipients. THE VISCOSITY REDUCTION PLATFORM ENABLING SUBCUTANEOUS DELIVERY Peter Balogh 1Merck Subcutaneous administration can improve patient convenience and reduce healthcare costs by avoiding the need for hospitalization. Yet in some cases, high protein concentrations make formulations far more viscous, prohibiting this route of administration. The presentation will introduce an excipient platform that makes it possible to combine excipients in a ways that can reduce protein viscosity. PROCESS RELATED IMPURITIES IN MRNA MANUFACTURING Kristina S Nemec, Urh Cernigoj, Jana Vidic, Andreja G Livk, Blaz Goricar, Klemen Bozic, Anze Martincic Celjar, Nina Mencin, Tomas Kostelec, Rok Sekirnik, Pete Gagnon, Ales Strancar 1 BIA Separations The recently demonstrated efficacy of mRNA-based Covid-19 vaccines has shown promise of this therapeutic format, but also highlighted the need for higher efficiency of mRNA production to meet enormous needs for global vaccine supply. Typical mRNA production process involves three key steps: 1) plasmid DNA production, linearization and purification, 2) in-vitro transcription reaction and 3) mRNA purification. Here we present a chromatographic toolbox and mRNA IVT synthesis for integrated mRNA production from pDNA to mRNA purification, including in-process analytics to address key process related impurities. 60 OP1 INFLUENCE OF THE BINDER JETTING PROCESS PARAMETERS AND BINDER LIQUID COMPOSITION ON THE RELEVANT ATTRIBUTES OF 3D PRINTED TABLETS Klemen Kreft1, Zoran Lavric1, Tijana Stanic2, Petra Perhavec2, Rok Dreu1 1 Faculty of Pharmacy, University of Ljubljana, Askerceva Cesta 7, 1000 Ljubljana, Slovenia 2 Lek Pharmaceuticals d.d., a Sandoz Company, Verovskova Ulica 57, 1000 Ljubljana, Slovenia 1. INTRODUCTION (polyvinyl pyrrolidone grade K 25) was sourced In the last decade, 3D printing has received a from Ashland (Wilmington, Delaware, USA). considerable interest from the pharmaceutical Glidant Syloid® 244 FP (silica) was sourced research community, primarily due to its ability from Grace GmbH (Worms, Germany). The to prepare personalized medication on demand. final powder blend was composed of 20.0 % One of the most advanced and promising 3D ketoprofen, 34.75 % Pharmatose® 125M, 34.75 printing technologies for pharmaceutical % Pearlitol® 100 SD, 10.0 % Plasdone® K-25 purposes is binder jet ing. It is a layer-by-layer and 0.5 % Syloid® 244 FP. Binding liquids manufacturing process, where the powder blend were ethanol-water mixtures in 10, 20 and 30 % is deposited in a fine layer. The particles are m/m concentration. then bound together by a binding liquid. This 2.2. 3D printing process repeats in layers several times to A custom-developed pharmaceutical 3D printer achieve the final tablet form [1]. Binder jet ed Picojet D220 (Spectra Laboratories, Ljubljana, tablets are porous in nature and therefore exhibit Slovenia), functioning as a standard binder instant disintegration, which is advantageous jet ing device, was used for sample preparation. for high-risk patient groups who have trouble Tablets with 15 mm diameter and 5 mm height with swal owing. The lack of any compression were designed and the information was force yields a fragile tablet structure, resulting transferred to the printer. 40 tablets were in troublesome handling and transportation. To prepared per each experiment batch. When the address this issue, several formulation printing process was finished, tablets were dried approaches have already been reported [1,2]. in a convective drying chamber (Kambic, However, not enough effort is devoted to Semic, Slovenia) at 60 °C for 150 minutes to understanding the printing process itself. In this LOD values below 2 %. study, key printing parameters with the 2.3. Design of experiments influence on the relevant properties of tablets A screening and optimization DoE study was were identified, while the binding liquid performed with use of software Modde 12.01 composition was also examined. (Sartorius, Göt ingen, Germany) and utilization 2. MATERIALS AND METHODS of 20 m/m % ethanol-water binding fluid. 18 2.1 Materials experiments were executed to evaluate the Active pharmaceutical ingredient ketoprofen influence of 5 process parameters on properties was sourced internal y from Lek d.d. stock. of 3D printed tablets. In addition, 9 experiments Fil ers Pharmatose® 125M (lactose were carried out at three tablet strength levels to monohydrate) was sourced from DFE Pharma assess the impact of the binding liquid (Goch, Germany) and Pearlitol® 100 SD composition on the final properties of tablets. (mannitol) from Roquet e Frères (Lestrem, France). Solid binder Plasdone® K-25 61 OP1 2.4. Analysis of tablets changed by the factor of two when adjusting Printed tablet samples were analysed for both printing parameters accordingly. hardness, friability, disintegration time, mass, mass uniformity and dimensions according to 3.2. Influence of binding liquid on tablet the European Pharmacopoeia procedures and properties with use of internal y validated equipment. Binding fluid composition could also affect tablet properties. Three strength levels of tablets 3. RESULTS AND DISCUSSION (best, medium, worst) were prepared by set ing 3.1. Influence of process parameters on tablet layer height and saturation accordingly. Within properties each strength level, tablets were prepared with Since binder jet ed tablets can be troublesome 10, 20 and 30 % ethanol-water mixture. The to handle, it is crucial to optimize the process amount of ethanol in binding liquid did not parameters to achieve the best mechanical influence the properties of tablets to the same properties. 5 printing parameters were analysed degree as process parameters (Fig. 2). We could in the screening phase. Doser speed, spreader argue that the potential difference in capil ary speed and printhead speed are related to the effect of employed binding liquids was of lesser manner and speed of powder and liquid importance in comparison to standard wet deposition, while layer height (thickness of each granulation as during binder jet ing procedure layer) and saturation parameter (number of powder layer height is thin and control ed. active nozzles) are connected to the amount of material in each layer. Wel -fit ing models (R2=0.65–0,89) with good predictability (Q2 values are close to R2) were obtained for al responses with exception of mass RSD and tablet height models (Fig. 1). Fig 2. Impact of ink composition on hardness, friability and disintegration time at three strength levels. 4. CONCLUSION An in-depth study of the binder jet ing 3D printing process revealed that the set ings of the layer height and saturation are significant for final properties of tablets. Binding fluid composition affects the properties to a lesser Fig 1. Summary of fit plot for the screening phase of extent. the DoE study Coefficient plots from screening phase showed, 5. REFERENCES that the doser speed, spreader speed and 1. Chang. S.Y., et al., Binder-Jet 3D Printing of printhead speed did not have any major Indomethacin-Laden Pharmaceutical Dosage influence on tablet properties. This Forms. Journal of Pharmaceutical Sciences, 2020. 109: 3054–3063 demonstrates that the printing process in very 2. van den Heuvel, K.A., et al., Evaluation of robust. On the other hand, layer height and Lactose Based 3D Powder Bed Printed saturation parameters both proved a significant Pharmaceutical Drug Product Tablets. Powder Technology, 2021. 390: 97–102. influence. The higher the saturation and the lower the layer height, the bet er the mechanical properties of printed tablets. Tablet hardness, friability and disintegration time can be 62 OP2 OPTIMIZATION OF RADIAL EXTRUSION AND PELLET COATING PROCESSES USING PAT APPROACHES Aljoša Gradišek1, Gregor Ratek1, Franc Vrečer1,2, Klemen Korasa1 1 Krka d. d., Novo mesto, Slovenia 2 University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia 1. INTRODUCTION In 2002, FDA lauched a new initiative to The mass was extruded using (Gea NICA™ implement risk-based modern approaches in IPS25; GEA; Germany) radial extruder. pharmaceutical development, manufacturing Extrudates were dried in fluid bed dryer (GEA and quality assurance. One of the key MP-1 S; GEA; Germany) and mil ed (Frewit technological approaches within this initiative Hammerwit 3; Frewit ; Switzerland) to is process analytical technology (PAT). FDA produce pel ets with median diameter 810 µm. describes PAT as a system for designing, Parameters of radial extrusion, extrusion speed, analyzing and control ing manufacturing screen size and water content in wet ed mass, through timely measurements of critical quality were optimized using DoE. and performance at ributes of raw and in- process materials and processes, with the goal 2.3. Pellet coating of ensuring final product quality. [1] Statistical Pel ets were coated in bot om spray fluid bed PAT tools include design of experiment (DoE), coater (GEA MP-1 S; GEA; Germany) to target that is a systematical approach to understanding weight gain of 20% of the starting pel et weight. and predicting end product properties. Another During coating of the first three batches, pel ets branch of PAT tools are process analyzers were periodical y sampled at predefined points among which NIR spectroscopy is one of the calculated from coating suspension most promising approaches for real to near-real consumption ranging from 4% to 28% weight time process monitoring. Use of PAT tools are gain. UV-VIS spectra were taken to measure the especial y important in case of chal enging content of tartrazine and to calculate the actual technological operations, such as radial coating quantity at each of the sampling points. extrusion and functional film coating, which Antaris Target analyser (ThermoFischer were thoroughly evaluated in the present study. scientific, USA) FT-NIR probe was used to [2] measure the spectra of previously mentioned samples at a spectral range of 5560-7400 cm-1. 2. MATERIALS AND METHODS Partial least squares regression (PLS) model for 2.1. Materials the prediction of film-coating quantity was Neutral pel ets were manufactured using created using Unscrambler version 11 microcrystal ine cel ulose (MCC; Vivapur® (AspenTech; USA). NIR spectral data was used 102; JRS Pharma, Germany) and polyvinyl- as independent variable and film-coating pyrrolidone (PVP; Kol idon® K-30; BASF; quantity as dependent variable. Ability to Germany). Suspension for film coating was predict the film-coating quantity was tested by prepared using polymer hydroxypropylmethyl- coating a batch where pel ets were periodical y cel ulose (HPMC; Pharmacoat® 606; Shin- sampled and NIR spectra were recorded at-line Etsu; Japan) and talc (Imerys talc; Italy). to determine the process end point. The final Sodium salt of tartrazine (Sigma Aldrich, USA) product was compared to previous batches by was added to determine film-coating quantity SEM measurements of coating thickness and by using UV-VIS spectroscopy. coating suspension consumption, assuming similar process yield. 2.2. Preparation of neutral pellets Wet ed mass for extrusion was prepared by high shear granulation (HSM; Gea UltimaGral™ 25; GEA; Germany) of MCC type 102 with PVP. 63 OP2 3. RESULTS AND DISCUSSION suspension consumption (Fig. 2), relatively low 3.1. Radial extrusion optimization with DoE RMSE of prediction (4.21%) and high R2 A response surface design with two quantitative (0.991) was observed. factors, quantity of granulation liquid and extrusion speed at three levels, and categorical variable extrusion screen size at two levels, was created and 13 experiments were performed with the goal to maximize the sphericity and minimize the width of particle size distribution and friability of pel ets. The desired properties (Fig. 1) were achieved using extrusion screen with dimensions 0.8 × 0.7 mm that gave us Figure 2. Coating quantity prediction graph extrudates with higher sphericity and narrower particle size distribution, compared to 0.5 × 0.5 Film-coating thickness of the test batch was mm screen. Higher water quantity and 110% of measured using SEM and compared to samples total dry mass used for preparation of extrusion from model calibration batches. The test batch mixture gave extrudates with lower friability, coating thickness was not statistical y different especial y in combination with a larger from samples with 100 and 105% of the target extrusion screen. Extrusion speed had no value and was thicker compared to the sample significant effect on the evaluated at ributes. with 90% of the target value. Multiple batches were produced at defined 4. CONCLUSION process parameters to produce enough pel ets for film-coating trials. In the present study, DoE was applied successful y to optimize critical process parameters for consistent production of pel ets. The PLS model for coating quantity prediction was calibrated using NIR spectral data as independent variable and coating quantity as dependent variable. The model enabled accurate film coating quantity prediction in near real time, especial y around our target film- Figure 1. Process parameter optimization graph coating quantity. Additional improvements like 3.2. Coating thickness prediction using the larger sample size for model calibration and NIR probe inclusion of more test batches would increase A PLS model for coating quantity prediction reliability of the prediction. In the future, use of was calibrated, based on differences in the in-line NIR probe that al ows real-time coating intensity of film-coating component NIR bands. quantity monitoring needs to be evaluated. A narrow sharp band of talc at wave number around 7180 cm-1 contributed the most spectral 5. REFERENCES variability. First principal component of the 1. FDA. Guidance for Industry PAT — A model explained 99.9% of the variability. R2 of Framework for Innovative Pharmaceutical the calibrated PLS model was 0.999, slope of Development, Manufacturing, and Quality the linear regression line was 0.999, and root Assurance, 2004. mean standard error (RMSE) was 1.23%. That 2. Korasa K., Vrečer F., Overview of PAT process showed good correlation between predicted and analysers applicable in monitoring of film coating unit operations for manufacturing of reference values throughout the whole model, solid oral dosage forms. European Journal of which was later used to predict coating quantity Pharmaceutical Sciences, 2018, Volume 111 of independent test batch, where film coating was stopped at the estimated coating quantity of ACKNOWLEDGMENT 98.5% of our target value. From coating This research has been supported by Krka, d. d., suspension consumption, theoretical film- Novo mesto. coating quantity of 100.2% of the target value was calculated. By plot ing individual predicted values throughout the coating process against the theoretical quantity calculated from coating 64 OP3 FROM BATCH TECHNOLOGY TO CONTINOUOS MANUFACTURING: FORMULATION OF GASTRORETENTIVE DOSAGE FORM BASED ON MELT FOAMING TECHNIQUE Gábor Vasvári1, Ádám Haimhoffer1,2, Ildikó Bácskay1,2 Ferenc Fenyvesi1 1 Department of Pharmaceutical Technology, University of Debrecen, Hungary 2 Institute of Healthcare Industry, University of Debrecen, Hungary 1. INTRODUCTION grade and purchased from Molar Chemicals, Variabilities of the digestive system chal enges while others were from Sigma-Aldrich. innovations in the development of drug delivery 2.2. Device setups and foaming systems. Motility and pH differences of GI juices affects the oral bioavailability of smal The batch apparatus was built from poly- molecules. Up to date, oral targeted delivery propylene tube, the jacketed vessel (60mL) was platforms can be considered the most effective closed with a valve (d:10mm) at the bot om. tools to increase the absorption rate of drugs Molten dispersions of MNZ were foamed with with poor solubility or permeability. Gastric air at atmospheric pressure with a whisker retention offers site-specific prolonged release agitator (Fig. 1A). QUICKfoamcel Lab®. is of active ingredients in the stomach. Low presented on Fig. 1B. Briefly, the molten density compositions can avoid the passage to dispersion is pumped from a melt container to the duodenum by floating on the gastric content. the foam cel (30 mL) where a roto-stator To produce such floating systems, several homogenizing tool (IKA®, S25 N 10G) technologies can be considered including hot disperses the injected air to create hot foam. melt extrusion, gas generating compositions or Final y, the hot foam can be dosed with a pinch HBS capsules. Extrusion is a continuous valve. process, but in most cases, high temperature or 2.3. Dissolution and mathematical analysis pressure is needed to reshape the solids, floating 900 mL of hydrochloric acid media, pH: 1.2 lag time exists for gas generating tablets and without pepsin was used for dissolution tests HBS capsules are susceptible to the churning (Erweka DT800, rotating paddle method, 75 movements. rpm and 37°C) API release was detected by Foamed molten dispersions are in the focus of UV/VIS spectrophotometry. Dissolution data our research since 2018.At the very beginning was fit ed to zero-order, first-order, and we have created an in-house apparatus for Korsmeyer–Peppas models. foaming batches of molten lipid dispersions, 2.4. In vivo studies later the concept was revised, redesigned and For the in vivo CT Imaging, 16-week-old, male upgraded into a novel, lab-scale apparatus for Fischer-344 rats (n=3; 250–300g, Animalab continuous production, the QUICKfoamcel Ltd.) were used. CT scans were acquired after Lab®. 30 and 120 mins. The pharmacokinetic study 2. MATERIALS AND METHODS (approval number: HB/06-ÉLB/1657-4/2019) included six female Beagle dogs, verapamil 2.1. Materials solution and verapamil molten foams were PEG 4000 and stearic acid, type 50 (SA) was administered oral y, PK parameters were used to create the foamed matrices. compared, gastric retention was checked by Metronidazol (MNZ) was used for the batch gastroscopy. technology, BaSO4 was the X-ray contrast agent for the in vivo imaging study while verapamil hydrochloride (VER) was used for the continuous production of in vivo pharmaco- kinetic study. These materials were Ph Eur. 65 OP3 the animals. VER capsules were present after 2 hours, while empty stomachs were observed after 4 hours. Figure 1. Batch apparatus (A) and the QUICKfoamcel Lab® (B). 3. RESULTS AND DISCUSSION 3.1. Density of the foams In case of the batch apparatus, 53°C was selected for foaming temperature. Dispersion containing 30 m/m% MNZ was successful y foamed when 5 or 10% SA was added to the Figure 2. Mean plasma levels of VER solution melt, densities decreased to 0.82 and 0.93 g/mL, (50mg/dose) and VER foamed capsules respectively. (120mg/dose). Process optimization was done with the 4. CONCLUSION QUICKfoamcel Lab® using using a Box– Melt foam techniques was designed and Behnken experimental design. Injected gas realized in batch technology, then transferred to volume (mL), rate (mL/s) and tool speed (rpm) continuous manufacturing. Molten dispersions were varied, while foaming cel temperature were successful y foamed, closed spheroid cel s was 56°C in al runs. It was found that 14 500 were present, 90% of them are smal er than 100 rpm is enough to decrease density below 1 microns. PEG and SA based matrix provided g/mL, while the optimal range for gas volume prolonged release for 10 hours, via matrix was 2.5–3.25 mL with the injection rates of erosion. CT scans and gastroscopy proved the 0.25-0.35 mL/s. Production speed was also retention of the matrices in rat and canine calculated, resulting 300–500 capsules/h stomachs. Compared to the VER oral solution, (capsule size 00). Capsules fil ed with melt a relative bioavailability of 99.3% was reached. foams containing 15 m/m% VER and 12.5% SA were prepared for the PK study with the average 5. REFERENCES density of 0.75 g/mL. 1. G, V., et al., Development and Characterisation 3.2. Dissolution properties of Gastroretentive Solid Dosage Form Based on MNZ foams containing 5 or 10 % SA released Melt Foaming. AAPS PharmSciTech, 2019. 20(290): 1-11. their content after 5 and 10 hours, fol owing 2. Á, H., et al., Process Optimization for the first-order drug release model. VER capsules Continuous Production of a Gastroretentive with 10, 12.5 or 15% SA released 85.3%, 79.4% Dosage Form Based on Melt Foaming. AAPS and 77.4% of its content in 10 hours. Data PharmSciTech, 2021. 22(187): 1-9. analysis revealed first-order drug release. In al 3. Á, H., et al., In Vitro and In Vivo Studies of a experiments, the PEG-SA based foamed Verapamil-Containing Gastroretentive Solid matrices released their API content due to the Foam Capsule. Pharmaceutics, 2022. 14(350): erosion of matrix, no swel ing was observed. 1-18. 3.3. In vivo studies ACKNOWLEDGMENT BaSO4 loaded foams were administered oral y Project no. TKP2021-EGA-18 has been to rats, CT scans proved the ability of the foam implemented with the support provided from to remain in the stomach for at least 2 hours to the National Research, Development and satisfy the desired needs of such formulations. Innovation Fund of Hungary, financed under Plasma VER levels of the PK study are the TKP2021-EGA funding scheme. presented in Fig. 2. Beside the blood sample analysis, gastroscopies were also performed on 66 OP4 THE INFLUENCE OF POLYMERIC BINDER TYPE AND CONCENTRATION ON FLOW AND DISSOLUTION PROPERTIES OF SYLOID® 244FP-BASED SMEDDS GRANULES Mila Kovačević1, Ilija German Ilić1, Alenka Zvonar Pobirk1 1University of Ljubljana, Faculty of Pharmacy, Department of Pharmaceutical Technology, Slovenia 1. INTRODUCTION Mesoporous carriers, such as Syloid® 244FP, hypromel ose (Pharmacoat® 603, Pharmacoat® are known for their high liquid load capacity 615 or Methocel® K100 LV). and ability to maintain characteristics of dry powders when loaded with liquid lipid-based 2.2. Preparation of SMEDDS granules by dispersions [1]. Therefore, they are a wet granulation convenient choice for solidification of self- SMEDDS granules were prepared manual y by microemulsifying drug delivery systems wet granulation method, fol owed by tray (SMEDDS) that are designed to improve the drying. Granulation dispersion containing solubility of poorly water-soluble drugs [2]. The microemulsion with SMEDDS/water ratio of loading of mesoporous carriers with SMEDDS, 70/30 and binding polymer (povidone or frequently results in sticky powders with poor hypromel ose) in concentration range 0.00-7.45 flow properties, which are inappropriate for % w/w, was added to mesoporous Syloid® further processing [1]. Thus, the aim of this 244FP. Granules with high and low binder study was to investigate the influence of concentration (1.85 and 7.45 %) were prepared different polymeric binders and their using laboratory high-shear granulator as wel , concentrations used in granulation dispersons to study the influence of method preparation on for SMEDDS solidifications on the quality granule characteristics. at ributes of granules, with particular focus on 2.3. Characterisation of SMEDDS granules flow and dissolution properties. Produced granules were evaluated for particle 2. MATERIALS AND METHODS size and size distribution, flow properties, self- microemulsifying properties and in vitro 2.1. Materials carvedilol release profile. Carvedilol (CTX Life Sciences LTD, India) was used as a model drug. 3. RESULTS AND DISCUSSION Liquid SMEDDS was composed of mixture of 3.1. The influence of polymer type on Capmul® MCM EP/NF (mono-diglyceride of SMEDDS granules characteristics medium chain fat y acids, Arbitec Corporation, Al povidone-based granules showed median USA), refined castor oil (Ph. Eur. Grade, Caesar diameter 600–800 µm and passable flow & Loretz GmbH, Germany), Kol isolv® PEG E properties (according to Carr index criteria in 400 (Sigma-Aldrich, USA) and Kol iphor® RH Ph. Eur.). Neither concentration nor type of 40 (polyoxyl 40 hydrogenated castor oil, binder had a major influence on these Sigma-Aldrich, USA). properties. On the other hand, hypromel ose- Syloid® 244 FP (Grace GmbH & Co. KG, based granules were smal er (d Germany) was used as mesoporous carrier for 50 value 300–600 µm), exhibiting bet er flowability, in SMEDDS solidification. Different binder types comparison to other polymer. Stil , the flow and concentrations were used in granulation time of all produced granules was 5.0-7.2 s/100 dispersion: vinylpyrrolidone-(co)polymers (PVP/VA 64, PVP K30 or K90) and 67 OP4 g, which is considered wel enough for further comparison to ones with lower polymer tableting. amount, as shown in Fig. 2. Regarding in vitro dissolution testing, SMEDDS loaded granules with low molecular 100 ) weight PVP K30 exhibited the fastest drug (% 80 release, in contrast to ones with high molecular 7.45% PVP K30 1.85% PVP K30 weight PVP K90, thus increasing molecular 60 7.45% PVP/VA 64 weight showing a negative influence on release 40 1.85% PVP/VA 64 profile (Fig. 1). The same trend could be 7.45% PVP K90 20 observed when hypromel ose was used as 1.85% PVP K90 Carvedilol released carvedilol binder, as from SMEDDS granules with low 0 molecular weight Pharmacoat® 603, 74 % of 0 100 200 time (min) carvedilol was released in 5 minutes, in comparison to 58 % for medium molecular Figure 2. In vitro carvedilol dissolution profile weight Pharmacoat® 615 and 41 % for granules (medium with pH 6.8) of SMEDDS granules with with high molecular weight Methocel® K100 high and low binder concentration (1.85 and 7.45%) LV (Fig. 1). in granulation dispersion. 3.3. The influence of granulation method on 100 ) SMEDDS granules characteristics (% 80 1.85% PVP K30 The rate of carvedilol release was affected by 1.85% Pharmacoat 603 granulation method, as the release was faster 60 1.85% PVP/VA 64 from al manual y prepared SMEDDS granules. 40 1.85% Pharmacouat 615 Contrary to this, the extent of released drug, 1.85% Methocel K100LV 20 wasn’t influenced by preparation method, as the Carvedilol released 1.85% PVP K90 drug was completely released until the end of 0 carvedilol testing period in both cases. 0 50 100 150 200 250 time (min) 4. CONCLUSION Figure 1. In vitro carvedilol dissolution profiles By incorporation of polymeric binders into the (medium with pH 6.8) of SMEDDS granules prepared using 1.85% of different binders in granulation granulation dispersion it was possible to dispersion. produce Syloid® 244FP-based free-flowing granules, with preserved self-microemulsifying 3.2. The influence of polymer concentration properties, responsible for improved in vitro on SMEDDS granules characteristics carvedilol release. Incorporation of higher When hypromel ose was used as binder in molecular weight polymers resulted in slower in granulation dispersion, the difference in vitro carvedilol release profile, while higher flowablity was noticed regarding concentration. binder concentration was related to faster drug Hence, SMEDDS granules with lower polymer release. amount were classified as good, in comparison 5. REFERENCES to fair flow properties of particles with 7.45% of polymer (according to the same Carr index 1. Mandić J. et al. Overview of solidification criteria). techniques for self-emulsifying drug delivery Concerning in vitro carvedilol properties, systems from industrial perspective. Int. J. SMEDDS granules with higher binder amount Pharm. 2017 533(2): 335–45. 2. Pouton CW. et al. Lipid formulations for oral showed slightly faster carvedilol release, in administration of drugs: non-emulsifying, self- emulsifying and ‘self-microemulsifying’ drug delivery systems. Eur J Pharm Sci. 2000 11: 93 68 OP5 PULMONARY DRY POWDER FOR INHALATION FOR TARGETED DRUG DELIVERY TO MACROPHAGES IN TUBERCULOSIS Mahwash Mukhtar1, Noemi Csaba2,3, Sandra Robla2,3, Rubén Varela‑Calviño4, Árpád Farkas5, Rita Ambrus1 1 Institute of Pharmaceutical Technology and Regulatory Affairs, Faculty of Pharmacy, University of Szeged, Szeged, Hungary 2 Department of Pharmacology, Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Santiago de Compostela, Spain 3 Center for Research in Molecular Medicine and Chronic Diseases, University of Santiago de Compostela, Santiago de Compostela, Spain 4 Department of Biochemistry & Molecular Biology, School of Pharmacy University of Santiago de Compostela, Santiago de Compostela, Spain 5 Center for Energy Research, Hungarian Academy of Sciences, Budapest, Hungary 1. INTRODUCTION freeze-dried to obtain INH-MC/HA The marketed oral therapies fail to deliver the nanopowder [1]. All the studies were compared anti-tubercular agent directly to the lungs. The with the INH-CS/HA nanopowder, synthesized conventional drug delivery systems are in a similar way using chitosan instead of MC. ineffective in the treatment of tuberculosis (TB) 2.3. In vitro aerodynamic profile due to the off-site drug release, inadequate drug Andersen cascade impactor (ACI) (Copley concentration at the targeted site and therefore Scientific Limited, UK), was used to determine cause systemic toxicity. As the causative agent the aerodynamic characteristics of the Mycobacterium tuberculosis resides in the nanopowder. Nanopowder fil ed into the alveolar macrophages, therefore targeted HPMC capsules was delivered to the ACI using nanotechnology-based dry powder for Breezhaler® device. The particles deposited on inhalation was fabricated to deliver the anti- each plate were dissolved in (2:1) tubercular isoniazid (INH) in the deeper lung methanol:water and emit ed dose (ED) was tissues. spectrophotometrical y analyzed. The flow rate 2. MATERIALS AND METHODS was set to 60 L/minute during an experiment. 2.1.Materials Mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF < 3 µm) were Isoniazid, Hyaluronic acid (HA), Chitosan (CS) analyzed using KaleidaGraph software [2]. 75–85 % deacetylated, Mannosylated chitosan (MC). 2.4.Studies on macrophages 2.2.Method to obtain nanopowder The cytotoxicity of nanopowders was evaluated An ionic gelation method was used to develop using an MTS assay on the primary the nanosuspension. Briefly, the developed macrophages The macrophage phenotypic polymer, MC was dissolved in 0.05 M glacial analysis was performed after incubation of acetic acid. The aqueous solution of HA was nanoparticles with macrophages and staining added dropwise to the MC solution along with with antibodies (CD83-APC and CD80-PE) [3]. the cross-linker, sodium tripolyphosphate with Also, 2,3-Indoleamine dioxygenase (IDO) continuous stirring. Later, INH solution in assay was performed to evaluate the tolerogenic methanol was added to the above mixture to effect of nanoparticles on macrophages [4]. yield the drug-loaded nanoparticles which were 69 OP5 3. RESULTS AND DISCUSSION 3.1. In vitro aerodynamic profile FPF < 3 μm was found to be promising which presents the drug deposition in the lower areas of the lungs, particularly in the alveolar region. MMAD was within the acceptable range for inhalation (Table 1). Table 1. Particle characteristics of the nanopowder Parameters INH-CS/HA INH-MC/HA nanopowder nanopowder Particle size 3.42 ± 6.5 302 ± 3.2 (nm) MMAD (µm) 2.46 ± 0.09 1.679 ± 0.1 FPF <3 µm (%) 50.90 ± 0.07 62 ± 0.09 Figure 2. % Quantification of the expression of macrophage maturation markers, CD80 and CD83 ED (%) 75 ± 0.88 87.70 ± 0.21 (a), IDO activity in macrophages cel culture (b) 4. CONCLUSION 3.2. Studies on macrophages The developed nanopowder for inhalation was The % cel viability was evidently but not found to be promising in terms of the primarily dependent on the increase in the aerodynamic deposition profile and cel ular concentrations of the samples (Fig. 1). Al the studies. This approach can be optimistic in samples presented a viability of more than 70 targeting macrophages in TB by exploiting the biocompatible polymers. %. 5. REFERENCES 1. Mukhtar, M., et al., Freeze-dried vs spray-dried nanoplex DPIs based on chitosan and its derivatives conjugated with hyaluronic acid for tuberculosis: In vitro aerodynamic and in silico deposition profiles. European Polymer Journal, 2021. 160: 110775. 2. Mukhtar, M., et al., Aerodynamic properties and in silico deposition of isoniazid loaded chitosan/thiolated chitosan and hyaluronic acid hybrid nanoplex DPIs as a potential TB Figure 1. Effect of nanoformulations on the viability treatment. International Journal of Biological of a primary culture of macrophages after 24 h Macromolecules, 2020. 165: 3007-3019. incubation. 3. Robla, S., et al., A chitosan-based nanosystem as pneumococcal vaccine delivery platform. The expression of T-lymphocyte costimulatory Drug Delivery and Translational Research, molecules (CD83 and CD80), the indicators of 2021. 11(2): 581-597. pro-inflammatory activated phenotype in 4. Crecente-Campo, J., et al . Design of polymeric macrophages, was evaluated by the incubation nanocapsules to improve their lympho-targeting capacity. Nanomedicine, 2019. 14(23): 3013- of NPs with macrophages for 2 h. The 3033. expression was higher for the MC/HA ACKNOWLEDGMENT nanoformulations (Fig. 2a). No tolerogenic activity was observed by the nanoparticles. This TKP2021-EGA funding scheme (Hungary). shows that the T-cel immune response was not suppressed, characteristic of the pro- inflammatory response, which is essential to the treatment of TB (Fig. 2b). 70 OP6 FLUORESCENT PROBES FOR DETECTION OF MISFOLDED PROTEINS IN BIOLOGICAL SAMPLES Damijan Knez1, Luka Rejc2, Lana Blinc3, Matic Rogan2, Bruno Aleksander Martek2, Ross Jansen van Vuuren2, Jerneja Kladnik2, Anže Meden1, Gabriela Molina-Aguirre4, Balazs Pinter4, Maja Bresjanac3, Stanislav Gobec1, Janez Košmrlj2 1 University of Ljubljana, Faculty of Pharmacy, Department of Medicinal Chemistry, Slovenia 2 University of Ljubljana, Faculty of Chemistry and Chemical Technology, Department of Organic Chemistry, Slovenia 3 University of Ljubljana, Faculty of Medicine, Institute of Pathophysiology, Slovenia 4 Department of Chemistry and Biochemistry, University of Texas at El Paso, Texas, USA 1. INTRODUCTION 2. MATERIALS AND METHODS Amyloid β (Aβ) has long been considered a 2.1. Design and synthesis of probes major contributor to the pathology of Alzheimer’s Disease (AD). Recent therapeutic Fluorescent probes were synthesized fol owing efforts with aducanumab, an antibody established procedures for the synthesis of developed to remove Aβ from the brain, have (F)DDNP analogues [3, 4]. demonstrated that this concept deserves 2.2. Spectral properties of the probes reevaluation. Timely diagnosis of AD is The spectral properties, i.e., absorption and chal enging because of the lack of reliable emission spectra, quantum yields in different detection methods [1]. Accurate information solvents (e.g. methanol, acetonitrile, with predictive diagnostic value currently relies dichloromethane) were determined as described on positron emission tomography (PET) previously [3]. imaging of cerebral deposition of Aβ and CSF markers (Aβ, tau, and phosphorylated tau). 2.3. In vitro binding experiments These analyses have gained acceptance and are Aβ1–42 fibrils were prepared in 10 mM HCl at 37 included in the diagnosis of AD; however, they °C, preformed tau-441 fibrils were acquired remain relatively inaccessible or invasive. To from rPeptide LLC. In vitro binding properties overcome this, new platforms and of probes in the presence of Aβ and tau-441 immunoassays have been developed in recent fibrils, non-aggregated Aβ, and BSA were years to measure Aβ in the periphery, since Aβ determined on 96- or 384-wel microplates in is elevated in early prepathological stages [2]. 150 mM HEPES buffer solution (pH = 7.4, 150 In addition, fluorescent probes that selectively mM NaCl) using Biotek Synergy H4 and Tecan stain Aβ deposits in brain samples have also Spark microplate readers. Kd values were been developed and could also be used to detect determined as described previously [5]. Aβ species in blood samples. Ex vivo 2.4. Binding to misfolded protein in human quantification of misfolded proteins in blood brain samples would be a minimal y invasive method for Human brain sections from AD patient and diagnosing AD and also for tracking disease healthy controls were stained with selected progression. The development of diagnostic probes [3], and examined under a fluorescence tools should consider the specificity of confocal microscope. detection and sensitivity to detect AD biomarkers at femtomolar concentrations in blood. 3. RESULTS AND DISCUSSION We focused on the design, synthesis, and 3.1. Synthesis of probes and DFT studies evaluation of fluorescent molecular probes to The fluorescent probes were designed to detect AD biomarkers, i.e., Aβ and tau protein, contain three building blocks: a) core by fluorescence spectroscopy in blood and brain framework with π-system, i.e., linker, b) tissue from AD patients. electron donor group, and c) electron acceptor 71 OP6 groups. Such structure enables the push-pul effect, which is often reflected in the fluorescence properties. The optical properties showed a clear influence of the core linker on the measured fluorescence emission maxima and quantum yields. DFT simulations revealed a charge-transfer excited singlet state responsible for the solvent/environment- dependent fluorescence of the probes. In addition, it was general y observed that these probes twist in the excited state with donor- acceptor groups nearly perpendicular to each Figure 1. Amyloid plaque (orange, granular) and other. neurofibril ary tangles (turquoise, filamentous), stained with probe 1. 3.2. In vitro binding properties Binding of probes to the Aβ 4. CONCLUSION 1–42 fibrils was accompanied by changes in fluorescence We have developed probes that differential y emission, which was fol owed to determine Kd stain amyloid plaques and neurofibrillary values (Table 1). The spectral changes in the tangles in human brain samples. The question of presence of monomeric Aβ1–42 indicated that the whether the probes are capable of detecting Aβ probes bound only to fibrils. The spectra in the species in blood samples from patients with AD presence of BSA indicated the degree of wil be the subject of further investigation. nonspecific binding, which was lower 5. REFERENCES compared with Aβ1–42 fibril binding. Two selected probes and ThT were subjected to 1. Vil emagne, VL., et al., Imaging tau and further assays, i.e., binding to tau441 fibrils and amyloid-β proteinopathies in Alzheimer disease experiments on human plasma. In plasma and other conditions. Nature Reviews samples spiked with Aβ Neurology, 2018. 14(4): 225-236. 1–42 fibrils, an increase in probe’s fluorescence emission was observed, 2. Chong, JR., et al., Blood-based high sensitivity measurements of beta-amyloid and indicating that the probes were able to detect phosphorylated tau as biomarkers of fibrils in a complex matrix. Alzheimer's disease: a focused review on recent Table 1. Spectral properties and binding affinities of advances. Journal of Neurology, Neurosurgery probes towards Aβ and Psychiatry, 2021. 92(11): 1231-1241. 1–42 fibrils. 3. Rejc, L., et al., Design, Syntheses, and in Vitro Aβ Evaluation of New Fluorine-18 Radiolabeled 1–42 fibrils Cpd Tau-Labeling Molecular Probes. Journal of λem,max Fold increase Medicinal Chemistry, 2017. 60(21): 8741-8757. [nm] of emission Kd [µM] 4. Petrič, A., et al., Dicyanovinylnaphthalenes for 1 595 328 ± 32 0.88 ± 0.22 neuroimaging of amyloids and relationships of electronic structures and geometries to binding 2 560 200 ± 2 0.36 ± 0.10 affinities. The Proceedings of the National Academy of Sciences, 2012. 109(41): 16492-ThT 485 17 ± 4 30.6 ± 5.0 16497. 5. Espargaró, A., et al., On the Binding of Congo Red to Amyloid Fibrils. Angewandte Chemie 3.3. Fluorescence and Confocal Microscopy International Edition, 2020. 59(21): 8104-8107. Selected probes bound to and distinguished between different types of AD-specific misfolded protein aggregates in human brain ACKNOWLEDGMENT samples as demonstrated by ultraviolet and confocal microscopy images (Fig. 1). Financial support from Slovenian Research Agency ARRS is acknowledged (P1-0208, P1- 0230, P3-0171 and J1-3018). 72 OP7 TARGETING CATHEPSIN X IN NEURODEGENERATIVE DISEASES WITH NOVEL TRIAZOLE-BENZODIOXINE INHIBITORS Urša Pečar Fonović1, Damijan Knez2, Ana Mitrović1, Anja Pišlar1, Martina Hrast2, Nace Zidar2, Jure Stojan3, Matic Proj2, Simon Žakelj4, Jurij Trontelj4, Bojan Doljak1, Boris Brus2, Tanja Jakoš1, Stanko Gobec2, Janko Kos1 1 Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, Slovenia 2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Slovenia 3 Institute of Biochemistry, Medical Faculty, University of Ljubljana, Slovenia 4 Department of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION determined using enzyme specific substrates. Cathepsin X is a cysteine cathepsin with Reversibility of binding was determined by the carboxymonopeptidase activity. It is expressed washout experiment, and by step-wise dilution mainly in immune and neuronal cel s and experiment. physiological y implicated in cel proliferation, 2.2 Cell assays migration, adhesion, etc. When up-regulated, After determining cytotoxicity with MTS assay, like in cancer and neurodegenerative disorders, the impact of selected compounds on migration it is involved in diverse pathological processes was tested in prostate cancer PC-3 cel model where it acts through cleavage of its distinct and on neurite outgrowth in pheochromocytoma substrates 1. In neuronal cel s cathepsin X PC-12 cel model. Real-time cel migration abolishes the neurotrophic activity of -enolase assay was performed on a RTCA DP by cleaving its C-terminus, and is involved in Instrument, xCELLligence System (ACEA the generation of plasmin. Therefore, cathepsin Biosciences) and measured continuously for 72 X regulates neuronal differentiation and neurite hours. Neurite outgrowth was measured with outgrowth and moreover it regulates neuronal Neurite Outgrowth Staining Kit 48 hours after survival and apoptosis. cel exposure to the compound. Cel F-actin Activity of cathepsin X is poorly control ed by content was determined with fluorescence microscopy using phalloidin-TRITC. endogenous cysteine peptidase inhibitors 3, and is thus a promising target for the 2.3. Synthesis development of new therapeutic agents. A concise series of analogues was synthesized Inhibitor AMS-36 which was designed based on by varying the benzodioxine and triazole the general clan CA cysteine protease inhibitor moieties. The central ketomethylenethio linker E-64 with epoxysuccinyl covalent warhead 4, was also modified to gain deeper insight into showed only limited selectivity towards SARs. cathepsin X, cross-reactivity with cathepsin B being the most problematic 5. 3. RESULTS AND DISCUSSION 3.1 Triazole-benzodioxine compounds 2. MATERIALS AND METHODS inhibit cathepsin X reversibly 2.1 Relative inhibition of cathepsins Fol owing the initial screening and the assays of 579 compounds from the in-house library, and the “analogues by catalogue” compounds, Ki subsequent 20 analogues of hit compounds values in low µM concentration were were screened for the inhibition of cathepsin X determined for the five most potent inhibitors. at 50 µM concentration using Abz-FEK(Dnp)- Al hit compounds selectively and reversibly OH substrate at 37 C. For compounds inhibited cathepsin X, and 1-(2,3-dihydro inhibiting cathepsin X by more than 50%, K benzo[ b][1,4]dioxin-6-yl)-2-((4-isopropyl-4 H- i was determined, and their inhibitory potencies 1,2,4-triazole-3-yl)thioethan-1-one, compound toward cathepsins B, L, S and H were Z9, was identified as the most potent inhibitor of the series. 73 OP7 3.2 Compound Z9 up-regulated neurite 4. CONCLUSION outgrowth in PC-12 cells and inhibited migration of cancer cells Based on an in-house screening of a compound New inhibitors were not cytotoxic at 10 µM library, reversible triazole-based cathepsin X concentration. Three of the inhibitors inhibited inhibitors were developed. Ki values were in the PC-3 migration, Z9 by more than 30%. Five low micromolar range, inhibitors showed high assayed inhibitors up-regulated neurite selectivity for cathepsin X, and performed wel outgrowth as measured with the kit, Z9 for 85%. in cel -based assays. Neurite outgrowth was shown also through the up-regulation of actin polymerisation after cathepsin X inhibition (Fig.1). 5. REFERENCES 1. Kos, J., et al., Intracellular signaling by cathepsin X: Molecular mechanisms and diagnostic and therapeutic opportunities in cancer. Seminars in Cancer Biology, 2015. 31: 76-83. 2. Pišlar, A., et al., Cysteine Cathepsins in Neurological Disorders. Molecular Neurobiology, 2014. 49: 1017-1030. 3. Pečar Fonović, U., et al., The Carboxypeptidase Activity of Cathepsin X is Not Controlled by Figure 1. After 2-hour inhibition of cathepsin X by Endogenous Inhibitors. Acta Chimica Slovenica, compound Z9, up-regulation of neurite outgrowth is 2019. 66(1): 58-61. shown by visualisation of actin polymerisation in PC- 4. Sadaghiani, A.M., et al., Design, synthesis, and 12 cel s. evaluation of in vivo potency and selectivity of 3.3 Structure activity relationships (SARs) epoxysuccinyl-based inhibitors of papain-family 52 analogues of Z9 were synthesized to explore cysteine proteases. Chemistry & biology, 2007. SAR, which ultimately point to the importance 14(5): 499-511. of the central linker for cathepsin X inhibition. 5. Pečar Fonović, U., et al., Identification and Variations of the triazole heterocycle had no characterization of the novel reversible and selective significant effect on inhibitory potencies, cathepsin X inhibitors. Scientific Reports, 2017. 7: whereas inhibition was diminished when the 11459. benzodioxine moiety was replaced with substituted phenyls. Five compounds were ACKNOWLEDGMENT analysed in more detail, and showed reversible binding and selective inhibition of cathepsin X. This study was supported by the Slovenian Their inhibitory activities were in the same Research Agency (Research Programs P1-0208 concentration range as that of Z9. and P4-0127, and Research project J4-8227). 74 OP8 CHALCOGEN CARBAMATES AS COVALENT CHOLINESTERASE INHIBITORS – SURVEYING GROUP 16 (VI) OF THE PERIODIC TABLE Anže Meden1, Damijan Knez1, Xavier Brazzolotto2, Fabrice Modeste2, Milica Denic2, Stanislav Gobec1 1 Department of Pharmaceutical Chemistry, University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, Ljubljana, Slovenia. 2 Département de Toxicologie et Risques Chimiques, Institut de Recherche Biomédicale des Armées, Brétigny sur Orge, France. 1. INTRODUCTION methylethan-1-amine. The compounds were Aryl carbamate alkaloids, such as analyzed to confirm identity and purity. physostigmine, were not only the first 2.2. In vitro characterization, crystallization, discovered cholinesterase inhibitors, but also and LC-MS proof of carbamoylation the first covalent, pseudo-irreversible The inhibitory potencies of the compounds cholinesterase inhibitors (ChEIs).[1] The ChEIs against the ChEs were determined using the found their use in medicine (Alzheimer’s method of El man, and for time-dependency disease, myasthenia gravis, glaucoma, etc.), measurements, the pre-incubation time was pest control, and, unfortunately, also as varied (1 min, 5 min, 15 min, and 30 min). The chemical weapons. The rates of carbamoylation crystal ization also fol owed a reported and decarbamoylation are governed by both procedure.[3] steric and electronic factors.[2] Review of the In LC-MS experiments, recombinant human literature has shown that besides the O-aryl BChE (approx. 5 µM) was incubated with 50 carbamate ChEIs, the chemical space of µM compound for 5 min, then 2 µL of the different carbamates remains largely mixture were injected on a bioZenTM 3.6 µm unexplored. Intact XB-C8 LC column 100 × 2.1 mm The group 16 (VIA) elements of the periodic (Phenomenex) on an Agilent 1290 Infinity table are also called chalcogens (“ore formers”). UHPLC coupled to an Agilent iFunnel Q-TOF The periodicity effects upon descending within 6550 HRMS, and eluted with MQ water/0.1% a group in the periodic table of elements, e.g., HCOOH – acetonitrile/0.1% HCOOH (linear the decrease in the hydrides’ p K a-s, the increase gradient). Data were treated using OpenChrom in the atomic radi , and the decrease in (v 1.4) and extracted spectra were deconvoluted electronegativities, have been wel studied with UniDec software (v 5.0.1). within the field of inorganic chemistry but are 3. RESULTS AND DISCUSSION quite neglected in medicinal chemistry. To elucidate the factors that determine the 2. MATERIALS AND METHODS carbamate leaving group prerequisites for 2.1. Synthesis covalent inhibition, we prepared a variety of The leaving groups were synthesized by dimethylcarbamates (Figure 1) based on a halogen-lithium exchange and quenching with previously reported, reversible, electrophile from 7-bromoindole, the butyrylcholinesterase-selective inhibitor dimethylcarbamoyl group was introduced with scaffold.[3] The covalent mechanism of binding N, N-dimethylcarbamoyl chloride and the rest of was tentatively determined by IC50 time-the molecule at ached via Mannich reaction dependency and kinetic experiments. For with formaldehyde and 2-cycloheptyl- N- selected dimethyl O-aryl carbamates, the carbamoylation of butyrylcholinesterase’s 75 OP8 Ser198 was also unambiguously confirmed by 5. REFERENCES mass spectrometry (71±1 Da mass increase due 1. Scheindlin, S., Episodes in the Story of to the dimethylcarbamoyl moiety and Physostigmine. Molecular Interventions, 2010. protonated His438), and crystal ization 10 (1): 4. (carbamoylated catalytic Ser198 was observed 2. Groner, E., et al., The Kinetics of Inhibition of in the crystal structure). Regarding different Human Acetylcholinesterase and leaving group chemotypes, oxime and enol Butyrylcholinesterase by Two Series of Novel Carbamates. Molecular Pharmacology, 2007. ether carbamates functioned as covalent ChEIs, 71 (6): 1610–1617. whereas aliphatic alcohol and trifluoroethanol 3. Meden, A., et al., From Tryptophan-Based carbamates did not. Amides to Tertiary Amines: Optimization of a To evaluate the periodicity effects within the Butyrylcholinesterase Inhibitor Series. European Journal of Medicinal Chemistry, chalcogens, we synthesized and evaluated thio-, 2022. 234: 114248. seleno- and tel uro-analogues of the O-aryl carbamate. The compounds indeed functioned as covalent inhibitors, but their potency was ACKNOWLEDGMENT decreased compared to their oxygen This research was funded by the Slovenian predecessor. To evaluate the steric Research Agency (ARRS), Research Core requirements, smal er fragments featuring the Funding N° P1-0208 and a young researcher same warhead motif were also tested, and to grant to A.M. X.B., F.N. and J.D. were evaluate the electronic effects, QM(/MM) supported by the French Ministry of Armed modelling was undertaken. Forces (Direction Generale de l'Armement and Service de Sante des Armees, NBC-5-C-4210). Authors would like to thank the SOLEIL synchrotron for long-term beamtime access (20181022 IBS BAG). Figure 1. Examples of compounds synthesized and evaluated. The blue-coloured carbamate moiety is transferred to the catalytic serine in the enzyme active site. The green-coloured moiety functions as the leaving group (LG), while the black-coloured portion of the molecule is responsible for recognition and confers selectivity for butyrylcholinesterase. The four non-radioactive chalcogens are highlighted within the periodic table of elements. 4. CONCLUSION To the best of our knowledge, despite the simplicity of the idea, this is the first time that al non-radioactive chalcogen carbamates, i.e., O-, S-, Se-, and Te-aryl carbamates, were prepared and reported as cholinesterase inhibitors. 76 OP9 IN VIVO EFFICIENCY OF A NEW LIPOSOMAL ADJUVANT SYSTEM BASED ON PORINS Selin Parmaksız1, Ece Türkmen1, Aykut Özkul2, Vanessa Rivero-Arredondo3, Louis Ontiveros-Padilla3, Maryam T. Hussein4, Neil Liam Andrew Forbes4, Constantino López-Macías3, Yvonne Perrie4, Sevda Şenel1* * 1Department of Pharmaceutical Technology, Faculty of Pharmacy, Hacettepe University, Turkey 2 Department of Virology, Faculty of Veterinary Medicine, Ankara University, Turkey 3 Medical Research Unit on Immunochemistry, Specialties Hospital, National Medical Centre “Siglo XXI”, Mexican Social Security Institute (IMSS), Mexico City, Mexico 4 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, United Kingdom *Correspondence: ssenel@hacettepe.edu.tr 1. INTRODUCTION C. Determination of humoral immune Liposomes have been shown to exert response immunostimulatory effects besides being used On 0, 8, 12 and 30 days the animals were bled as a delivery system for vaccines [1]. The size, via the submandibular route. IgM and IgG charge, bilayer rigidity and composition of levels were determined by ELISA at 492 nm. liposomes al ow for tailored antigen delivery RESULTS AND DISCUSSION and have an impact on stimulation of immune The properties of the liposomal adjuvant system response [1-3]. On the other hand, Salmonella and the adjuvanted formulations are given Typhi (S. Typhi) porins have been shown to Table 1. exert adjuvant effect on antibody responses to Table 1. Animal study groups several antigens [4]. In this study, we have Groups Particle Zeta combined neutral and cationic liposomes with Size (nm) Potential (mV) porins and evaluated immune response against 1 Saline solution - - a model antigen, OVA in immunized BALB/c 2 Porins - -20,83±0,55 mice. 3 OVA - -24,36±1,16 MATERIALS AND METHODS 4 Porins+OVA - -22,75±0,59 a. Preparation of liposomal formulations 5 Neutral Liposome 524,0 ± 20,4 -6,05±0,81 6 Porins+ Neutral 466,5 ± 28,9 -9,96±0,74 Liposomes were prepared by the film-hydration Liposome method [3]. Lipids were obtained from Avanti 7 OVA+ Neutral Liposome 662,8 ± 14,5 -5,49±0,29 Lipis, USA. DSPC (distearoyl-sn-glycero-3- 8 Porins+ OVA+ Neutral 481,7 ± 5,8 -8,62±2,28 phosphocholine), Cholesterol and TDB (1,2- Liposome 9 Cationic Liposome 407,7 ± 3,9 39,73±0,85 trehalose 6,6-dibehenate) were used for neutral 10 Porins+ Cationic 423,1 ± 7,8 19,13±2,09 liposomes and DDA (dimethyl-dioctadecyl Liposome ammonium) and TDB were used for cationic 11 OVA+ Cationic 310,8 ± 96,2 16,77±1,10 liposomes. 1 mg/mL endotoxin-free ovalbumin Liposome 12 Porins+ OVA+ Cationic 383,5 ± 72,5 14,67±1,31 (Invivogen, France) and 0,2 mg/mL porins Liposome (purified from S. Typhi 9,12, Vi: d ATCC 9993) [4] were added to the lipid film. IgM levels: Our results showed that the combination of porins with cationic or neutral b. Immunisation liposomes induced higher anti-porins and anti- 36 female, 6 to 10 weeks old BALB/c mice were OVA IgM antibody levels compared to that divided into twelve groups (n=3) (Table 1). porins, OVA, porins+OVA and empty Mice were immunized intraperitoneal y on days liposomes on Day 8 (p <0.001) (Fig. 1). 0 and 15. Saline solution was used as a control. Cationic liposomal adjuvant induced higher The administration volume was 100 µl. Al anti-porins IgM than neutral liposomal animal care and experimental protocols were adjuvants, whereas with OVA, anti-OVA IgM approved by animal experimentations local levels were higher with neutral liposomal ethics board (protocol no: 2015/75-14). systems after first immunization. After booster, 77 OP9 IgM levels were found to be similar with (p<0.001). IgG levels were not induced with cationic and neutral liposomal adjuvants and any groups after the first immunization. After these levels were slightly higher than that of booster, IgG level with porins+OVA without porins, OVA, porins+OVA and empty adjuvant was observed to increase significantly liposomes on Day 30 (p>0.5). when compared to adjuvanted groups (p<0.0001). A 1.0 4. CONCLUSION 0.8 0.6 We have showed that our adjuvant system D 492 0.4 O prepared by combination of porins with 0.2 liposomes resulted in significantly increased 0.0 immune responses. The charge of the liposomal Saline solution Porins Neut. Lip. system (neutral and cationic) was found to have Porins+Neut. Lip. Cat. Lip. Porins+Cat. Lip. a significant impact on the immune responses. B 0.5 It was observed that neutral liposomal adjuvant 0.4 systems were able to stimulate only the early- 0.3 D 492 stage IgM immune response, whereas cationic 0.2 O liposomal systems stimulated both the early- 0.1 0.0 stage IgM and the long-term IgG immune responses. Cationic liposomal adjuvant system Saline solution OVA Porins+OVANeut. Lip. OVA+Neut. Lip. based on combination of liposomes and S. Typhi Porins+OVA+Neut. Lip.Cat. Lip. OVA+Cat. Lip. Porins+OVA+Cat. Lip. porins are suggested as a promising Day 0 Day 8 Day 12 Day 30 adjuvant/delivery system that exerts long- Figure. 1. Anti-porins (A) and anti-OVA (B) IgM lasting immune responses responses (n=3) 5. REFERENCES A [1] Perrie, Y., et al. Advanced Drug Delivery, 2015; 2.0 99:85-96. 1.5 [2] Carstens, M. G., et al. Vaccine, 2011;29(29- 1.0 D 492 30):4761-70. O 0.5 [3] Henriksen-Lacey, M., et al. J. Control. Rel, 0.0 2010;145(2):102-8. [4] Pérez-Toledo M., et al. Frontiers in Immunology. Saline solution Porins Neut. Lip. 2017; 9(8):230. Porins+Neut. Lip. Cat. Lip. Porins+Cat. Lip. B 2.0 ACKNOWLEDGMENT 1.5 This project was supported by TÜBİTAK- 1.0 D 492 Turkey (SBAG-215S995) and UK GCRF O 0.5 Networks in Vaccines Research and 0.0 Development which was co-funded by the MRC and BBSRC, through BactiVac-Network Saline solution OVA Porins+OVANeut. Lip. OVA+Neut. Lip. project BVNCP3-21. Porins+OVA+Neut. Lip.Cat. Lip. OVA+Cat. Lip. Porins+OVA+Cat. Lip. Al authors have equal contributions. Day 0 Day 8 Day 12 Day 30 Figure 2. Anti-porins (A) and anti-OVA (B) IgG responses (n=3) IgG levels: A significant increase in IgG levels was observed with the combination of porins with cationic liposomes after booster when compared to that obtained with the porins alone and the combination of porins with neutral liposomes (p<0.0001). Anti-OVA IgG levels were induced with cationic liposomal systems higher than with neutral liposomal systems 78 OP10 IN VIVO EVALUATION OF DOXORUBICIN AND ELACRIDAR CO-LOADED PLGA/SILICA HYBRID NANOPARTICLES AGAINST MULTIDRUG RESISTANT BREAST CANCER Hayrettin Tonbul1, Adem Şahin2, Süleyman Can Öztürk3, Gözde Ultav4, Güneş Esendağlı5, Yılmaz Çapan6 1Department of Pharmaceutical Technology, Faculty of Pharmacy, Inonu University, Turkey 2Department of Pharmacy Service, Vocational School of Health Services, Bilecik Seyh Edebali University, Turkey 3Laboratory Animals Research and Application Centre (HUDHAM), Hacettepe University, Turkey 4Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Inonu University, Turkey 5 Department of Basic Oncology, Hacettepe University Cancer Institute, Turkey 6Department of Pharmaceutical Technology, Faculty of Pharmacy, Lokman Hekim University, Turkey 1. INTRODUCTION bearing models that obtained with Dox resistant Although doxorubicin (Dox) is very effective in EMT6/AR1 breast cancer cel line. several cancer treatment, being a P- 2. MATERIALS AND METHODS glycoprotein (P-gp) substrate limits its effectiveness. The multidrug resistance (MDR) 2.1. Materials observed in many cancers is frequently Al chemicals were obtained from Sigma associated with overexpression of P-gp and Aldrich (MO, USA). Al cel culture some other membrane transporters. Various consumables obtained from Lonza (Basel, approaches have been developed to overcome Switzerland). Al other used reagents were the growing resistance to these drugs including analytical or reagent grade. Dox. One of them is the co-delivery of Dox with 2.2. Preparation and Characterization of a P-gp inhibitor substance such as elacridar PLGA/Silica HyNPs (Elc). Elc is a third generation P-gp inhibitor To produce HyNPs, first of al folic acid (FA) that has been one of the most studied inhibitors conjugated Dox loaded MSNs were obtained because of the BCRP (breast cancer resistant with previously optimized method [2]. After protein) and P-gp inhibition ability [1]. Another that, elacridar and MSNs were loaded to PLGA- promising approach about overcoming MDR PEG NPs with double emulsion method. For resistance is developing a nanoparticulate drug this, 50 mg PLGA-PEG, 7,5 mg MSN and Elc delivery system. Mesoporous silica were dispersed in 1 ml dichloromethane nanoparticles (MSN) have at racted high (DCM). 200 µL distil ed water added to the interest for use as drug delivery system due to mixture and sonicated for 1 min. After that possibility to obtain very smal size, high obtained emulsion system were sonicated storage capacity and excel ent col oidal another 1 min after adding 4 mL %1 PVA stability. PLGA nanoparticles (NPs) is one of solution. Final y, obtained system poured to 20 the most investigated nanoparticles due to their mL 0.3 % PVA solution and mixed overnight to several advantages including excel ent evaporate the DCM. Obtained nanoparticles biocompatibility, biodegradability, FDA and centrifuged and washed several times. Both EMA approval, possibility to control ed release MSN and HyNPs average particle size and PDI and surface modification. Furthermore, were measured with Horiba Nanopartica SZ- combining these two type nanoparticles in a 100v2 and TEM analysis (FEI Tecnai BioTwin) single entity and obtain hybrid nanoparticles (HyNPs) hold big potential in the treatment of 2.3. In vivo experiments with tumor-bearing several diseases including cancer [2]. mice In this study, folic acid modified Dox loaded In vitro evaluation of HyNPs were previously MSN were prepared and these nanoparticles shown and cel culture results show that were co-loaded with Elc to pegylated PLGA obtained system readily uptake and show highly nanoparticles to obtain hybrid nanoparticles. cytotoxic effect on ZR-75-1, T-47 and Effectiveness of developed drug delivery EMT6/AR1 cel lines [2]. For in vivo studies, system were evaluated with mouse tumor- female Balb/C mice were used and breast 79 OP10 cancer tumor model were developed with Dox Results show that accumulation of dye in liver resistant EMT6/AR1 cel line. Mice were significantly decrease in HyNP+ formulation divided to 8 groups as I. PBS, II. blank HyNPs, where there is no dye in tumor in dye solution III. Dox solution, IV. Caelyx® (marketed group. liposomal dox formulation), V. Dox+Elc solution VI. Caelyx+Elc solution, VII. Only Dox loaded HyNP formulation (HyNP-) VIII. Dox and Elc loaded HyNP formulation (HyNP+). Formulations were applied to mice at 0, 4, 8, 12, 16 and 20th days as IV with 5 mg/kg Dox and 5,18 mg/kg Elc concentration. Formulation effect on tumor growth were evaluated. Moreover, in vivo biodistribution studies were conducted using animal imaging device (Vilber Newton 7.0, France). 3. RESULTS AND DISCUSSION 3.1. Characterization of PLGA/Silica HyNPs DLS results show that average particle size of FA conjugated MSNs and HyNPs were found 61.3±5.9 and 248.1±11.8, respectively which is corelated with TEM results (Figure 1) Figure 2. A. Change in tumor size in mice during 24 days. B. Real image of tumors in mice after sacrification in 24th day. After 24 days, elacridar founded groups showed lesser tumor growth among the al groups, where HyNP+ formulation showed least tumor Figure 1. TEM image of MSN (A) and HyNP (B). growth. 3.2. In vivo experiments with tumor-bearing 4. CONCLUSION mice After 24 hours from injection of Indocyanine Results show that unique hybrid nanoparticle green (ICG, dye) solution and ICG loaded formulation that combine MSN and PLGA HyNP+, accumulation of dye in the organs nanoparticles hold big potential in MDR shown in Figure 1. resistant breast cancer treatment. 5. REFERENCES 1. Tonbul H., et al. Combination drug delivery with actively-targeted PLGA nanoparticles to overcome multidrug resistance in breast cancer. Journal of Drug Delivery Science and Technology, 2019, 101380. 2. Tonbul, H., Development of doxorubicin and elacridar loaded PLGA/silica hybrid nanoparticles and evaluation of activity against breast cancer, Haccet epe University Graduate School of Health Science PhD Thesis, 2019. ACKNOWLEDGMENT This work was supported by The Scientific and Technological Research Council of Turkey Figure 1. Biodistribution of ICG (dye) solution and (TUBITAK), Project Number: 216S999 and ICG loaded HyNP+ formulation A. Real image B. Research Fund of the Inonu University, Turkey MFI value after 24h from IV injection to mice. Project Number: TCD-2021-2229. 80 OP11 NANOFIBERS FOR LOCAL DELIVERY OF TWO BACILLUS STRAINS WITH ANTIBACTERIAL ACTIVITY Nina K. Grilc, Tomaž Rijavec2, Anže Zidar1, Aleš Lapanje2, Petra Kocbek1, Julijana Kristl1, Špela Zupančič1 1 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia 2 Department of Environmental Sciences, Jožef Stefan Institute, Ljubljana, Slovenia 1. INTRODUCTION containing 4% (w/v) polymeric dispersions of Dysbiotic biofilm-related infectious diseases PEO and ALG to yield the nanofibrous mats hinder the efficacy of conventional treatment with different ALG content (0, 40 and 80 %). with antibiotics and contribute to the spread of 2.3. Nanofiber characterisation antibiotic resistance (1). Probiotics represent a Fabrication of nanofibers and incorporation of promising alternative strategy for the treatment spores into their structure was studied by of dysbiosis-related diseases. Live therapeutic scanning electron microscopy (SEM). The bacterial strains used as probiotics should be preservation of bacterial viability was evaluated wel -characterised and exhibit antibacterial based on the colony forming unit (CFU) counts potential (2), while the products containing in the serial y diluted dispersion of dispersed appropriate strains must retain their viability nanofibers (compared to the theoretical loading and enable their precise local administration of the starting spore dispersion). and release at the target site which can be Spore-loaded nanofibrous samples were placed achieved with bioadhesive nanofibers (3). into phosphate buffer (pH 6.8) and spore release The aim of this study was the incorporation of from nanofibers was studied based on the CFU two genetical y characterised Bacillus strains counts in serial y diluted aliquots taken from the into bioadhesive nanofibers intended for medium at different time points. periodontitis treatment. The nanofibers were 2.4. Characterisation of the studied probiotic designed to retain the viability of probiotics and potential of bacterial strains provide their control ed release. The probiotic The studied strains were genotypical y potential of the biomaterial was evaluated based characterised. Their antibacterial activity was on their antibacterial properties. evaluated by studies of growth kinetics of select 2. MATERIALS AND METHODS periodontopathogens in the medium containing metabolites of the studied probiotics. 2.1. Materials Polyethylene oxide (PEO) 900 kDa and 2 MDa, 3. RESULTS AND DISCUSSION Nutrient broth and MnSO4×H2O were from 3.1. Nanofiber characterisation Sigma-Aldrich. Sodium alginate (MW 138 Successful incorporation of spores into al three kDa) Protanal LF 10/60 was from FMC nanofiber formulations was revealed by SEM BioPolymer and select agar from Invitrogen. which also revealed the fabrication of smooth 2.2. Culturing, sporulation of bacteria and non-beaded fibers (Fig. 1). Spore loadings in the their incorporation into nanofibers nanofibers were also confirmed by CFU Both studied bacterial strains ( Bacillus strains concentration in the dispersed nanofibers which 27.3.Z and 25.2.M, isolated from healthy oral revealed the preservation of viability during the microbiota) were sporulated by culturing in electrospinning process as wel as six months manganese-supplemented media (nutrient storage, achieving the loading of more than 7 broth). Incorporation of spores of individual log(CFU/mg). This indicates that pre- strains or their combination into nanofibers was electrospinning sporulation of both strains is an carried out by electrospinning of the spore- appropriate strategy for viability preservation 81 OP11 compared to the fabrication of nanofibers concurrent use. Antibacterial action of the containing bacteria in their vegetative state. investigated strains against selected periodonto- The release of spores from the bulk nanofibrous pathogens ( Aggregatibacter actinomycetem- product was studied in order to elucidate the comitans, Fusobacterium nucleatum, potential of the different formulations to Porphyromonas gingivalis) was also confirmed provide prolonged release of the bacterial experimentally. strains to the targeted epithelium with dysbiotic biofilm. Both studied strains exhibited comparable release kinetics from nanofibers with the same composition (Fig. 2). Figure 1. Spore-loaded nanofibers: (a) 40 % ALG + 27.3.Z bacteria, (b) 80 % ALG + 25.2.M bacteria. Figure 2. Release of spores 27.3.Z from nanofibers. Fol owing the initial burst release in the first The smal er figure shows spore release in the first 3 hours in more detail. hour, the remaining spores were gradual y released over the next three to eight hours for 4. CONCLUSION PEO and PEO/ALG nanofibers, respectively. The developed spore-loaded nanofibers are a The burst release is at ributed to rapid promising approach for periodontitis treatment dissolution of hydrophilic polymers resulting in and are capable of delivering high loadings of the dispersion of nanofibers from the surface viable bacteria in a control ed manner. The use and outer layers of the nanofibrous mat. The of wel -characterised strains represents a later prolonged release can be at ributed to the foundation for the development of a complex entrapment of spores in the swol en matrix of biomaterial with desired probiotic properties, the inner layers of the nanofibrous mat as PEO such as antibacterial efficacy against relevant and ALG are known to swell (4), likely closing pathogens. Meanwhile, the combination of the the interfiber pores. Thus, spore release is strains aims toward a higher complexity of the limited by polymer erosion. The kinetics of local y delivered microbial community. spore release from nanofibers was dependant on 5. REFERENCES PEO/ALG ratio with higher ALG content 1. Del Pozo, J.L., Biofilm-related disease. Expert prolonging the spore release. The release of Review Anti-infective Therapy, 2018. 1:51-65. spores from nanofibers can therefore be 2. Gupta, G., Probiotics and Periodontal health. control ed with the ratio of the two polymers, Journal of Medicine and Life, 2011. 14(4):387- whereas the use of ALG provides the feature of 94. 3. Brako, F., et al. Making nanofibres of bioadhesion (3), desired to prolong nanofiber mucoadhesive polymer blends for vaginal retention at the targeted oral cavity surfaces. therapies. European Polymer Journal, 2015. Vol. 70:186-196. 3.2. Probiotic potential of the studied 4. Christe, S.M., Sodium Alginate with PEG/PEO bacterial strains Blends as a Floating Drug Delivery Carrier – In The probiotic potential of both studied bacterial vitro Evaluation. Advanced Pharmaceutical strains (27.3.Z and 25.2.M) was evaluated by Bul etin, 2016. 6(3):435–442. genotypic characterisation and genomic se- ACKNOWLEDGMENT quencing, revealing that the strains did not This work was supported by the Slovenian match completely in the genes coding for Research Agency [Programme J1-9194], antibiotics, corroborating the rationale for their European Commission, SurfBio project [grant no. 952379]. 82 OP12 DEVELOPMENT OF CYCLOSPORINE A-LOADED MICELLES EXIBITING A PROMISING ANTIVIRAL ACTIVITY AGAINST SARS-COV-2 Fabiola Guareschi1, S. Pescina1 F. Buttini1, M. Brandolini2, V. Sambri2,3, E. Del Favero4, L. Cantù4, C. Ricci4, F. Sonvico1 1 Department of Food and Drug, University of Parma, Parma, Italy 2 Unit of Microbiology, The Great Romagna Hub Laboratory, 47522 Pievesestina, Italy 3 Department of Experimental, Diagnostic and Specialty Medicine—DIMES, Alma Mater Studiorum—University of Bologna, 40138 Bologna, Italy 4 Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Milan, Italy 1. INTRODUCTION analyzed by Smal Angle X-ray Scat ering The immunosuppressive peptide drug (SAXS, ID02 high-bril iance beamline, ESRF, Cyclosporine A (CSA) has been shown to have Grenoble, France). an inhibitory activity against the viral 2.3. In vitro activity of the micelles against replication of several coronaviruses, but its SARS-CoV-2 efficacy against SARS-CoV-2 has been The in vitro tests were performed on Vero E6 hypothesized but not demonstrated [1–3]. cel s. The cytotoxicity of the micel es was test CSA-loaded vitamin E polyethylene glycol by treating the cel s for 2 hours, then they were succinate (TPGS) micel es were developed for washed with PBS and maintained in (Minimum intranasal administration and were evaluated for Essential Medium) MEM with 2% Fetal Bovine their potential antiviral activity against SARS- Serum (FBS) for 72 hours. Six different concentrations of CSA were tested: 64 µM, 32 CoV-2 in vitro. µM, 16 µM, 8 µM, 4 µM and 2 µM [1]. Seven 2. MATERIALS AND METHODS protocols have been fol owed to understand at 2.1. Materials what level of the viral replication cycle the Cyclosporine A was from Metapharmaceutical micel es acted: A. Pre-treatment, B. Treatment (Barcelona, Spain); vitamin E polyethylene contextual to the infection, C2. Treatment 2 glycol succinate was from Recordati S.p.A hours after the infection; C6. Treatment 6 hours (Milan, Italy); sodium chloride was from VWR after the infection, D. Pre-treatment associated International (Leuven, Belgium); acetonitrile to a Treatment 2 hours after the infection, E. 3 and trifluoroacetic acid were of analytical post-treatments; F. Pre-treatments associated to grade. 3 post-treatments. Viral concentrations of 0.005 2.2. Preparation and characterization of the m.o.i and 0.0005 m.o.i were chosen, and the blank and CSA-loaded micelles Omicron BA.1 was selected as viral variant. CSA-loaded micel es (0.1, 0.25 and 0.5 mg/mL) 3. RESULTS AND DISCUSSION were prepared by directly adding CSA to the 3.1. Characterization and stability study of blank formulation prepared by dissolving TPGS the blank and CSA-loaded micelles in a NaCl 9 g/L solution [4]. The micel es were Al the micel es showed stable particle size characterized at time 0 and after 1 month stored never exceeding 15 nm, narrow PDI and null at 25°C for particle size, PDI and Zeta potential surface charge, suitable for avoiding the binding by Dynamic Light Scat ering (DLS) and for to mucin glycoproteins [5], thus favouring CSA encapsulation efficiency (EE%) by HPLC. mucopenetration. The SAXS analysis The molecular structure of the micel es and highlighted the tendency of micel es to assume their interaction with mucus were further a more spherical shape with increasing CSA 83 OP12 encapsulation, and a high rate of mucodiffusion. micel es a potential strategy for both the The CSA EE% was in al the cases higher than prevention and the treatment of the COVID-19 95%, with a slight reduction after a month at disease. 25°C. 5. REFERENCES 3.2. In vitro antiviral activity of the micelles 1 De Wilde, A.H. et al . , Cyclosporin A inhibits against SARS-CoV-2 the replication of diverse coronaviruses, The toxicological study pointed out a reduction Journal of General Virology, 2011. Vol. of the cytotoxicity with increasing loading of 92(11): 2542–2548 CSA in the micel es. The 0.5 mg/mL CSA- 2 Ma-Lauer, Y. et al., Influences of loaded micel es indeed exhibited a cytotoxicity cyclosporin A and non-immunosuppressive only at the highest concentration of CSA (64 derivatives on cellular cyclophilins and viral nucleocapsid protein during human µM) used, while the 0.1 mg/mL CSA-loaded coronavirus 229E replication, Antiviral ones were already toxic at 16 µM. These results Research, 2020. Vol. 173: 104620. highlighted that TPGS, as a surfactant, exhibits 3 Pfefferle, S. et al . , The SARS-Coronavirus-an intrinsic cytotoxicity. host interactome: Identification of cyclophilins as target for pan-Coronavirus As shown in Table 1, both the 0.5 and the 0.25 inhibitors, PLoS Pathogens, 2011. Vol. mg/mL CSA-loaded micel es exhibited the 7(10): e1002331. highest antiviral activity at the lowest CSA 4 Pescina, S. et al., Preliminary investigation concentration (2µM) tested, and they turned out on simvastatin-loaded polymeric micelles in to be more active when used fol owing the view of the treatment of the back of the eye, protocol E and F, respectively. The 0.1 mg/mL Pharmaceutics, 2021. Vol. 13(6): 855. micel es showed the highest antiviral activity 5 Araújo, F. et al., Chemical modification of when a CSA concentration of 8µM and protocol drug molecules as strategy to reduce interactions with mucus, Advanced Drug F were used. Delivery Reviews, 2018. Vol. 124: 98–106. Table 1. Antiviral activity of the CSA-loaded ACKNOWLEDGMENT micel es against SARS-CoV-2. We thank Sara Nicoli and Davide 0.5 mg/mL 0.25 mg/mL 0.1 mg/mL D’Angelo (University of Parma, Food micelles micelles micelles and Drug Department) for the support (2µM CSA) (2µM CSA) (8µM CSA) provided during the development of the 3 post- pre-treatments pre-treatments treatments associated to 3 associated to 3 CSA-loaded drug delivery systems post- post- prepared for this study, and for sharing treatments. treatments. with us their knowledge regarding the Viral 0.005 0.0005 0.005 0.0005 0.005 0.0005 potential antiviral action of CSA. concentration m.o.i m.o.i m.o.i m.o.i m.o.i m.o.i Antiviral 117% 136% 122% 155% 115% 122% activity 4. CONCLUSION The very low particle size, high CSA encapsulation efficiency and great long-term stability make the developed TPGS micel es a promising peptide-drug delivery system. Moreover, the promising results obtained by testing the antiviral activity make the developed 84 OP13 HDAC6 INHIBITION IN CYSTIC FIBROSIS: IN VIVO PROOF-OF-CONCEPT STUDY ANTI-INFLAMMATORY PROFILE, EFFECTS ON BACTERIAL LOAD, FORMULATION AND BIODISTRIBUTION STUDIES. Margherita Brindisi 1, Simona Barone 1, Emilia Cassese 1, Alice Rossi 2, Nunzio Del Gaudio 3, Alvaro Feliz Morel 4, Gessica Filocamo 4, Camilla Montesano 5, Alessandra Bragonzi 2, Lucia Altucci 3, Vincenzo Summa 1 1University of Naples Federico II, Dept. of Pharmacy, Via D. Montesano 49, 80131 Naples, Italy; 2Infections and Cystic Fibrosis Unit, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; 3University of Campania Luigi Vanvitelli, Dept. of Precision Medicine, Via de Crecchio, 80138 Naples, Italy; 4Exiris s.r.l., Via di Castel Romano, 100, 00128 Castel Romano, Italy 5Sapienza University of Rome, Dept. of Chemistry, Piazzale Aldo Moro 5, 00185 Rome, Italy. 1. INTRODUCTION After 3- or 7-days post infection mice were Compel ing new support has been provided for monitored weight loss and sacrificed with an histone deacetylase isoform 6 (HDAC6) as a overdose of Avertin. BALF was col ected and common thread in the generation of the lung was aseptical y excised to evaluate dysregulated proinflammatory phenotype in bacterial burden and inflammatory response in cystic fibrosis (CF). HDAC6 also plays a crucial the lung homogenate and bronco-alveolar role in bacterial clearance or kil ing as a direct lavage fluid (BALF). consequence of its effects on CF immune responses. Inhibiting HDAC6 functions thus eventual y represents an innovative and 3. RESULTS AND DISCUSSION effective strategy to tackle multiple aspects of 3.1. Results and Discussion Sub-headings CF-associated lung disease.[1] In this study, we We embarked a careful compound selection by provided the first in vivo PoC of the efficacy of performing a thorough analysis of scientific a selective HDAC6 inhibitor in contrasting the literature and relevant patents in the field. We CF-associated proinflammatory phenotype and thus identified compound 1 as the most suitable effectively reducing bacterial load in treated candidate to be engaged in in vivo PoC studies animals. based on its high HDAC6 inhibitory potency and relevant selectivity over other HDAC 2. MATERIALS AND METHODS isoforms (Fig. 1). We re-profiled compound 1 2.1 Methods in house on its HDAC1/6 potency and its ability in selectively increasing levels of acetylated C57BL6/NCrl male (8-10 weeks old) purchased tubulin in HeLa and A549 cel s. We also by Charles River were anesthetized by an i.p. evaluated solubility of 1 in the vehicle selected injection of 2.5% Avertin (0.015 ml/g body for the planned aerosol administration. weight) and infected by intratracheal (i.t.) Gratifyingly, compound 1 demonstrated no injection of 4-51x105 P. aeruginosa MDR- toxicity in mouse and was able to dose- RP73 clinical strain embedded in agar dependently reduce the total cel counts and beads.[8,9] To evaluate the efficacy of F2F- neutrophils in bronchoalveolar lavage fluid 2020187-00X mice were treated soon after the (BALF), when local y administered (5, 10 and infection, by aerosol administration using Penn 20 mg/kg) in a mouse model of chronic Century device with different doses (5 mg/kg, Pseudomonas aeruginosa (PA) infection using 10 mg/kg and 20 mg/kg or with vehicle a Penn-Century MicroSprayer® Aerosoliser. (PBS+4%DMSO)). Mice were treated daily for We also performed a cytokines/chemokines 3 or 7 days. Mice were sacrificed at two profiling by Bioplex assay, which highlighted different time point: after 3 days and after 7 relevant changes in the levels of interleukins days. (eg. Il-1a, IL-1b, IL-6) and other inflammatory markers, thus confirming the potential of 1 in 85 OP13 effectively reverting the pro-inflammatory Quantitative determination of 1 in plasma phenotype. Moreover, we proved that 1 is able samples col ected from treated mice showed to reduce bacterial load in the same model at the that the compound is not distributed in the body three tested doses, as determined by the even after a 7-day treatment, thus supporting the reduction of colony-forming units (Fig. 1). safe profile of 1 in our administration set ings. Figure 1. (A) General workflow for proof-of-concept (PoC) compound identification; efficacy of selected compound 1 on (A) bacterial load and (B,C) inflammatory cel count in a murine model of Pseudomonas Aeruginosa chronic infection. 4. CONCLUSION Our study is of particular significance since it demonstrates for the first time the utility of selective HDAC6 inhibitors as innovative therapeutic option for CF, using a relevant in vivo model. Our data pave the way to the development of novel HDAC6 inhibitors specifical y tailored for chronic administration in CF patients, thus improving the CF- associated inflammatory phenotype and promoting an effective immune response against infections. 5. REFERENCES [1] Barone, S.; Cassese, E.; Alfano, A. I.; Brindisi, M.; Summa, V. J. Med. Chem. 2022, 3080–3097. ACKNOWLEDGMENT The Authors kindly acknowledge Fondazione Fibrosi Cistica (FFC Grant 20-2020) for financial support. 86 OP14 NEW COVALENT REVERSIBLE INHIBITORS OF SARS-CoV-2 MAIN PROTEASE Sveva Pelliccia 1, Paola Storici 2, Elisa Costanzi 2, Enzo Tramontano 3, Angela Corona 3, Carmen Cerchia 1, Vincenzo Summa 1 1Department of Pharmacy, University of Naples, Federico II via D. Montesano 49, Napoli, 80131, Italy 2 Protein Facility, Elettra Sincrotrone Trieste S.C.p.A., SS 14 - Km 163, 5 in AREA Science Park, Trieste, 34149, Italy 3 Department of Life and Environmental Sciences, University of Cagliari, Monserrato, 09042, Italy 1. INTRODUCTION synthesis, enantiomerical y pure starting In 2019, SARS-CoV-2 caused worldwide the materials were employed. current outbreak named COVID-19. Despite 2.2. Method multiple countermeasures implemented and The knowledge of both catalytic mechanism approved DNA, RNA and protein subunits and substrate specificity of the Mpro triggered vaccines an additional step forward has been the idea to exploit multicomponent reactions made thanks to approved drugs targeting the (MCRs) as a fast and versatile synthetic tool CoV RdRp (e.g. Remdesivir i.v., Molnupiravir toward novel Mpro inhibitors. Accordingly, the p.o.) and the CoV 3CL Protease (Nirmatrelvir) Passerini reaction-amine deprotection-acyl although they suffer of modest efficacy or migration (PADAM) oxidation route, was suboptimal PK properties. It is an urgent global employed for the development of novel smal need to identify new direct-acting antiviral peptidomimetic compounds with a ketoamide drugs (DAAs) against this pathogen and new warhead behaving as covalent reversible emerging viruses, in order to prevent the inhibitors (Figure 1) [2]. progression to severe disease or new pandemic. 3. RESULTS AND DISCUSSION In particular, the main protease (Mpro) of SARS- CoV-2 is a cysteine protease playing the 3.1. Results and Discussion essential role in viral replication, thus being The peptidomimetics prepared were evaluated identified as a solid target for the development in SARS-CoV-2 Mpro biochemical and antiviral cel -based assays, showing IC of effective antiviral drugs [1]. The most 50 and EC50 in nanomolar/low micromolar range. Furthermore, favored P2−P1−P1′ sequence is Leu-Gln-Ser X-ray co-crystal structures of (or Ala and Gly instead of Ser), and the protease−inhibitor complexes were determined exclusive cleavage of polypeptides by Mpro after as a part of this study, revealing the molecular a glutamine (Gln) residue, that no known human determinants of the interaction with the Mpro. protease displays, al ows the identification of novel selective drugs. 2. MATERIALS AND METHODS 2.1. Materials A series of dipeptides exploring the P1’ region through the use of -ketoamide moiety as “warhead” which unlike the aldehyde can be derivatized using different alkyl/aryl groups, preserving the reported 5-membered ring derivative of glutamine (γ-lactam) in P1 that enhanced the inhibition up to 10- fold. For the 87 OP14 ACKNOWLEDGEMENTS PON R&I 2014-2020-Asse IV- REACT-EU “Green technologies” is gratefully acknowledged. Figure 1. A. The PADAM strategy for the α-hydroxy- β-amino amides. B. General structure of our synthesized α-ketoamides. 4. CONCLUSION The knowledge of both catalytic mechanism and the substrate specificity of the Mpro combined with the possibility to exploit the milestone Multicomponent reactions (MCRs), using the PADAM oxidation route, has led us in the synthesis of novel covalent reversible inhibitors. SARS-CoV-2 Mpro biochemical and antiviral cel -based assays, showed IC50 and EC50 in nanomolar / low micromolar range. Two crystal structures of protease−inhibitor complexes were determined as part of this study, revealing the molecular determinants of the interaction with the Mpro and providing key hints for further optimization. 5. REFERENCES [1] Cannalire, R.; Tramontano, E.; Summa, V. A Focus on Severe Acute Respiratory Syndrome (SARS)Coronavirus (SARS-CoVs) 1 and 2. Book Series: Methods and Principles in Medicinal Chemistry Chapter 18, New Drug Development for Known and Emerging Viruses, First Edition. Wiley 2022. [2] Banfi, L.; Guanti, G.; Riva, R. Passerini multicomponent reaction of protected a- aminoaldehydes as a tool for combinatorial synthesis of enzyme inhibitors . Chem. Commun., 2000, 985–986. 88 OP15 SYNTHESIS AND BIOLOGICAL EVALUATION OF QUINOLINE AND ANTHRANILIC ACID DERIVATIVES AS POTENTIAL QUORUM SENSING INHIBITORS Ivana Perković1, Tanja Poljak2, Maja Beus3, Kirsi Savijoki4, Pekka Varmanen4, Anja Kučević1, Ivan Džajić1 1 University of Zagreb, Faculty of Pharmacy and Biochemistry, Croatia 2 Selvita Ltd., Croatia 3 Institute for Medical Research and Occupational Health, Croatia 4 Department of Food and Nutrition, Faculty of Agriculture and Forestry, University of Helsinki, Finland 1. INTRODUCTION compounds for anti-QS and bactericidal Quorum sensing (QS) is a mechanism of activities as described in previous report [2]. bacterial cel -to-cel communication and gene 3. RESULTS AND DISCUSSION expression co-ordination; an important strategy performed by bacteria to enhance its virulence 3.1. Results and Discussion – Chemistry and promote host damage [1]. Thus, inhibition The synthesis of title compounds was divided in of QS is widely investigated as a novel approach two parts. First, the building blocks for the final to combat bacteria which would act by blocking reaction step were obtained. The quinoline their ability to cause disease and is considered building blocks bearing terminal amino group as an alternative approach to conventional were prepared in the reaction of 4,7- antibacterial therapy [2]. In the present study, chloroquinoline and ethylenediamine or 1,4- we report the synthesis of novel compounds diaminobuthane. The anthranilic acid building composed of two distinctive structural motifs, blocks were obtained in multiple reaction steps, anthranilic acid and 4-amino-7-chloroquinoline first, from the carboxylic acid, corresponding derivatives linked with oxadiazole or hydrazide was obtained, which was then semicarbazide. The compounds were screened converted to the corresponding 3- H-1,3,4- for their anti-QS and antibacterial activity using oxadiazole-2-ones in a reaction with CDI. In the gram-negative Chromobacterium violaceum as reaction of two building blocks the the reporter strain. corresponding 1,4-disubstituted semicarbazides were obtained (Fig. 1), which in the presence of 2. MATERIALS AND METHODS triphenylphosphine and carbon tetrachloride 2.1. Synthesis of novel compounds formed 1,3,4-oxadiazoles (Fig. 2). A series of oxadiazole linked compounds 3.2. Results and Discussion – Anti-QS and composed of anthranilic acid and 7-chloro- bactericidal activity quinoline moieties were synthesized together The 1,3,4-oxadiazole and semicarbazide with their precursors. The 4-amino-7- derivatives were screened for their anti QS and chloroquinoline part of the molecule varied in bactericidal activity. Quercetin (Q) was used as the length of the alkyl chain on the 4- the positive control for QS and azithromycin as aminoquinoline group (2 or 4 carbon atoms) and the positive control for bactericidal activity (cel anthranilic acid was substituted at various viability). Inhibitory activity was tested at 400 positions of the phenyl ring with halogen atoms μM concentrations and for the most potent (F, Cl or Br). The compounds were obtained by compounds at 100 μM and 40 μM as well. Six using standard synthetic procedures. out of total of twelve tested compounds 2.2. Anti-QS and bactericidal activity inhibited the violacein production in C. screening violaceum to the same extent as Q. However, The Chromobacterum violaceum ATCC31532 two compounds exerted also a strong (ATCC; Wesel, Germany) was used as the bactericidal effect on the reporter. Dose- indicator reporter strain to screen the title concentration-response analyses with the most promising compounds indicated that each compound with concentrations at 400 and 100 89 OP15 μM reduced the violacein production by more than 50 % compared to the control cel s with DMSO, while the viability of the reporter under the same conditions was marginally affected. Figure 1. Semicarbazide derivatives Figure 2. 1,3,4-oxadiazole derivatives. 4. CONCLUSION We have successful y prepared and characterized a series of 9 novel 1,3,4- oxadiazole derivatives with short/long alkyl chain 4-amino-7-chloroquinoline and anthranilic acid moieties and evaluated their potential as OS inhibitors together with their semicarbazide precursors (three compounds). The most active compounds come from the series of short chain 1,3,4-oxadiazoles. 5. REFERENCES 1. Ilangovan, A., et al., Structural Basis for Native Agonist and Synthetic Inhibitor Recognition by the Pseudomonas aeruginosa Quorum Sensing Regulator PqsR (MvfR). PLOS Pathogens, 2013. 9:e1003508. 2. Beus, M., et al., Chloroquine fumardiamides as novel quorum sensing inhibitors. Bioorganic and Medicinal Chemistry Letters, 2020. 30(16): 127336. ACKNOWLEDGMENT The authors acknowledge the financial support by University of Zagreb (support for 2021) and Tampere Tuberculosis Foundation (support for 2020-21). 90 OP16 EXPANDING THE CHEMICAL SPACE OF N-PHENYLPYRROLAMIDES AS DNA GYRASE B INHIBITORS Peter Peršolja1, Martina Piga1, Tihomir Tomašič1, Janez Ilaš1, Lucija Peterlin Mašič1, Anamarija Zega1, Danijel Kikelj1, Nace Zidar1 1 University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia Correspondence: nace.zidar@ffa.uni-lj.si 1. INTRODUCTION generated knowledge gives hope that a The growing threat of antibacterial resistance is candidate might reach clinical use in the near intensifying the need for antibiotics with novel future [2]. modes of action [1]. Fluoroquinolones are effectively used for the treatment of infections 2. MATERIALS AND METHODS by inhibiting the bacterial topoisomerase subunits GyrA and ParC, but they also suffer 2.1. Materials from the ever-expanding antibiotic resistance The research was conducted at the Department of Pharmaceutical Chemistry, Faculty of and certain toxicity drawbacks. However, Pharmacy, University of Ljubljana. Al inhibition of GyrB and ParE subunits provides chemicals and solvents used were acquired a viable alternative, as demonstrated by from reputable sources. novobiocin [2]. 2.2. Methods DNA gyrase and topoisomerase IV are enzymes We focused on further expanding the essential for the replication of the DNA double knowledge of N-phenylpyrrolamides and their helix in bacteria [3]. Both are constructed as activity as DNA gyrase B inhibitors. N- heterotetrametric complexes consisting of two Phenylpyrrolamides primarily exhibit high subunits responsible for the cleavage and affinity for the GyrB subunit, while they also transport of DNA (DNA gyrase, 2x GyrA; often display good inhibitory values towards the topoisomerase IV, 2x ParC) and two subunits ParE subunit [4]. responsible for the hydrolysis of ATP (DNA In this work, we aimed to advance the gyrase, 2x GyrB; topoisomerase IV, 2x ParE) development of an in-vitro effective [1, 3]. antibacterial agent against “ESKAPE” Since novobiocin was withdrawn from the pathogens. To achieve this goal, we planned to market, no other GyrB inhibitor managed to further explore the influence of different key reach clinical use. Since then, several other groups on the inhibitory properties of our compounds have been discovered that can compounds. utilise this unexploited mechanism of action [1- 4]. Due to discouraging physiochemical properties, pharmacokinetic issues, and high at rition rates in antibiotic R&D, no other candidate has yet reached the market [1]. Effective antibacterials against the “ESKAPE” pathogens are urgently needed. Recently, substantial research has been taking place in both industry and academia. The amount of 91 OP16 Figure 1. Representative N-phenylpyrrolamide 4. CONCLUSION inhibitor of DNA gyrase B with indicated substrate-enzyme interactions The synthesised compounds were tested for their inhibitory activity against DNA gyrase B. As depicted in Figure 1, we aimed to optimize Additional y, the most potent compounds were the interactions with the eastern part of the tested against a range of Gram-positive and molecule by introducing groups capable of Gram-negative bacteria, many of which belong forming ionic or hydrogen bonds with the to the “ESKAPE” pathogens panel. positively charged Arg136 residue and/or cation-π interactions with the Glu50-Arg76 salt bridge. When targeting the lipophilic floor, our 5. REFERENCES goal was to enhance the hydrophobic and H- [1] Zidar, N., et al., New N-phenyl-4,5- bond interactions, while improving the dibromopyrrolamides as DNA gyrase B inhibitors. physiochemical properties of our compounds. MedChemComm, 2019. 10: 1007-1017. [2] Durcik, M., et al., ATP-competitive DNA gyrase The final compounds were ful y characterized and topoisomerase IV inhibitors as antibacterial using 1H and 13C NMR, HRMS, IR agents. Expert opinion on therapeutic patients, 2019. spectroscopy, melting point analysis, and their 29(3): 171-180. purity was confirmed to be above 95% by [3] Zidar, N., et al., New N-phenyl-4,5- HPLC. Final y, they were evaluated for their dibromopyrrolamides and N-Phenylindolamides as inhibitory properties against DNA gyrase B ATPase inhibitors of DNA gyrase. European Journal of Medicinal Chemistry, 2016. 117: 197-211. from E. coli in the DNA supercoiling assay. [4] Benedet o, D. T., et al. An optimised series of substituted N-phenylpyrrolamides as DNA gyrase B inhibitors. European Journal of Medicinal 3. RESULTS AND DISCUSSION Chemistry, 2019. 167: 269-290. Our synthetic work focused on the generation of a library of analogues bearing diverse side ACKNOWLEDGMENT groups at the ortho position in respect to the amido-substituted central benzene ring (Figure The authors acknowledge the financial support 2, R1). The focus was on maximizing the from the Slovenian Research Agency, Projects J1-3031 and J1-3021. interactions with the lipophilic floor of the enzyme via the introduction of bulkier substituents. Additional y, we aimed to construct analogues with diverse carboxylic acid bioisosters to optimise the interactions with the positively charged roof of the enzyme’s active site (Figure 2, R2). Figure 2. General structure of proposed new inhibitors 92 OP17 Rational development and process optimization of an amorphous solid dispersion generic drug product prepared by hot-melt extrusion (a case study) Ognen Jakasanovski1, Katja Berginc1, Vid Puž1, Žiga Jeraj1, Ivana Gazić Smilović1, Petra Perhavec1, Katarina Zajc Kreft1, Polonca Kralj1, Biljana Janković1 1 Sandoz Development Center, Lek Pharmaceuticals d.d., Slovenia 1. INTRODUCTION 2.4. In vivo dog PK study A significant number of drug substances in To determine the bioequivalence of the generic development are poorly water soluble and have drug product and the originator product, a PK low bioavailability (BCS II and IV). Hot-melt study was performed. 90% confidence limits for extrusion (HME) is a promising technology that the geometric means ratio of Cmax and AUC0-48 enables enhanced solubility and bioavailability parameters were determined. Generic drug of drug substance through drug amorphization product test sample was prepared according to that does not require the use of organic solvents. HME parameters stated in Table 1. An immediate release film coated tablet generic Table 1. HME process parameters used in product with poorly soluble (BCS IV) drug preparation of test sample for in vivo study on dogs substance based on a solid dispersion with Barrel Barrel Barrel Barrel Screw speed Feed rate copovidone was prepared using hot-melt Parameter temp. temp. temp. temp. (zone 1) (zone 2) (zone 3) (zone 4-8) (rpm) (kg/h) extrusion process. Since generic product Test bioequivalence testing involved steady-state sample for dog PK 25 70 150 215 250 3.0 clinical study on patients, study in vitro drug release methods with sufficient discriminatory power 3. RESULTS AND DISCUSSION and bio-relevance are important for the 3.1. Hot-melt extrusion design of assurance of bioequivalence. Experiments 2. MATERIALS AND METHODS DoE model with suitable fit was obtained for influence of HME process on non-sink drug 2.1. Hot-melt extrusion design of release rate, for time points in which experiments precipitation was observed (after 60 minutes). A Design of Experiments (DoE) was performed Only barrel temperature had significant to study the influence of extrusion process influence on the drug release rate, with higher parameters (barrel temperature, feed rate, screw barrel temperatures assuring higher average speed) on drug release (ful factorial 23, drug release (Figure 1). interaction model). Experiments were performed on Leistritz ZSE 18 HP-PH hot-melt extruder. 2.2. Drug release methods Non-sink dissolution (Dissolution in smal volume): Apparatus 2, 150 rpm, 100 mL SGF/FeSSIF pH 5.0 Microflux dissolution QC release method 2.3. Characterization techniques Figure 1. DoE model overview for influence of Hot stage polarized light microscopy (HS-extrusion barrel temperature ( Temp) on drug release PLM) and differential scanning calorimetry after 60 minutes (non-sink dissolution method). (DSC) were used for detection of residual crystallinity in extrudates. Based on further experiments (Table 2), influence of feed rate on drug release in non- sink dissolution test was additional y observed. Sample with lowest feed rate (Sample 6) 93 OP17 assured highest amount of drug released at the compartment of the microflux system (Figure 3, end of the test, likely due to the increase of Figure 4). material residence time (prolonged exposure to high temperature inside the hot-melt extruder). Table 2. Influence of hot-melt extrusion process parameters on drug release in non-sink dissolution test (samples S1-S9). Parameter S1 S2 S3 S4 S5 S6 S7 S8 S9 % diss. at 160 min. 61.0 62.6 65.4 69.2 83.7 94.5 88.7 87.5 77.2 Temp. zone 3 (°C) 130 130 130 160 160 170 130 130 170 Temp. zone 4 (°C) 150 150 150 220 220 220 150 150 220 Figure 3. Non-sink dissolution of test sample and Temp zone reference product used in dog PK study. Geometric 5-8 (°C) 220 220 220 220 220 220 220 220 220 means ratios of Cmax and AUC0-48 parameters are Screw speed 200 300 400 300 400 200 200 200 300 stated above. (rpm) Feed rate (kg/h) 3.0 3.0 3.0 4.5 4.5 2.0 3.0 3.0 4.5 3.2. Precipitation in non-sink dissolution Hot-stage microscopy and DSC characterization confirmed that no residual crystal ine drug substance is present in the extrudate samples which exhibited precipitation, indicating that precipitation is Figure 4. Summary of microflux dissolution results likely caused by other incoming drug substance for donor and receiver compartment for test sample properties. Drug substance quality was shown (blue) and reference product (red). to affect the degree of precipitation, as incoming 4. CONCLUSION drug substance from same supplier with the same polymorphic form obtained from lab-scale Based on DoE and additional experiments, it and ful -scale synthesis process had different was concluded that extrusion barrel temperature extent of precipitation. had strongest influence on the extent of drug precipitation in non-sink media. An effect of Small Vessel dissolution feed rate on precipitation was also observed. 100 Lab scale API synthesis Although the dog study test sample exhibited Full-scale API synthesis Reference product precipitation in non-sink dissolution it was 50 found to be bioequivalent to the reference released% product, suggesting that non-sink dissolution method is over discriminatory. 0 0 50 100 150 Time (min) The gained knowledge from the in vitro Figure 2. Non-sink dissolution of samples prepared methods, as wel as the in vivo dog PK study using lab-scale and ful -scale drug substance, as wel serve as a valuable guidance for the hot-melt as reference product. extrusion process optimization for bioequivalence study. 3.2. In vivo study on dogs Despite precipitation of test sample observed on 5. REFERENCES in vitro experiments (non-sink dissolution, / donor compartment of microflux studies), there was no impact of precipitation on ACKNOWLEDGMENT bioequivalence observed on in vivo dog PK The authors would like to thank associates from study (PK parameters for generic product and Novartis Technical Research & Development and reference were comparable). This finding was Faculty of Pharmacy, University of Copenhagen. also confirmed with results in the receiver 94 OP18 USE OF ADDITIVES TO CONTROL THE POLYMORPHISM OF SOLID LIPID FORMULATIONS: A SIMPLE WAY TO FACE A COMPLEX PROBLEM Serena Bertoni1, Elena Simone2, Nadia Passerini1, Beatrice Albertini1 1 Department of Pharmacy and Biotechnology, University of Bologna, Italy 2 Department of Applied Science and Technology (DISAT), Politecnico di Torino, Italy. 1. INTRODUCTION addition of 10% w/w of liquid lipids (LL) [3]. Solid lipid formulations have recently received This study aims to investigate the effect of a interest due to their ability to modulate drug wide variety of additives with different release, as wel their intrinsic safety and chemical composition and amount on the sustainability. Despite their advantages, crystal ization and polymorphism of tristearin. challenges within the production and storage of The ultimate goal is to obtain a stable solid lipid-based products exist, due to their formulation in terms of polymorphic complex solid state behavior. Solid transitions, able to achieve a control ed release triacylglycerides (TAGs), the most common of hydrophilic drugs with unaltered dissolution class of natural, edible lipids, have the ability to profiles during storage. form different crystal structures (polymorphism). TAGs polymorphism is based 2. MATERIALS AND METHODS around three main forms, which are defined 2.1.Materials according to the different subcel structure: Different oral-approved additives were added to hexagonal (α-form), orthorhombic (β’-form) tristearin (Dynasan®118) at different amounts. and triclinic (β-form), from the less stable to the Additives included fat y acids: oleic, lauric, most stable structure, respectively [1]. Upon myristic, palmitic and stearic acids; fat y acid cooling during melting-based manufacturing esters, such as isopropyl myristate (IM) and processes, TAGs general y crystal ize in the ethyl oleate (EO); sorbitan esters (Span®); metastable α-form. Polymorphic other LL: medium chain trigycerides (MCT), transformations into more stable structures then vitamin E acetate (Vit E), glyceryl monooleate happen during downstream processes and/or (GMO), soya lecithin. storage, affecting the release profile and the 2.2.Methods overal performance of the formulation [2]. At present, lit le at ention has been given to the As example of melting-based process, we effect of additives on the crystal ization of lipid employed spray congealing, a simple solvent- systems during melting-based manufacturing free process based on the atomization of the processes. molten lipid with obtaining of solid spherical microparticles (MPs). The effect of the additives on the crystal ization, polymorphism, phase transition behavior and crystal structure of tristearin was studied by DSC, X-ray diffraction analysis (PXRD), polarized light microscopy (PLM) and scanning electron microscopy (SEM). In order to estimate the kinetics of polymorphic transformations characteristic of each additive/tristearin mixture, selected samples were analysed at the Austrian SAXS beamline at Elet raǦSincrotrone Trieste. Final y, the most promising MPs Figure 1. DSC analysis of MPs with 5% w/w of LL formulation were loaded with a model after 7 days form production (heat flow endo up). hydrophilic drug (caffeine) at 30% w/w and in We have recently shown that polymorphic vitro release tests in different media (water, transitions of tristearin can be modified by FaSSGF, FaSSIF) were performed. 95 OP18 3. RESULTS AND DISCUSSION 4. CONCLUSION 3.1. Effect of additives on tristearin The use of small amount of lipid additives is an crystallization and polymorphism interesting approach to achieve TAGs Differently from solid additives, liquid crystallization in the β-form. In particular, additives promoted the transition of tristearin addition of 5% w/w of IM, EO, oleic acid and from the α-form to the stable β-form with a MCT al owed the complete conversion of the kinetic varying from few minutes to months, formulation into the stable polymorphic form depending both on the additive structure and its within 1 week from production, concurrently amount (Fig. 1). The polymorphic with a control ed release of hydrophilic drugs in transformation was monitored by synchrotron media simulating fasted state gastric and SAXS/WAXS studies and the results confirmed intestinal conditions. From the industrial that liquid additives, specifical y IM and EO, viewpoint, this approach might be were the most effective in accelerating the advantageous as any polymorphic change wil transformation. Isothermal crystal ization be accelerated, hence enabling the production of studies suggested that LL mostly caused the stable lipid formulations. polymorphic transformation via solid state, i.e. after completion of α-crystal ization, as the initial fast α-crystal ization was fol owed by a second exothermic event at ributed to the evolution into the β-form. 3.2. Effect of additives on tristearin structure PLM studies showed that the most “active” LL in favouring α→β transition were included in the β-tristearin crystal lat ice favoring the compaction and maximizing interactions, leading to a more compact crystal ine network. Differently, the other liquid additives determined a heterogeneous microstructure with larger spherulities. PLM, DSC and PXRD studies suggested that solid fat y acids have partial miscibility with tristearin and crystal ize separately forming two different solid phases. 3.3. Effect of additives on drug release The presence of LL strongly enhanced the drug release with respect to pure tristearin in the β form. The release profiles of MPs with 5% of Figure 2. In vitro release profiles of CAF from MPs LL increased passing from water to the fluid with 5% of selected LL in FASSGF and FASSIF and SEM images of MPs with EO (yel ow curves) simulating the fasted gastric conditions col ected after the test. (FaSSGF) and further enhanced in fasted state intestinal environment (FaSSIF). Notably, MPs 5. REFERENCES determined a gradual and sustained release over 1. Chapman, D. The Polymorphism of Glycerides. 5 hours in both biorelevant media, as shown in Chemical Reviews, 1962. 62: 433–456. Fig. 2. MPs col ected after release studies in 2. Lopes, D. G., et al., Microphase separation in water and in FaSSGF, analysed by SEM, solid lipid dosage forms as the cause of drug showed mostly intact particles with spherical release instability. International Journal of shape, while partial y fragmented MPs with Pharmaceutics, 2017. 517: 403–412. eroded surface were observed after the test in 3. Bertoni, S., et al., Liquid lipids act as FaSSIF. This may indicate that, beside a polymorphic modifiers of tristearin-based formulations produced by melting technologies. diffusion mechanism, which is common to al Pharmaceutics, 2021. 13(7):1089. media, the lipid degradation/digestion become important in the intestinal environment. 96 OP19 FABRICATION OF 3DP COLON-TARGETED TABLETS INCLUDING FLUTICASONE WITH ENHANCED SOLUBILITY BY γ-CYCLODEXTRIN Diren Sarisaltik-Yasin1, Cennet Duran1, Sevgi Takka2 1Department of Pharmaceutical Technology, Dicle University, Turkey 2Department of Pharmaceutical Technology, Gazi University, Turkey 1. INTRODUCTION extruded at 125⁰C and 70 rpm in a single-screw Oral y colon-targeted drug delivery systems extruder (Noztec Pro HME, UK). have recently gained importance in reducing Table 1. The formulation components side effects of corticosteroids used in the local treatment of colonic diseases such as Components (%) F1 F2 F3 F4 ES100* 64 64 64 64 inflammatory bowel diseases (IBD). However, GCD - 7,5 7,5 7,5 there are limitations such as lower fluid FLT 5 5 3,75 2,5 volumes, and longer transit times in colon- Talc 10 2,5 3,75 5 targeting of corticosteroids which may cause CAMH 7 7 7 7 inter/intra-individual variability. This makes it Gl 4 4 4 4 EC 3 3 3 3 difficult to adjust the dose. These problems can MgS 7 7 7 7 be solved with personalized 3DP tablets specifical y designed for the course of the 2.3. 3D Printing of Tablets disease (1). The filaments were printed by an FDM 3D Printer (Craftbot 3, Hungary). The printing Fluticasone propionate (FLT) is a second- parameters were set as fol ows: nozzle generation corticosteroid with lower side effects temperature: 165⁰C; build plate temperature: and more potent local effects compared with the 70⁰C; extrusion speed: 30 mm/s, travelling first generations. However, the very low speed: 120 mm/s, layer height: 0,2 mm; the solubility of FLT poses a major chal enge in number of shel s: 4, shel thickness: 0.8 mm; formulation development. infil density: 50%, infil pat ern: paral el line. The aim of this study is to develop colon- 2.4. Characterization of Filaments and 3D targeted tablets of FLT to improve its solubility Tablets with γ-cyclodextrin using the FDM 3DP. Diameters were measured by a caliper. 2. MATERIALS AND METHODS Mechanical properties of filaments were examined by Texture Analyzer ( Stable Micro 2.1.Materials Systems, TA.XTPlus ). Morphological FLT is supplied by Ontario Chemicals. Citric properties of filaments and tablets were acid monohydrate (CAMH), magnesium determined by SEM (FEI Quanta 250 FEG, The stearate (MgS), and talc (Tc) were donated by Netherlands). Drogsan. Eudragit S100 (ES100), Ethocel (EC), γ-cyclodextrin (GCD), Gelucire (Gl), and 2.5. In Vitro Dissolution Studies triacetin (TA) were gifted from Evonik, Dissolution tests were carried out in buffer Dupont, Barentz, Gat efosse, and BASF, media of pH 1,2 for 2 hours, pH 6,8 for 4 hours, respectively. and pH 7,4 for 18 hours, respectively, by using the USP apparatus-2 (Pharmatest, Germany). 2.2. Extruding the Filaments Drug release studies were performed in ES100 was plasticized with 50% of TA. The dissolution media with and without SLS (2%). plasticized ES100 and other excipients (Table 1) were mixed and the formulations and 97 OP19 The dissolution profiles were evaluated by In the second method, doses were adjusted by similarity factor (f2). printing tablets in different sizes and weights 3. RESULTS AND DISCUSSION (200 mg, 300 mg, and 400 mg) by using the F4 filament. Due to the dissolution rate decrease 3.1. The Printability of Filaments with an increasing tablet size and weight, dose The formulations were successfully extruded to proportionality was not observed in this produce filaments and the filaments were method. This may be related to the smal er printed without failing (F1-F4) (Fig 1). The surface area/mass ratio of larger tablets. diameters of the filaments were within the acceptable limits as 1.70-1.80 mm. The surfaces 120 of the filaments were smooth and non-sticky. )100 The mechanical properties of the filaments were suitable for 3DP. The structural integrity and 80 homogeneity of the tablets were demonstrated 60 F1 200 mg F2 200 mg by SEM. 40 F3 200 mg F4 400 mg Dissolved FLT (% 20 F4 200 mg F4 100 mg 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (h) Figure 1. Three Dimensional Printing (3DP) Tablets Figure 2. In vitro Release Profiles of FLT-loaded 3D Tablets 3.2. In Vitro Dissolution Studies 4. CONCLUSION The dissolution profiles (Fig. 2) of the two formulations with (F2) and without GCD (F1) In conclusion, FDM-3DP was successful y were similar (f utilized to fabricate colon-targeted 3D tablets of 2>50). The GCD-dependent solubility improvement was not observed in the FLT with enhanced solubility for the dissolution media containing SLS %2 since personalized therapy of IBD. Additional y, the FLT was completely released from both F1 and dimensions and weight of the tablets have been F2. Therefore, the dissolution tests for F1 and shown to be crucial in tailoring the dose. F2 were repeated in the dissolution medium 5. REFERENCES without SLS. Thereby, the ratio of dissolved 1. Charbe N.B., et al., Application of FLT increased tenfold within 24 h (from 0.48% three-dimensional printing for colon targeted to 4.9%). drug delivery systems , International Journal of Pharmaceutical Investigation, 2017. 7(2): 47-3.3. Dose Proportionality in Formulations 59. The dose proportionality was investigated in 2. Buyukgoz, G.G., et al., Exploring tablet design two different ways (2). In the first, the loading options for tailoring drug release and dose via percentage of FLT in the formulations was fused deposition modeling (FDM) 3D printing . International Journal of Pharmaceutics, 2020. altered by keeping the tablet weights constant 591(11987): 1-13. (200 mg). In this method, 5 mg, 7,5 mg and 10 ACKNOWLEDGM ENT mg doses of FLT were loaded to the formulations F3, F4 and F2, respectively. The f This study was supported by a grant of 2 factors revealed that there was no significant TUBITAK (SBAG-219S197). difference in the dissolution profiles of F2, F3, and F4 tablets of the same size and weight (200 mg) (f2>50). 98 OP20 A USEFUL METHOD FOR ROUTINE MONITORING OF ENDOCRINE DISRUPTORS IN SURFACE WATERS BY SPE-LC-MS/MS Andrej Grobin1, Robert Roškar1, Jurij Trontelj1 1Department of biopharmaceutics and pharmacokinetics, University of Ljubljana, Faculty of Pharmacy, Slovenia 1. INTRODUCTION 2.2. LC-MS/MS Analysis Endocrine disruptors (EDs) are one of the most Chromatographic separation was performed important environmental contaminants with using an Agilent Infinity 1290 system with a natural and synthetic steroid hormones being Kinetex Biphenyl column at 45 °C. 0.2 mM among the most active endocrine disrupting ammonium fluoride in MQ water and methanol compounds, while bisphenols also exhibit were used as a mobile phase in a gradient strong endocrine effects [1]. These effects are method. The Agilent 6460 Triple Quadrupole important in terms of decreasing aquatic was used for MS/MS analysis. Two wildlife populations, and their biodiversity, and chromatographic and MS/MS methods were may also pose a risk for negative effects on used, one for the analysis of analytes in their human fertility rates which are a serious modern non-derivatized form, and another for their public health issue [2]. Various EDs are highly derivatized form, where applicable. effective and can present serious risks even at 2.3. Sample Preparation very low amounts while being present in the Strata X Polymeric reversed phase 60 mg / 3 mL environment simultaneously [3]. Therefore, it is SPE cartridges were used for extraction. 200 necessary to study the environmental presence mL of unfiltered surface water samples were of a broad selection of substances which can loaded onto the cartridges, achieving a cause combined effects in organisms with concentration factor of 1000. Aliquots of each suitable sensitivity. Our aim was therefore the sample were derivatized with dansyl chloride, development and validation of a method while the rest of the sample was used for non- suitable for monitoring EDs from the derivatized sample analysis. aforementioned groups of substances in surface 2.4. Sample Collection waters with suitable quantitation limits and 1 litre of sample was col ected at a standardized simple sample preparation. depth of 1 meter in precleaned sampling bot les. For method validation purposes, several samples with different matrices were selected (a 2. MATERIALS AND METHODS lake sample, river samples, a stream, and a 2.1 Materials channel sample). Analytical standards of estrone (E1), estradiol (E2), estriol (E3), chlormadinone acetate, cyproterone, dienogest, drospirenone, ethinyl 3. RESULTS AND DISCUSSION estradiol (EE2), gestodene, norethindrone, 3.1. Method Sensitivity Optimization norgestrel, tibolone (Carbosynth), testosterone By using simple sample preparation, the (Fluka), progesterone (Pion UK), BPAP, BPE, sensitivity was adequate for analytes ionizing in BPF (TCI Europe), BPB (Fluorochem), positive mode and inadequate for those ionizing corticosterone, BPA, BPAF, BPC, BPG, BPS in negative mode. Therefore, al the analytes and BPZ (Sigma-Aldrich) were used. ionizing in negative mode were successful y derivatized with a simple procedure using dansyl chloride, improving ionization by a 99 OP20 factor of 2.3 to 453 (Fig. 1). Thus, limit of quantitation (LOQ) values lower than ng/L were achieved for most analytes. Figure 2. A combined MRM chromatogram of a river sample for non-derivatized analytes. The time segment from 2 minutes onwards is magnified on a smal er scale (right Y-axis) for better visibility. 4. CONCLUSION Figure 1. Derivatization signal gain by comparing The developed methodology was based on a signals pre- and post-derivatization of al analytes simple SPE extraction, and two LC-MS/MS eligible for derivatization. methods, and was extensively validated. The 3.2. Method Validation environmental y relevant LOQs were The developed method was ful y validated in experimental y confirmed in samples with terms of selectivity, linearity, limits of concentrations lower than 1 ng/L. This was quantitation, accuracy, precision, extraction achieved with a simple dansyl chloride efficiency, matrix effect, and stability. The derivatization which improved signals for al achieved LOQ values in the range below 1 ng/L derivatized analytes with the least amount of were determined as the lowest calibrators with sample manipulation necessary utilizing a mid- suitable accuracy, precision, signal to noise, and ranged LC-MS/MS system. The method signal to blank. The method has a wide linear therefore provides a reliable tool for the routine range, achieves great repeatability and is monitoring of important endocrine disruptors to accurate. To further underline the accuracy of provide relevant information on their presence the method, a standard addition principle was in the environment. used on samples from three significantly 5. REFERENCES different surface water matrices. The results confirmed the method’s accuracy without the 1 Delfosse V., et al., Structural and need to routinely fortify samples. mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of 3.3. Method Application bisphenol A substitutes. Proceedings of the National The method was applied to a selection of Academy of Sciences, 2012. 109 (37): 14930– 14935. surface water samples to determine the occurrence of EDs and their concentration 2 Gunnarsson L., et al., Pharmacology beyond the patient – The environmental risks of levels in the environment. The samples were human drugs. Environment International, 2019. 129: col ected from the upper course of the Sava 320–332. River and its tributaries. A representative 3 Drakvik E., et al., Statement on advancing chromatogram is presented in Fig. 2. E1, E3, the assessment of chemical mixtures and their risks progesterone, EE2, drospirenone, BPA, BPF for human health and the environment. Environment and BPS were the most commonly found International, 2020. 134: 105267. analytes in concentrations up to 670 ng/L. ACKNOWLEDGMENT This research was financial y supported by the Slovenian Research Agency (ARRS) [P1- 0189]. 100 OP21 STUDY OF DIFFERENT SOURCES OF NITROSAMINE TRACES IN PHARMACEUTICALS BY HIGHLY SENSITIVE HYPHENATED MASS SPECTROMETRY TECHNIQUES Nejc Golob1,2, Rok Grahek1, Robert Roškar2 1Lek Pharmaceuticals d.d., Sandoz Development Center Slovenia, Ljubljana, Slovenia 2University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia 1. INTRODUCTION 2.1. Materials Nitrosamines have been identified as mutagenic 2.1.1. Standards/reagents components that may be present in foods, cosmetics and in water for many years [1]. They Standards of nitrosamines NDMA, NDEA and became the focus of pharmaceutical regulatory NDMA-d6 were used for quantitation. authorities when Tetrahydrofuran (THF) and dichloromethane N-nitrosodimethylamine (NDMA) was first detected in sartan medicines, (DCM) were used as extraction solvents. in 2018. Later other nitrosamines were Diethylamine (DEA) was used for experiments identified as contaminants, linked to the usage on nitrosamine formation in a lidding foil. of certain solvents, reagents and catalysts which 2.1.2. Drug products are either secondary amines or secondary amine Commercial 20 mg FCTs were plate-sealed in precursors which can react with nitrosating blister packs during SPME (solid-phase agents ultimately yielding nitrosamines. Most microextraction) sampling of nitrosamine lately, drug substances that are secondary vapours. amines, have been recognized as a risk for N- 2.1.3. Blister material/printing ink nitrosylation [2]. The significance of these Lidding foil without print with polyester (PES) findings and the regulatory requirements have primer, lidding foil without print with NC been addressed by European Medicines Agency primer and printing ink Verde color were used (EMA), the U.S. Food and Drug Administration for experiments on nitrosamine formation in a (FDA) as wel as other regulatory authorities lidding foil. NC lidding foils were used for plate [1]. Since the acceptable daily intake limits for and rol er sealing, during SPME sampling of nitrosamines are extremely low (ng per day nitrosamine vapours. range), the trace analysis of these compounds requires analytical methods with highly 2.2. GC-MS/MS method sensitive instruments. Therefore most of the GC-MS/MS method for nitrosamine analysis methods which were published use hyphenated was an optimized FDA method [3]. For the mass spectrometry techniques (gas SPME method, 50/30 µm chromatography-mass spectrometry and liquid DVB/Carboxen/PDMS fiber was used. chromatography-mass spectrometry). During 2.3. Sample preparation our work we observed a case wherein N- Powdered tablets were extracted with DCM. nitrosamines at very low levels were detected in Lidding foils were shredded and extracted with the finished dosage form but could not be found THF. Printing ink/DEA was added onto the in the bulk drug product [1]. Findings of the lidding foil which was after 20 min shredded investigation emphasized nitrocel ulose (NC) and extracted with THF. Empty blister cavities blister material as a nitrosating agent for were extracted with THF. SPME sampling of secondary amines, present in printing inks, nitrosamines vapours was performed on two yielding N-nitrosamines. These were shown to types of blistering equipment, operating with evaporate from a lidding foil and contaminate plate sealing and roller sealing technology. drug product in an adjacent blister cavity. 3. RESULTS AND DISCUSSION 2. MATERIALS AND METHODS 101 OP21 3.1. Formation of nitrosamines in a lidding in the same time, and that total air flow and air foil with addition of printing ink or DEA movement through the equipment were Printing ink Verde color as a source of restricted when compared to rol er sealing. In secondary amines (present as non-intentional y addition, drug product (20 mg FDF) blistered added substances (NIAS)) was added onto both with plate sealing equipment was analyzed and lidding foils. On subsequent re-analysis, there contamination confirmed. Moreover, NDMA was a significant increase in nitrosamines and NDEA traces were also confirmed in content, with NDEA being predominant, in a cavities of empty blisters that were plate-sealed, lidding foil containing NC. Higher NDEA in contrast to the ones that were rol er-sealed. increased levels in comparison with NDMA This is in line with the observation that more levels were expected because printing ink that nitrosamine vapours were produced during was used for the experiment contained higher plate sealing. amounts of DEA than dimethylamine (DMA). Additional y, DEA was added onto both lidding Table 1. Results of GC-MS/MS analysis of 20 mg FDF and blister cavities, sealed using plate sealing foils. A significant increase in NDEA content and rol er sealing technology (SPME experiment). was detected in a lidding foil containing NC, NDMA NDEA whereas no NDEA formation was observed in a Sample (ng per tablet (ng per tablet lidding foil without NC. These experiments /cavity) /cavity) confirmed that NC as a primer in lidding foil 20 mg FDF acts as a nitrosating agent for secondary amines (plate sealing) < 1.0 (0.8)* < 1.0 (0.5)* present in the printing ink, resulting in Blister cavity nitrosamine formation in a lidding foil. (plate sealing) < 1.0 (0.2)* < 1.0 (0.2)* Blister cavity 3.2. SPME sampling of nitrosamine vapours (roller sealing) < LOD (0.2) < LOD (0.2) at blistering equipment *estimated values below LOQ Since nitrosamines NDMA and NDEA have relatively low boiling points, the possibility of 4. CONCLUSION their evaporation from lidding foil and Results of this investigation provided an insight subsequent contamination of drug product in into an additional source of N-nitrosamine blister cavities with their vapours was contamination of drug products, which may considered. This assumption was supported by lead to trace quantities of N-nitrosamines in the the information that elevated temperatures > finished dosage form. Switching to NC-free 200 °C are used during the blistering/sealing blister material should be considered to avoid process. Several SPME fibers were exposed potential N-nitrosamine traces in finished near the blistering equipment (plate dosage forms. sealing/rol er sealing) and nitrosamines adsorbed during the blistering of drug products. 5. REFERENCES In both cases lidding foils containing NC and 1. Golob, N., et. al., Nitrocellulose blister material nitrosamines were used for blistering of drug as a source of N-nitrosamine contamination of products. Results confirmed traces of NDMA pharmaceutical drug products. International Journal and NDEA during blistering operation in of Pharmaceutics, 2022. 618(121687). vicinity of both types of equipment, with 2. EMA, Q&A for MAHs/applicants on the CHMP significantly higher amounts detected in the Opinion for the Article 5(3) of Regulation (EC) No 726/2004 referral on nitrosamine impurities in case of plate sealing technology. The highest human medicinal products. 2022. Available on: values were detected closest to the sealing https://www.ema.europa.eu/en/documents/referral/n region. These results indicated that less NDMA itrosamines-emea-h-a53-1490-questions-answers- and NDEA vapours were produced during marketing-authorisation-holders/applicants-chmp- blistering with rol er sealing equipment, opinion-article-53-regulation-ec-no-726/2004- although lidding foils used for the experiment referral-nitrosamine-impurities-human-medicinal- contained approximately the same amounts of products_en.pdf. Accessed July 2022. nitrosamines in both cases, and moreover, 3. FDA, Combined Direct Injection NDMA, NDEA, higher sealing temperature was used during NEIPA, NDIPA and NDBA Impurity Assay by GC- rol er sealing. This could be partial y explained MS/MS. 2019. Available on: https://www.fda.gov/media/123409/download. by the fact that during plate sealing a larger area Accessed July 2022. of lidding foil was exposed to high temperature 102 OP22 SELECTIVE DETERMINATION OF WHEY PROTEINS BY A CHROMATOGRAPHIC ANALYTICAL APPROACH Nika Osel1, Timeja Planinšek Parfant1, Albin Kristl1, Robert Roškar1 1Department of Biopharmacy and Pharmacokinetics, University of Ljubljana, Faculty of Pharmacy, Slovenia 1. INTRODUCTION 2.2. Methods Whey has been considered a waste material for We were focused on the development of many years. However, due to its rich complementary chromatographic methods. composition, mainly due to its protein content, Various ion-exchange (IEX), size-exclusion it is gaining in importance [1]. The evaluation (SEC), and reversed-phase (RP) of the isolation and purification processes of chromatographic columns obtained from six individual whey proteins (WPs) from whey, the manufacturers were tested (Agilent, Vydac, development of formulations with selected Phenomenex, Sigma-Aldrich, BIA Separations, WPs, and the evaluation of their stability require and Waters). An Agilent 1100/1200 HPLC the development of appropriate analytical system, equipped with a diode array detector support which presents a special chal enge due (DAD) was used. The chromatographic to the structural complexity of proteins [2]. analytical approach was further extended by Moreover, the concentration range of WPs in two-dimensional liquid chromatography whey is relatively wide and individual WPs (2D-LC; Waters). exist in multiple genetic variants or isoforms. 3. RESULTS AND DISCUSSION The development of analytical methods is It was observed that LPO can be selectively further complicated by the complex and determined in the presence of other WPs at a variable composition of whey-based samples different wavelength by using a DAD. LPO has [1,3]. Therefore, the main purpose of our work a heme moiety in its structure and therefore was to develop a chromatographic analytical absorbs at 405 nm (Fig. 1). approach that would al ow a selective 䣯 䣃 䣗 evaluation of WPs in various samples. The most common WPs and WPs with positive effects on 5 0 0 human health were included in the study. 4 0 0 2. MATERIALS AND METHODS 3 0 0 405 nm 280 nm 2.1. Reagents and chemicals 2 0 0 Reference standards of lactoferrin (Lf), 1 0 0 α-lactalbumin (α-LA), β-lactoglobulin (β-LG), bovine serum albumin (BSA), IgG, and 0 䣰 䣯 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 lactoperoxidase (LPO) were obtained from Figure 1. Absorption spectrum of LPO. Sigma-Aldrich. HPLC-grade acetonitrile was purchased from Honeywel . Al mobile phases WPs differ in their isoelectric point (pI) values. and sample solutions were prepared using the Therefore, an IEX method using a weak cation Mil i-Q water obtained through a Mil i-Q A10 exchange 0.1 mL column CiMAC (BIA Advantage water purification system. Al other Separations) was developed. WPs were eluted chemicals used were of analytical grade. by a salt gradient at a pH of 7.0. The IEX method al ows selective determination of Lf and LPO in the presence of more acidic WPs (e.g. β-LG, IgG, α-LA) (Fig. 2). 103 OP22 䣯 䣃 䣗 䣯 䣃 䣗 1 4 0 α-LA (pI ~ 4,4) 8 0 β-LG 1 2 0 β-LG (pI ~ 5,4) α-LA 1 0 0 IgG (pI 5–8) 6 0 Lf (pI 8–9) 8 0 LPO 4 0 6 0 LPO (pI ~ 9,6) 4 0 2 0 2 0 0 0 0 1 2 3 4 5 䣯 䣫䣰 䣯 䣫䣰 0 2 .5 5 7 .5 1 0 1 2 .5 1 5 Figure 2. Selective determination of Lf and LPO by Figure 4. RP-HPLC chromatograms of β-LG, α-LA, IEX. and LPO. The developed SEC method can be used in 4. CONCLUSION stability studies for the evaluation of the The development of an appropriate analytical aggregation and fragmentation behaviour of methodology is necessary for the evaluation of individual WPs [4]. However, not al WPs can WPs in complex samples. A combination of be separated using the SEC method since some various chromatographic methods coupled with of them (LPO, Lf, BSA) have a similar different detectors al ows the selective molecular weight (Fig. 3). Therefore, due to its evaluation of WPs in various samples. In the poor selectivity, this method is unsuitable for future, the analytical approach wil be extended the analysis of complex samples containing with mass spectroscopy, which wil al ow a WPs. more reliable and selective evaluation of WPs 䣯 䣃 䣗 in complex samples. 50 5. REFERENCES Lf (~80 kDa) 40 BSA (~ 66 kDa) 1. Smithers G.W., Whey-ing up the options – 30 Yesterday, today and tomorrow. International LPO (~78 kDa) Dairy Journal, 2015. 48: 2–14. 20 2. ICH Q5C, 1995. 10 3. Guo M., et al., Chemistry of Whey Proteins. 0 Whey Protein Production, Chemistry, 0 2 4 6 8 䣯 䣫䣰 Functionality, and Applications (1st edition), Figure 3. Size-exclusion chromatograms of Lf, BSA, 2019. and LPO. 4. Osel N., et al., Stability-Indicating Analytical The most chal enging was the development of Approach for Stability Evaluation of the RP-HPLC method as the separation (Lf and Lactoferrin. Pharmaceutics, 2021. 13(7): 1065 BSA) or the appropriate chromatographic peak (2021). shape (IgG) of selected analytes was difficult to ACKNOWLEDGEMENT achieve. The optimized RP-HPLC methods This research was co-funded by Slovenian were used for the evaluation of the selected WPs Research Agency, grant number [P1 0189] and in relatively simple samples (Fig. 4). For more by Support of Research and development complex samples, a 2D-LC method which was projects (TRL 3–6): LAKTIKA. based on a combination of SEC and RP-HPLC methods was developed. The 2D-LC method enabled the separation of al selected WPs. However, it is less appropriate for routine use since it is a relatively sophisticated technique. 104 OP23 PREPARATION OF BUCCAL FILMS IN PARKINSON’S DISEASE Krisztián Pamlényi1, Géza Regdon Jr.1, Dániel Nemes2, Ildikó Bácskay2, Katalin Kristó1 1 Institute of Pharmaceutical Technology and Regulatory Affairs, University of Szeged, Eötvös u. 6., H-6720 Szeged, Hungary 2 Department of Pharmaceutical Technology, University of Debrecen, Nagyerdei krt. 98., H-4032 Debrecen, Hungary 1. INTRODUCTION 2.2. Preparation of buccal films Parkinson’s disease (PD) is the second most The films were prepared at room temperature common movement disorder. It affects 2-3% of with the solvent casting method. As the first the population over the age of 65 years [1]. PD step of preparation, SA was dissolved in is caused by the death of dopaminergic neurons distil ed water and mixed at room temperature. in the substantia nigra area of the human brain PR was incorporated in the polymer solution. [2]. Enzymatic and non-enzymatic oxidation of As the third step, HPMC was added to the dopamine generates reactive oxygen species, solution with mixing. In the fourth step, GLY which induces apoptotic cel death in dopamine was added to the solution. Mixing was stopped neurons. and it was placed in an ultrasonic cleaning unit Current treatment of PD focuses on replacing (Elmasonic S 30 H, Germany) for 30 minutes to the dopamine level from an external source help the air bubbles disappear from the solution. (Levodopa-L-DOPA) or on applying a The solution was cast on a glass surface in Petri dopamine agonist API, which can stimulate the dishes, then it was dried at room temperature dopamine receptors in the central nervous (24.4±0.5 °C). Table 1 shows the different system. compositions of the prepared polymer films. In Parkinson’s disease, the advantages of buccal Table 1. Composition of prepared PR films films include that the patients do not have to HPMC GLY PR swal ow the dosage form, so they can be used in Sample SA (w/w %) (w/w %) (w/w %) (0.7 mg) the case of swal owing problems, which is a 1 1 2 1 + common symptom in this disease. 2 1 2 2 + In this project, we applied pramipexole 3 1 2 3 + dihydrochloride (PR) as an active agent in the 4 1.5 1.5 1 + polymer film system, which can also be used on the buccal mucosa to improve the success of 5 1.5 1.5 2 + current Parkinson’s therapy with PR compound. 6 1.5 1.5 3 + 7 2 1 1 + 2. MATERIALS AND METHODS 8 2 1 2 + 2.1. Materials 9 2 1 3 + Sodium alginate (SA) (Biochemica GmbH, 2.3. Mechanical properties of films Germany) (10,000 – 600,000 g/mol) and HPMC The thickness of the polymer films was (Metolose® 2208, Shin Etsu Chemical Co., Ltd., measured with a screw micrometre (Mitutoyo Japan) were used as film forming agents in the Co. Ltd, Japan), sensibility was 0.001 mm [3]. polymer film. Glycerol (GLY) 85% (w/w%) Tensile strength was measured with a texture was added to the film as a plasticizer (Ph. analyser developed in our department, with a Eur.8.). Pramipexol dihydrochloride (PR) (Ph. needle-like probe. [3]. Eur. 8.) was the API, which was a gift from KRKA, d.d., (Novo Mesto, Slovenia). 105 OP23 Mucoadhesion was measured with the same texture analyser with a rod-like probe and 100 different set ings [3]. 80 60 2.5. Dissolution test ipexole 40 Films with a size of 4 cm2 were measured in the ount of dissolved pram 20 dissolution test. The dissolution test was Am 0 performed in a 100 mL glass lab beaker, 50 mL 0 10 20 of phosphate buffer (pH=6.8) was used as Time (min) dissolution medium, its temperature was 37 °C. Figure 1. Dissolution of pramipexole from the Aliquots of 5 mL were analyzed with UV-different polymer films in 20 minutes spectrophotometry at λ=262 nm wavelength. 3.3. Cytotoxicity test 2.6. Cytotoxicity test It can be said that most formulations had cel Biocompatibility was studied by flow viability higher than 87%. Buccal film Sample cytometry. TR-146 buccal mucosa cel s were 8 had the lowest impact on cel viability as treated in a 1 mil ion cel /mL concentration with 87.1% of the treated cel s survived. 1 mL of PR solution (PR films dissolved in HBSS). After 30 minutes of incubation, the 4. CONCLUSION cel s were stained with propidium iodide. Non- cel ular events were excluded and the ratio of It can be concluded that we can formulate stained and non-stained cells was calculated. buccal films with PR. The films have adequate thickness, flexibility, breaking hardness, and 3. RESULTS AND DISCUSSION mucoadhesivity is also appropriate for use on the buccal mucosa. Al films can release 100 % 3.1. Mechanical properties of films The thickness of the films can be increased by API in less than 20 minutes from the films fit the amount of SA, so the concentration of SA for the purpose. can influence the thickness of the films. It can be concluded the GLY can increase film 5. REFERENCES thickness. Films with a higher GLY 1. Poewe, W., et al., Parkinson Disease. Nature concentration have higher breaking hardness Reviews Disease Primers, 2017. 3: 1-21 values. SA can increase the breaking hardness 2. Naoi, M., et al., Cell death of dopamine neurons in aging and Parkinson’s disease. Mechanisms of the films. Every composition has great and of Ageing and Development, 1997. 111 (2-3): adequate mucoadhesive properties. The in vitro 175-88 mucoadhesion force of al samples is larger than 3. Pamlényi, K., et al., Formulation and 15 N. Optimization of Sodium Alginate Polymer Film as a Buccal Mucoadhesive Drug Delivery 3.2. Dissolution test System Containing Cetirizine Dihydrochloride. As it can be seen in Figure 1, al the amount of Pharmaceutics, 2021. 13 (5) 619-37 the API can dissolve from each composition in the first 20 minutes. The dissolution of the API ACKNOWLEDGMENT is faster from the films which contain a low GLY concentration. The authors thank Krka, d. d., Novo Mesto, Slovenia for supplying pramipexole dihydrochloride. This research work was supported by the ÚNKP-21-3 New National Excel ence Program of the Ministry for Innovation and Technology from the Source of the National Research, Development and Innovation Fund. 106 OP24 DESIGN AND EVALUATION OF COMPOSITE FILM FORMULATIONS USING QUALITY BY DESIGN FOR PAEDIATRIC USE Genada Sinani1, Mohammed Kalayi1, Buket Aksu1 1 Department of Pharmaceutical Technology, Altinbas University, Türkiye 1. INTRODUCTION were al owed to dry at 45 °C for 48 h. After Lidocaine is widely used to treat different drying, the films were careful y peeled off and painful conditions like oral ulcers, teething, or stored wrapped in an aluminium folium in a gingivitis in children. However, it is stated in desiccator until further use in characterization various reports that the use of oral viscous studies. lidocaine solution in infants and young children 2.3. Formulation development and can result is serious side effects, including heart optimization of films using QbD approach and brain problems, due to overdose or when The main purpose of applying QbD approach the drug is accidental y swal owed [1]. Thus, for the development and optimization of there is a need to develop paediatric-friendly polymeric film formulations was to investigate oral formulation for anaesthetic drugs. The use the optimum amounts of polymers and glycerol of buccal films for lidocaine delivery could to be used in the formulation of composite films provide a safe and easy way for treating mouth with ideal mechanical characteristics. Thus, disorders in children [2]. Herein, composite amount of HPMC (X1), SA (X2) and glycerol film formulations containing hydroxypropyl (X3) were selected as independent variable, methylcel ulose and sodium alginate as while tensile strength (Y1) and percentage polymers were develop and optimized by QbD elongation (Y2) that are indicative of approach to ensure their use as potential mechanical properties of polymeric films were paediatric dosage forms. selected as dependent variables. QbD approach 2. MATERIALS AND METHODS for the optimization of the film formulations was performed by using MODDE Pro version 12.1 2.1. Materials software. Different 30 film formulations were Hydroxypropyl methylcel ulose (HPMC) investigated in triplicate using Texture (Ashland, Germany), sodium alginate (SA) Analyzer (TA.XT.Plus C Stable Micro (Sigma Aldrich, Switzerland), lidocaine Systems, UK). hydrochloride (Doğa İlaç, Istanbul, Türkiye), 2.3. Characterisation of optimum composite glycerol (Tekkim Kimya, Bursa, Türkiye). Al film formulations other chemicals were of analytical grade. Fol owing development and optimisation 2.2. Preparation of polymeric films studies drug loaded formulations were prepared The polymeric films were prepared by solvent as described earlier and the effect of drug casting method, as described earlier with loading on mechanical characteristics of films modifications [3]. Different amounts of HPMC was further investigated. In addition, optimum and SA polymers at different ratios were added composite films formulations were in distil ed water to make a total polymer characterised in terms of weight uniformity and concentration of 2-3% (w/v%) and stirred thickness, folding endurance, pH, drug overnight to al ow the formation of a uniformity and drug release profile. homogeneous solution. Further, glycerol used 3. RESULTS AND DISCUSSION as plasticizer was added at concentrations varying 40-60% (w/v% polymer weight) and 3.1. Formulation development and stirred for 2 h. The formulation was stored optimisation. overnight in refrigerator to remove the air Considering that the formulation composition is bubbles and cast on glass petri dishes. The films closely related with mechanical properties such 107 OP24 as tensile strength and percentage elongation of further studies. It might also help to increase the films, their evaluation is important to ensure availability of high-quality medicines designed appropriate production, handling, and use of for children. polymeric films. In this study, the design space (Figure 1.) was determined to investigate and 5. REFERENCES assure the development of drug formulation 1. Curtis, L.A., et al., Are one or two dangerous? with desired quality. As expected, the careful Lidocaine and topical anesthetic exposures in selection of the amount and type of polymer as children. The Journal of emergency medicine, wel as the amount of plasticizer is necessary to (2009). 37(1), 32-39. ensure the formation of uniform films with good 2. Kot ke, D., et al., Development and evaluation mechanical properties. It was observed that of mucoadhesive buccal dosage forms of lidocaine hydrochloride by ex-vivo permeation increasing the amount of HPMC in formulation studies. International Journal of Pharmaceutics, resulted in formation of films showing (2020). 581, 119293. increased tensile strength, whereas increasing 3. Morales, J.O., & McConvil e, J.T., Manufacture the amount of glycerol general y resulted in and characterization of mucoadhesive buccal decreased tensile strength. It was observed that films. European Journal of Pharmaceutics and blending of the polymers improved the Biopharmaceutics, (2011). 77(2), 187-199. mechanical properties compared to films prepared using only one polymer. Figure 1. Design space graph of optimal parameters for tensile strength and percentage elongation at break 3.2. Evaluation of characteristics of optimised composite film formulations To evaluate the suitability of optimised composite films formulations to be used for lidocaine delivery in paediatric patients further characterisation studies were performed. Films showing thickness of approximately 0.1 mm, good physical appearance, weight and drug uniformity were prepared. The pH values measured for the films showed that they were compatible with the buccal mucosa. Prolonged drug release was observed depending on formulation characteristics. 4. CONCLUSION The optimised composite polymeric film formulations developed in this study using QbD approach for buccal administration films might be promising to be used as effective and safe patient-friendly formulations in children after 108 OP25 DEVELOPMENT OF A PEDIATRIC ORALLY DISINTEGRATING TABLET DOSAGE FORM BY MASKING THE BITTER TASTE OF ORNIDAZOL WITH EUDRAGIT E PO Gülbeyaz YILDIZ TÜRKYILMAZ1, Çağlar AKGÜNLÜ2, Ozan ÜNSALAN3, Mehmet Ali EGE2, Çisem ALTUNAYAR ÜNSALAN4, Hatice Yeşim KARASULU2 1 Department of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, Ege University, Izmir, Turkey 2 Department of Pharmaceutical Technology, Faculty of Pharmacy, Ege University, Izmir, Turkey 3 Department of Physics, Facuty of Science, Ege University, Izmir, Turkey 4 Central Research Testing and Analysis Laboratory Research and Application Center, Ege University, Izmir, Turkey 1. INTRODUCTION İbrahim) was used as the reference drug. Ornidazole (ORN) is an antiprotozoal with a 2.2. Coating with spray dryer sharp bit er taste [1] and an important drug in For the coating study, many experiments were antibacterial therapy against anaerobic bacteria. carried out by changing the pump speed from Pediatric use is limited to the iv dosage form the spray dryer conditions to 20% or 40% and only that can only be administreted to the changing the ORN:E-EPO ratio to 1:0.5-4 and hospitalized patient. Therefore, it is not possible ethanol (EtOH) as a solvent to 50 or 60 in the to continue treatment at home and the antibiotic feed solution content. The optimum spray must be changed. In this study, the development drying condition that masked the bit er taste was of a dosage form suitable for pediatrics is the determined and production was carried out mainly goal in terms of ensuring the under these conditions. Quantification, sustainability of the treatment. One of the flowability, bulk angle, density, Carr’s index, dosage forms applicable to pediatrics is the moisture determination, particle size oral y disintegrating tablet (ODT) form. The measurement and raman analysis were problem here is the bit er taste of the active performed. The amount of ORN was ingredient. The secondary main aim of the study determined according to the pharmacopoeia is to mask this bit er taste. One of the bit er taste method [3]. masking techniques is coating the active ingredient by spray drying. The bit er-tasting 2.3 Development of ODT formulation active substance should not be released or The coated powder, which was taken to contain dispersed from the coated material that wil 125 mg of ORN, together with different ratios disperse in the mouth. For this purpose, of super dispersant, fil er and lubricants were Eudragit EPO (E-EPO) polymer, which is mixed in a cubic mixer and the formulation slightly soluble in saliva pH, was chosen as the suitable for compressing was determined. coating. Notched tablets were printed with a 1 cm diameter seal using the direct compression During the study, in vitro taste masking test was method. Quantification of the final product was performed with the digital taste transmit er made using the validated method. Thickness- prototype [2]. After appropriate masking, the diameter, hardness, brit leness, dispersion ODT formulation was prepared from the coated determination, content uniformity, wet ing time active substance. Since the dose is administered and water absorption capacity and dissolution per kg, notched tablets were compressed test were performed. A comparative in vitro considering ease of dosage. Developed ODTs dissolution study and similarity test with the were evaluated with in vitro quality control tests market preparation was performed. determined by pharmacopeias and guidelines. The stability of the formulation was fol owed 2. MATERIALS AND METHODS for 3 months at 25 ºC ± 2; RH 60% ± 5 and 40 2.1. Materials ºC ± 5; RH 75% ± 5. ORN, E-EPO supplied by Deva İlaç. All other materials are pharm grade, al consumables used in the analysis are in HPLC grade. Ornisid® Fort 500 mg film-coated tablet (Abdi 109 OP25 3. RESULTS AND DISCUSSION F1 F2 F3 F3.1 F4 F11 3.1. Coating with spray dryer SA (g) - - - - - 1 The taste scale of the formulations performed Method with spray drying under the conditions in Table EtOH % 50 60 60 60 60 60 1 were measured (Figure 1). According to the Raman spectra, when the polymer ratio was İnlet / 120 / 120 / 120 / 120 / 120 / 120 / oulet (⁰C) 40 51 56 68 58 68 higher, taste masking was higher. It was decided to produce in F3 conditions, where the bit er PR (%) 40 40 40 20 40 40 taste is the least and the yield is high. Result 3.2. Development of ODT formulation Yield (%) 68 56 72 59 55 65 The formulation suitable for production was determined as F3E according to Hausner ratio Taste More bit er Bitter Less bit er Bitter Bitter Sour bit er (HR) and compressibility limit (CI). SA=Sitric acid; PR=Pump rate F3E was found suitable relative to EP/TF with a disintegration time of 80 seconds [4]. Wet ing Tablo 2. HRs and CIs of F3 formulation and powder time and water absorption capacity were found mixtures prepared from F3 formulation to be bet er with values of 30.5±2 seconds and F3 F3A F3B F3C F3D F3E 75.1±1.1%, respectively, compared to similar ORN (mg) 125 125 125 125 125 125 ODT forms performed with E-EPO [1]. Al other characterization test results were E- EPO (mg) 375 375 375 375 375 375 evaluated according to the pharmacopoeia and Lubricant1 (mg) - 1 1 2 2 9 found to be appropriate. Super dispersant2 Dissolution studies were performed at pH 1.2, (mg) - - - 2 2 5 4.5 and 6.8 (both salivary and intestinal media). Filler3 (mg) - - - - 80 86 As a result of the comparative dissolution study with the Ornisid®, f HR 1,85 1,5 1,6 1,6 1,42 1,3 2 values were below 50 in al results except pH 6.8, showing that the CI (%) 46 50 33 40 29 25 formulation was not similar. 1Magnesium stearat; 2Col idon® CL-F; 3.Avicel It was observed that its stability continued for 3 pH-102 months in long and accelerated stability 4. CONCLUSION environments. In this study, successful results were obtained in As a result, the taste of ORN was masked in a terms of taste masking by coating ornidazole, single operation compared to other taste which has a sharp bit er taste, with Eudragit E masking methods with E-EPO and an ODT dosage PO at a ratio of 1:3. The proposed ODT form suitable for pediatric use was developed [1,5]. formulation has the potential to close both an economic and therapeutic gap in the pharmaceutical market with in vivo tests. 2 5. REFERENCES 1. Shishu, Kamalpreet, Kapoor, V. R. Development of taste masked oral formulation of ornidazole. Indian J Pharm Sci, 2010. 72(2):211-5 2. Turkish Patent and Trademark Office Application No: 2021/018046 Figure 1. Raman spectra of coated powders such as 3. Ornidazole Monograph in EP 2019 F2, F3, F11 formulations 4. European Pharmacopoeia (EP) 2019 Table 1. Spray drying trials and results 5. Chivate A, Sargar V, Nalawade P, Tawde V. Formulation and development of oral dry F1 F2 F3 F3.1 F4 F11 suspension using taste masked Ornidazole ORN (g) 1 1 1 1 1 1 particles prepared using Kol icoat(®) Smartseal E-EPO (g) 1 2 3 3 4 0.5 30 D. Drug Dev Ind Pharm. 2013 Jul;39(7):1091-7. 110 OP26 NANOCELLULOSE-BASED ORODISPERSIBLE FILMS AS A POTENTIAL DRUG DELIVERY SYSTEM Katarina Bolko Seljak, Mirjana Gašperlin, Mirjam Gosenca Matjaž Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION The use of nanocel ulose, a ful y sustainable and also prepared. Afterwards, hydrogels were biodegradable polymer, in drug delivery has evenly distributed across glass plates using been gaining at raction. In particular, ZUA applicator, dried at 60˚C to form films and nanocrystal ine cel ulose (NCC) can be cut into even pieces. inexpensively isolated from renewable cel ulosic biomass. Depending on the input Rheological properties were measured with cel ulosic material and hydrolysis conditions, Physica MCR301 rheometer (Anton Paar). the geometrical dimensions of the isolated NCC Mechanical properties of 10 cm long films were can vary greatly. Nevertheless, its measured with Instron 5567 compression and biocompatibility, biodegradability and low tensile tester at 100 mm/min speed. cytotoxicity are broadening its possibility of use in biomedical applications [1]. 3. RESULTS AND DISCUSSION In drug delivery research, the focus has been shifting from traditional dosage forms towards Since NCC properties can vary depending on those offering more tailor-made options. the source and manufacturing procedures, films Among these, oral drug delivery films are were developed using NCC from two different gaining interest due to faster rate of drug manufacturers – gNCC was supplied in the form absorption, improved bioavailability and bet er of water gel, while pNCC was presented as compliance among patients facing swal owing spray-dried powder. deficiencies, such as children and elders. While al hydrogels exhibited behaviour of non- Moreover, their potential for delivering proteins newtonian liquids prior to solvent casting (data and peptides has been at racting further not shown), alg-pNCC sample displayed the attention [2]. highest viscosity at 12.05 Pa×s. Overal , pNCC In this research, we present the use of NCC as a al owed for formulations with higher viscosities novel reinforcing material in orodispersible compared to gNCC formulations, although the films (ODF), while stil al owing for unhindered longer polymer chains are at ributed to the lat er disintegration. (Table 1). Table 1. NCC properties according to manufacturer’s specifications. 2. MATERIALS AND METHODS NCC Crystallinity Fibre width Fibre length 2.1. Materials gNCC 90,3 % 10- 15 nm 150 -300 nm Nanocrystal ine cel ulose from wood pulp- pNCC 88 % 7,5 nm 150 nm gNCC (Nano-crystalcel , Slovenia) and pNCC (Cel uforce, Canada), sodium alginate Protanal LF 10/60 (FMC Biopolymer), pectine (Sigma Frequency sweeps confirmed weaker gel Aldrich) were used in the present study. structure of gNCC samples compared to pNCC, as storage modulus (G’) and loss modulus (G’’) 2.2. Methods Hydrogels of 1 % NCC with 2 % of alginate values crossed (Fig. 1). At lower radious (alg) were prepared. Alternatively, 0,6 % of frequencies, the G’ of pNCC was 24 x the G’’ NCC was blended with 1,2 % of pectin (pec). 3 value compared to 3 x for gNCC. % of glycerol was added to al hydrogels. Variations with addition of 0,14 % of Ca2+ were 111 OP26 100 10 Figure 2. Disintegration behaviour of alg-gNCC film in contact with water (at 0, 2 min and 20 min from left to right). alg-gNCC (G') alg-gNCC (G' ) log G' [Pa] log G" [Pa]1 alg-gNCC-Ca (G') The addition of Ca2+ ions did not at ribute to alg-gNCC-Ca (G' ) rheological and mechanical properties of ODFs alg-pNCC (G') alg-pNCC (G' ) in a meaningful way; it did however prolong alg-pNCC-Ca (G') their disintegration time due to formation of egg alg-pNCC-Ca (G' ) box structure (data not shown). 0,1 0,1 1 10 100 While longer polymer chain lengths usual y ω [rad/s] at ribute to higher viscosities and stronger Figure 1. Frequency sweep test of alg-NCC films. hydrogels, this was not the case for the Adequate mechanical properties enable investigated NCCs. Overal , rheological handling and application of ODFs. Al properties of hydrogels prior to solvent casting developed ODF exhibited excel ent elongation were bet er predictors of mechanical properties at break (Table 2). pNCC films, in particular of resulting ODFs and their disintegration alg-pNCC were the most elastic (E ̴ 3,65 Mpa). behaviour. Moreover, tensile strength of al films was in the range of 2 MPa or higher. 4. CONCLUSION The ability of NCC from renewable cel ulose Table 2. Mechanical properties of prepared films: sources to form multi-polymeric ODF films Tensile strength (TS), Young modulus (E), with adequate rheological and mechanical Elongation at break (emax). properties was demonstrated. ODF SAMPLE TS [MPa] E [MPa] emax [%] Nanocel ulose-based ODF therefore present a alg-gNCC 2.601 ± 0,44 0.557 ±0.21 97,9 ± 11.1 novel step towards improved sustainability in drug delivery while catering to specific alg-gNCC-Ca 2.051 ± 0.56 0.711 ±0.47 50.7 ± 13.8 demographic populations. Future research alg-pNCC 5.902 ± 1,11 3.653±1.25 90.3 ± 10.2 should explore potential of nanocel ulose-based alg-pNCC-Ca 1.727 ± 1,38 1.634±0.99 38.4 ± 14.3 ODFs for carrying and delivering active pek-gNCC 3.029 ± 0.48 0.499 ±0.12 31.97 ± 5.25 pharmaceutical ingredients. pek-gNCC-Ca 3.643 ± 0.72 0.532 ±0.12 26.63 ± 4.13 pek-pNCC 2.688 ± 0.44 0.461 ±0.27 31.97 ± 6.02 5. REFERENCES pek-pNCC-Ca 4.269 ± 0.98 0.618 ±0.09 42.84 ± 6.80 1. Pachuau LS. A Mini Review on Plant-based Nanocellulose: Production, Sources, When ODFs came in contact with water, Modifications and Its Potential in Drug alginate and pectine containing films behaved Delivery Applications. Mini Reviews in similarly (data not shown). Regardless of the Medical Chemistry. 2015;15(7):543-52. choice of natural polymer in ODFs, the use of 2. He M, et al. Recent advances of oral film as platform for drug delivery. International Journal gNCC over pNCC resulted in shorter of Pharmaceutics. 2021 Jul 15;604:120759. disintegration time when in contact with water (example in Fig. 2), which is of importance for potential drug delivery applications. ACKNOWLEDGMENT The research was financial y supported by the Slovenian Research Agency (research core funding number P1-0189). 112 OP27 EXCIPIENTS ROLE IN FORMULATING USER FRIENDLY ORAL DOSAGE FORMS Pawel Balcerzak1, Liliana Miinea2, Elena Draganoiu3 1Lubrizol Advanced Materials, Inc., Belgium; 2Lubrizol Advanced Materials, Inc., USA 3Lubrizol Canada Limited, Canada 1. INTRODUCTION K100M; magnesium hydroxide; anhydrous Designing user friendly oral dosage forms col oidal silica; magnesium stearate. implies use of effective formulation techniques 2.2. Methods and versatile excipients to al ow for desired Metformin HCl formulation development therapeutic effect and patient compliance. Carbopol® polymers (carbomers) are Metformin HCl formulations (500/1000 mg), crosslinked polyacrylic acid polymers proven to were developed by aqueous high shear wet be highly effective in control ing drug release at granulation using intra-granular and extra- low usage levels.1 Furthermore, there are granular ingredients listed in Table 1. The reported studies regarding their mucoadhesive formulations were tested for physical properties.2 Our work has been focused on parameters, dissolution profile, assay and using Carbopol® polymers to design user impurities. Dissolution was carried as per USP friendly oral dosage forms. Test 4. Stability studies were conducted as per Metformin HCl remains the first line of ICH guidelines at accelerated (ACC) conditions treatment of type 2 diabetes. Due to increased (40°C/75% RH). frequency of dysphagia in diabetes patients, it is Nitrosamine impurities analysis was necessary to formulate stable compositions with performed. higher drug loading (500/1000 mg) and reduced tablet size. Regulatory agencies have issued Table 1. Ingredients in Metformin HCl ER tablets guidance regarding control of potential Intra-granular Extra-granular carcinogenic nitrosamine impurities in drug Magnesium hydroxide products that has resulted in global recal s, Hypromel ose K100M metformin HCl being one of the most affected. Metformin HCl Carbopol® 971P NF Work has been conducted to develop smal size, Hypromel ose K100M polymer high dose metformin HCl extended release (ER) Carbopol® 971P NF Carbopol® 71G NF polymer polymer tablet compliant formulations. Magnesium hydroxide Anhydrous col oidal Cold and cough oral liquid formulations can silica benefit from a mucoadhesive excipient that Magnesium stearate would enable the formulation to coat the mucous membrane, providing soothing benefits in addition to potential improved retention and Cold and cough formulation development absorption. We have evaluated cold and cough Mucoadhesive liquid formulations were formulations with mucoadhesive polymers in prepared by dissolving acetaminophen, comparison with commercial formula. dextromethorphan and sweetener in deionized water fol owed by mixing with an aqueous 2. MATERIALS AND METHODS dispersion of Carbopol® polymer. The inclusion 2.1. Materials level of Carbopol® polymer in the formulation was varied (0 - 1%). The mucoadhesive Metformin HCl; acetaminophen (APAP); properties of the formulations were tested using dextromethorphan (DXTR); Carbopol® 971P a device adapted from in-vitro oesophageal NF and 71G NF polymers; hypromel ose retention model (IVOR).3 113 OP27 3. RESULTS AND DISCUSSION 3.1. Metformin HCl ER tablets The 500 mg and 1000 mg strength tablets were successful y formulated at smal sizes (achieving 20-30% size reduction compared to commercial formulations of the respective strengths). Smal er tablet size enables more tablets per batch, resulting in increased productivity and cost savings (analytical testing, inventory, packaging and storage). The tablets complied to the USP monograph and were stable under accelerated conditions when Figure 2. Impact of Carbopol® 971P NF polymer packed in HDPE bottles (Fig. 1, Table 2). (CBP971P NF) on mucoadhesion of oral liquids 4. CONCLUSION Smal size, high dose stable metformin HCl ER tablet formulations were successful y developed using Carbopol® polymers. The tablets complied to Nitrosamines (NDMA) impurities as per US FDA guidance. Cough and cold mucoadhesive liquid formulations were designed to ensure prolonged Figure 1. Metformin HCl ER tablets dissolution in pH 6.8 phosphate buffer (initial and six month contact with mucosal membranes, providing accelerated conditions - 6 M ACC) patient compliance and product differentiation. Carbopol® polymers have been used as key Table 2. Metformin HCl ER tablets - six month excipient to formulate smal er extended-release accelerated conditions (6 M ACC) stability tablets and liquid product for mucosal delivery, proving to be versatile excipients to design user Test USP Metformin Metformin specs 500 mg 1000 mg friendly drug products. Assay (%) 90- 100 99.9 97.1 5. REFERENCES Single max impurity (%) 0.1 0.04 0.06 1. Lubrizol Bul etin 01 Polymers for Total Pharmaceutical Applications Lubrizol impurity (%) 0.6 0.10 0.16 Pharmaceutical Excipient Resource Hub 2. Draganoiu, E., Properties of mucoadhesive polymers and their use in tablets and other Selected tablet formulations were tested at an dosage forms. Tablets and Capsules, 2016. 14 FDA approved testing laboratory as per US (5) 17-23. FDA guidelines and were found to be compliant 3. Young, S. A., et al., A novel in-vitro apparatus for al eight nitrosamine impurities.4 for evaluating the mucoadhesion of liquid and semi-solid formulations. Journal of Pharmacy 3.2. Cold and cough formulation and Pharmacology, 1998. 50(Supplement): 167. In our study it was demonstrated that the 4. US FDA Guidance “Control of Nitrosamine presence of Carbopol® polymer prolonged the Impurities in Human Drugs” retention of the formulation on the substrate https://www.fda.gov/regulatory- simulating oral mucosa (artificial membrane information/search-fda-guidance- hydrated in a mucin solution; Fig. 2). documents/control-nitrosamine-impurities- Additional y, increasing the polymer human-drugs concentration resulted in longer retention. 5. Lubrizol Mucoadhesive Polymers in Pharmaceutical Formulations These results correlate wel with similar https://www.lubrizol.com/- findings on using Carbopol® polymer to /media/Lubrizol/Health/Literature/Mucoadhesi formulate other liquid, semi-solid and film ve-Polymers-in-Pharmaceutical- dosage forms.5 Formulations.pdf 114 OP28 A CHITOSAN BASED BIOADHESIVE GEL FOR DELIVERY OF COMBINED ANTIMICROBIALS WITH ENHANCED ACTIVITY IN LOCAL TREATMENT OF SKIN INFECTIONS IN HUMANS AND ANIMALS Ece Türkmen1, Selin Parmaksız1, Şeyma Nigiz2, Meral Sağıroğlu2, Sevda Şenel1 * 1 Department of Pharmaceutical Technology, 2 Department of Pharmaceutical Microbiology, Hacettepe University, Faculty of Pharmacy, Turkey Correspondence: ssenel@hacettepe.edu.tr 1. INTRODUCTION concentration in 2% v/v acetic acid. 2% w/v Bacterial and fungal skin infections are CHX and 2% w/v MN was incorporated into the common both in humans and animals, gel. 4% Tween 20® and %1 Tween 80® were especial y in companion animals such as cats used as surfactants and 1% propylene glycol, and dogs. Use of systemic antimicrobial drugs 4% ethanol were added as co-solvents. Tris- has resulted in increased antimicrobial EDTA (TE) at 16:1 (w:w) ratio was resistance (AMR). Under “One Health” incorporated into the gels in order to enhance approach, among the strategies to overcome the antimicrobial activity. AMR is topical delivery of combination of antimicrobials, which allows enhanced efficacy Cell viability studies at lower concentrations of antimicrobials. MTT assay was performed on L929 mouse Chlorhexidine digluconate (CHX) and fibroblast cel s to determine cel viability. miconazole nitrate (MN) are the most Antimicrobial activity commonly used drugs for topical treatment [1]. Antimicrobial activity of the gel formulations In this study, we have used chitosan, which is a against S. pseudintermedius and M. bioadhesive biopolymer exerting antimicrobial pachydermatis were evaluated using broth activity itself [2] to prepare the gels for delivery microdilution assay according to Clinical and of combined antimicrobials, MN and CHX. Laboratory Standards Institute (CLSI) Antimicrobial activity of the formulations was guidelines [3,4]. Inoculum for S. investigated against S. pseudintermedius and M. pseudintermedius was adjusted to 2x108 pachydermatis. CFU/mL, broth dilution was performed with 2. MATERIALS AND METHODS Muel er-Hinton Broth and incubated at 35qC for 24 h. Inoculum for M. pachydermatis was 2.1. Materials adjusted to 7.5x106 CFU/mL and broth dilution MN was generously provided by IE Ulagay- was performed with Saboraud dextrose broth Menarini Group (Turkey) and chitosan was (SBD) containing 1% Tween 80, and incubated generously provided by Koyo Co., LTD Japan. a t 35qC for 72 h. CHX, Tris base, EDTA and Tween 80® were purchased from Sigma-Aldrich (Germany). 3. RESULTS AND DISCUSSION Tween 20® was purchased from BDH White, homogenous opaque gels were prepared Laboratory Supplies Poole, England and with suitable adhesivity, spreadability and propylene glycol from Merck Mil ipore viscosity for topical application on skin as (Germany). Muel er-Hinton broth was described previously (5). The release profiles purchased from Merck (USA). Al chemicals of the drugs were also shown to be different, due used were analytical grade unless otherwise to the different solubility properties of the stated. drugs, with high released amount of CHX, whilst a lower release with MN, which has low 2.2. Methods solubility, however it was shown that the drug Preparation of formulations was accumulated on the surface [5]. Gel formulations were prepared using base 3.1. Cell viability chitosan (degree of deacetylation: 85%, The results obtained for cel viability are shown Molecular weight: 235 kDa) at 3% (w/v) in Fig. 1. The developed formulation has been 115 OP28 shown to be biocompatible without causing any 4. CONCLUSION decrease in cel viability at al concentrations. In this study, we have demonstrated that the The viability obtained with the formulation was developed bioadhesive gel formulation is higher than the CHX which has cytotoxic effect biocompatible and provides enhanced on cells and the CHX-MN combination. antimicrobial activity against S. pseudintermedius and M. pachydermatis, in addition to other pathogens tested previously, S. 0,625 µg/mL 1,25 µg/mL 2,5 µg/mL 120 aureus and M. canis [5] and can be used safely for the topical treatment of skin infections in 100 ) humans and animals. Besides its safety and 80 enhanced activity, due to its bioadhesive and drug release properties, providing slow release 60 as wel as accumulation on the skin, it wil be 40 Cel viability (% possible to decrease the frequency of 20 application, thereby reduce the amount of the antimicrobials used for treatment, thereby 0 resulting in a significant contribution to the combat against AMR. 5. REFERENCES Figure 1. Cel viability of the formulations after 24h (n=5) ( CHX and MN at a concentrations of 2.5, 1.25 1.Muel er, R., S., et al. Vet Dermatol. and 0.625 µg/mL). 2012;23(4):330-e62. 3.2. Antimicrobial Activity 2.Şenel S. React Funct Polymers, 2020;147:104452. MIC values obtained against S. 3. CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; pseudintermedius and M. pachydermatis are Approved Standard, CLSI Document M27-A3, 3rd given in Table 1. Combination of CHX and MN ed. 2008:PA, USA. was shown to increase the antimicrobial activity 4. CLSI, Reference Method for Broth Dilution against S. pseudintermedius. The antimicrobial Antifungal Susceptibility Testing Filamentous activity of the formulation was found to be Fungi, Approved Standard, CLSI document M38- similar to that obtained with the combination of A2. 2nd ed. 2008:PA, USA. CHX and MN. Antimicrobial activity against 5. Türkmen, E., et al., Development of a bioadhesive M. pachydermatis was found to be enhanced system for combined antimicrobial delivery for significantly with the developed formulation topical treatment of skin infections. 20th IPTS, 21-23 when compared to that obtained with February 2022, Ankara-Turkey. combination of CHX and MN. Further ACKNOWLEDGEMENT enhancement in microbial activity was observed This study was supported by Hacet epe with incorporation of Tris-EDTA (p<0.05). University Scientific Research Coordination Unit (Project number: THD-2020-18516). Table 1. MIC values against S.pseudintermedius and M. pachydermatis Authors are also thankful to Prof. Dr. Barış MIC (µg/mL) Sareyyüpoğlu, Veterinary Faculty, Ankara S. M. University for clinical isolate of S. pseudintermedius pachydermatis pseudintermedius and Assoc. Prof. Dr. Kemal CHX 0.33 9.77 Metiner, Veterinary Faculty of Istanbul MN 0.33 2.44 University for clinical isolate of M. CHX-MN 0.20 2.44 TE pachydermatis. 1000: 62.5 1000:62.5* CHX-MN-TE 0.39 0.31 Chi (3%) 7.32 14.65 Chi-CHX 0.24 4.89 Chi-CHX-MN 0.20 2.44 Chi-MN-TE 0.98 2.03 Chi-CHX-MN-TE 0.20 1.02 * no inhibition was observed 116 OP29 TRANSCRIPTOME ANALYSIS REVEALS INVOLVMENT OF THIOPURINE S-METHYLTRANSFERASE IN OXIDATION-REDUCTION PROCESSES Alenka Šmid1, Miha Štajdohar2, Dunja Urbančič1, Nataša Karas Kuželički1, Riin Tamm3, Andres Metspalu3, Irena Mlinarič-Raščan1 1Department of Clinical Biochemistry, University of Ljubljana, Faculty of Pharmacy, Slovenia, 2Genialis d.o.o., Slovenia, 3Estonian Genome Center, University of Tartu, Estonia 1. INTRODUCTION TotalPrep RNA Amplification Kit was used to Thiopurine S-methyltransferase (TPMT) is transcribe 200 ng RNA which was then involved in deactivation of thiopurine drugs hybridized to the Il umina HT12v3 arrays which are used in the treatment of acute according to the manufactures protocols. TPMT lymphoblastic leukemia, inflammatory bowel activity was measured in RBC hemolysates disease, and some other autoimmune disorders using the HPLC method as previously described and represents a major determinant of (1). thiopurine-related toxicities. Despite its wel - 2.3. Data collections and data fusion known importance in thiopurine metabolism, We model ed gene associations with 6 data sets the understanding of its endogenous role is that describe the involvement of TPMT in lacking. The aim of the present study was to intracel ular processes by using a data fusion gain insight into the molecular processes approach with penalized matrix tri-factorization involving TPMT by applying a matrix (2). The model considered 3 types of objects: factorization-based data fusion approach to genes, GO terms, and subjects, on which six analyse blood-based whole-genome expression data sets (matrices) were built, each relating a data from healthy volunteers combined with pair of object types. Protein–protein gene ontologies and protein-protein interactions interactions were included as constraints databases. between corresponding genes. Proteins from the 2. MATERIALS AND METHODS STRING database were used to build the constraint matrix Θ2. Degrees of interactions 2.1. Subjects and sample collection were normalized to values between 0 and -1. In In this study, which was approved by the case of no known interaction, the value was 0. Research Ethics Commit ee of the University of Hierarchical structure of GO (Θ3) was included Tartu, Estonia, we analysed blood samples of by reasoning over has_part, part_of and is_a 1017 volunteers, donors to the Estonian relations in the GO graph. A constraint between Biobank. Three aliquots of blood were used: a pair of GO terms was set to -0.2hops, where one for DNA extraction, one which was drawn hops was the length of the shortest path between into Tempus Blood RNA Tube for RNA the two GO terms. extraction, and one for hemolysate preparation in which TPMT activity was measured. The 3. RESULTS AND DISCUSSION demographic data of the donors (including age, 3.1. TPMT gene association network built by and gender) were extracted from the Biobank data fusion approach database. The cohort consisted of 50.2% males The model of TPMT gene association network and 49.8% females, with a mean age of 37.8 was built for al subjects independently of their years. TPMT genotype-phenotype status. The network 2.2. TPMT genotyping, RNA analysis and includes genes in the nodes that are connected TPMT activity measurement based on the distance function, defined as the Genotyping of the TPMT activity-deficient average Euclidean distance between genes in TPMT*3B (rs1800460), *3C (rs1142345) and the reconstructed relation matrices ෡ͳǡ͵ and *2 (rs1800462) al eles was carried out by ෡͵ǡͻ. The part of the gene association network TaqMan Genotyping Assays. Ambion closest to TPMT is presented in Figure 1. 117 OP29 membrane respiratory chain NADH dehydrogenase (NDUFS1 and NDUFB4), 2) have a significant role in cytoplasmic NADPH production (IDH1), or 3) utilise NADPH as the cofactor in their enzymatic reactions (other genes). NADPH is a critical molecule for the maintenance of antioxidant cel capacity through the regeneration of reduced pools of glutathione in the transsulfuration pathway. This pathway is tightly connected with and dependant on one-carbon metabolism and methylation reactions. TPMT as methyltransferase utilizes SAM as a methyl donor and directly determines the methylation capacity of cel s, which could in turn influence the redox capacity modulated by Figure 1. Part of a gene network showing genes transsulfuration pathway. clustered around TPMT constructed from the model for al subjects. Nodes represent genes and arches relations between genes. TPMT is represented by a 4. CONCLUSION green node. The genes in the red circle were analysed by DAVID functional annotation tool. In this study we used a matrix factorization- based data fusion approach in which we integrated whole genome gene expression data, 3.2. Analysis of the gene network suggests gene ontology and protein–protein interaction that TPMT is closely related to oxidation- data and applied it to investigate TPMT-related reduction processes gene network and gain new insight into its Genes most similar to TPMT based on biological role. The constructed model of calculated distances (Figure 1, encircled red )— TPMT-related gene network revealed that which among others contained PAICS, PPAT, TPMT is closely related to genes involved in GMPS and MTHFR—were analysed with oxidation reduction processes and may thus DAVID’s functional annotation clustering tool. play an important role in regulating cel redox Table 1. Functional Annotation Clustering of TPMT capacity. gene network using DAVID tools (the first top cluster). 5. REFERENCES Cluster 1 Enrichment Score: 1. Milek, M., et al. Post-translational stabilization 3.8923084803846 044 of thiopurine S-methyltransferase by S- adenosyl-L-methionine reveals regulation of Category Term # P B TPMT*1 and *3C allozymes. Biochemical SP_PIR_KEYW oxidoreductase 9 1.26 1.30 Pharmacology, 2012. 83, 969-76 ORDS E-06 E-04 2. Zitnik, M. & Zupan, B. Matrix factorization-GOTERM_BP_F GO:0055114~oxi 9 8.90 5.11 based data fusion for gene function prediction AT dation reduction E-06 E-04 in baker's yeast and slime mold. Pacific SP_PIR_KEYW nadp 5 8.44 0.00 Symposium on Biocomputing, 2014. 400-11. ORDS E-05 1738 ACKNOWLEDGMENT GOTERM_CC_F GO:0000267~cel 4 0.28 0.66 AT fraction 4677 3387 This work was supported by the Slovenian Research Agency grant no. J3-3615 and by the Estonian Ndufb4, Cbr1, Mthfr, Gmpr2, Cyp51a1, Sqle, Idh1, Cyp26a1, Ndufs1 Research Council (Grant IUT20-60) and Estonian Science Foundation (ETF9293). Several enriched annotation clusters were identified, among which the most prominent one is related to oxidation-reduction processes (presented in Table 1). The cluster contains genes that 1) form the part of the mitochondrial 118 OP30 MICRODIALYSIS AND NANOFILTRATION ALLOW TO DISTINGUISH MOLECULARLY DISSOLVED FROM COLLOID-ASSOCIATED DRUG CONCENTRATIONS DURING BIOMIMETIC DISSOLUTION TESTING OF SUPERSATURATING FORMULATIONS Florentin Lukas Holzem a, Jeannine Petrig Schaffland c, Martin Brandl a, Annette Bauer- Brandl a, Cordula Stillhart b a Department of Physics, Chemistry & Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark b Pharmaceutical R&D, Formulation & Process Sciences, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland c Roche Pharmaceutical Research & Early Development, Pre-Clinical CMC, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland 1. INTRODUCTION was performed with three alternative sampling Many new drug entities are poorly water- approaches in paral el: the classical bench soluble and thus require solubility-enhancing centrifugation approach was used to assess total formulations to ensure sufficient bioavailability. dissolved concentrations, while a nanofiltration On the other hand, it is more and more accepted method and a microdialysis setup were tested that not all “dissolved” states of a drug for their ability to discriminate molecularly and contribute equal y to enhanced absorption, i.e. col oid-associated drug concentrations over an increase in apparent solubility does not time. For comparison, a single-stage dissolution necessarily go in paral el with an increase in setup was performed, where a marketed PCZ molecularly dissolved drug, the lat er being suspension was dispersed in biomimetic media regarded as the key driving force for absorption. with increasing amounts of solubilizing agents to understand their effect on the concentration Our study aimed to provide time-resolved of molecularly dissolved drug. information on the dissolution, supersaturation, and precipitation behavior of molecularly 3. RESULTS AND DISCUSSION dissolved drug as released from an amorphous It was demonstrated that the microdialysis setup solid dispersion and a surfactant-containing al owed to fol ow the molecularly dissolved crystal ine suspension of Posaconazole (PCZ), drug concen-tration in a time-resolved manner a weakly basic and poorly water-soluble drug. during the single-and two-stage dissolution tests Thereby, we aimed to gain a deeper mechanistic with marginal delays. understanding of enabling formulation principles and possibly establish a dynamic Interestingly, the PCZ concentrations measured biophar-maceutical assessment tool for by the nanofiltration approach differed from molecularly dissolved drug released from both, the molecularly dissolved (assessed by enabling formulations. microdialysis) and apparently dissolved (assessed by centrifuge) PCZ concentrations, 2. MATERIALS AND METHODS indicating that nanofiltration may allow to 2.1. Materials differentiate between different col oid- associated (apparently) dissolved drug species. For details on chemicals and media, pls check the published article Moreover, it was shown that the release of the molecularly dissolved drug from an amorphous 2.2. Methods solid dispersion did not correlate at al with the A two-staged dissolution test, with media results obtained by the centrifugation method: transition from simulated gastric fluid (SGF) to While this conventional sampling revealed a fasted state simu-lated intestinal fluid (FaSSIF), classical spring and parachute concentration / 119 OP30 time-profile with a high degree of (apparent) supersaturation, the concentration of molecularly dissolved drug (assessed by the microdialysis setup) indicated an initial short decline of PCZ concentration, fol owed by a prolonged (moderate) molecular super- saturation. This observation may give rise to a re- thinking of the current mechanistic understanding of how amorphous solid dispersions enhance oral bioavailability. 4. CONCLUSION In essence, the current study provides data which indicate a benefit of both the microdialysis sampling and nanofiltration approaches for the in vitro biopharmaceutical assessment of enabling drug formulations. 5. REFERENCES The abstract is taken from the fol owing publication: Holzem, F.L., et al., Microdialysis and nanofiltration allow to distinguish molecularly dissolved from colloid-associated drug concentrations during biomimetic dissolution testing of supersaturating formulations European Journal of Pharmaceutical Siences, 2022. 174: article 106166 For further references pls check the published articleś reference list. ACKNOWLEDGMENT This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skło- dowska-Curie grant agreement No 955756 ITN InPharma). Furthermore, the authors are grateful for fruitful discussions enabled by funding from European Union’s Horizon 2020 framework program (COST action UNGAP CA162050) and Nord- forsk program Nordic University Hub (project #85352). 120 OP31 MEMANTINE LOADED PLGA NANOPARTICLES FOR ALZHEIMER’S DISEASE Gizem Tezel1,2, Tuba Reçber3, Sıla Ulutürk4, Selin Seda Timur1, Emirhan Nemutlu3, Sıla Gülbağ Pınar2 Güneş Esendağlı4, Hakan Eroğlu1 1 Department of Pharmaceutical Technology, Faculty of Pharmacy, Hacettepe University, Turkey 2 Department of Pharmaceutical Technology, Faculty of Pharmacy, Süleyman Demirel University, Turkey 3 Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, Turkey 4Department of Basic Oncology, Hacettepe University Cancer Institute, Turkey 1. INTRODUCTION Double emulsion method was used for the Alzheimer disease (AD) is one of the most preparation of Mem HCI loaded PLGA common neurodegenerative diseases in the nanoparticles. For the formation of organic world. It is a neurodegenerative disease of the phase (o) a predetermined amount of PLGA was central nervous system and is classified under dissolved in ethyl acetate. For the formation of dementia. According to a predictive statistics aqueous phase (w1) Mem HCI was dissolved in 6.2 mil ion Americans having age of 65 and deionised water containing PVA or TPGS in older are living with AD today [1]. The major different concentrations. w1 phase was poured boundary for the treatment of AD is the Blood into oil phase dropwise under magnetic stirring Brain Barrier. Tight junctions of the BBB, limit and for primary emulsion (w1/o) formation and the passage of active pharmaceutical sonication energy was applied. For the second ingredients from systemic circulation to the emulsification step deionised water containing brain. Nanotechnology brings new insights for PVA or TPGS (w2) was dispersed in w1/o access to the BBB with localized and active emulsion and sonication was applied again to drug delivery. form w1/o/w2 secondary emulsion. After solvent evaporation NPs were centrifugated at 13.750 Memantine HCI (Mem HCl) is a N-methyl D- RCF for 30 minutes. aspartate (NMDA) receptor antagonist which is used for the non-competitive inhibition of 2.3. In-Vitro Drug Release NMDA receptors and used to provide In-vitro drug release of Mem HCI loaded NPs neuroprotection [2]. Chemical structure of was studied in a dialysis bag technique under Memantine does not contain chromophore sink conditions for 72 h. Firstly, a volume of 5 group. For this reason, analytical method ml of formulation was placed directly into a development for quantification of Mem HCI by dialysis bag (cel ulose membrane, 12–14 kDa, UV detection is quite difficult [3]. size 25mm 100ft 16 mm diameter, Spectra por/4) and each bag was placed in 100 ml of In the present study Mem HCI loaded PLGA isotonic phosphate-buffered saline (PBS), pH nanoparticles were prepared and characterized. 7.4, at 37 °C. The samples were analysed by LC-MS method for Mem HCI quantization LC-MS. developed and validated. 2.4. LC-ESI-MS/MS Method 2. MATERIALS AND METHODS The LC-ESI-MS/MS system (Shimadzu, Japan) 2.1. Materials consists of an LC system (Shimadzu LC- PLGA (50:50, 24.000-38.000MA) and TPGS 20AXR) combined with a triple quadrupole (D-α-Tocopheryl polyethylene glycol tandem mass spectrometer (Shimadzu 8030 succinate) were purchased from Sigma Aldrich. MS/MS). Chromatographic analysis parameters Mem HCI obtained as a generous gift from Ali were as fol ows: At 40°C, C18 column (GL Raif Pharmaceuticals (Turkey) and al the other Sciences, 50 x 3.0 mm, 2.1 um), gradient chemicals used were analytical grade. elusion method used which has two phases %0.1 Formic acid in water (phase A) and %0.1 2.2. Preparation of the Mem HCI loaded Formic acid in Methanol (phase B). 0.4 ml/min Nanoparticles flow rate was used, injection volume was set as 1 µL. Total analysis time was 5 minutes. MRM mode was used for MS analysis. 121 OP31 2.5. In-vitro Cell Culture Studies 3.3. Analytical Method Validation Cytotoxicity test of Mem HCI carried out with Analytical resolution of Mem HCI peak was MTT test in HEK293T cel s. After that Mem presented in Figure 2. HCI loaded nanosystems and empty nanosystems applied to same cel s. Mem HCI applied in 300 µM, 150 µM, 75 µM, 37,5 µM, 18,75 µM, 9,3 µM concentrations to HEK213T. 3. RESULTS AND DISCUSSION 3.1. Optimization of NP Parameters Mean average particle size (Z-AVE) and Figure 2. Memantine HCI resolution in LC-MS polydispersity index (PI) of NPs were 3.4. In-vitro Cell Culture Studies determined by photon correlation spectroscopy Mem HCI MTT test results in HEK293 were showed (PCS) using a Zeta Sizer Nano ZS (Malvern). in Figure 2. Studies showed that the particle size increased with increasing polymer concentration. But this increase can be control ed with optimizing sonication parameters. Particle size decreased with increasing surfactant concentration. Lastly with the use of %0.2 TPGS, encapsulation efficacy (EE) was increased. F11 showed highest EE which was %55. Formulation parameters of Mem HCI loaded PLGA nanoparticles shown in Table 1. Table 1. Formulation parameters of Mem HCI loaded PLGA nanoparticles Figure 3. Mem HCI MTT test results in HEK293 4. CONCLUSION In this study sustained release of encapsulated Mem HCl was demonstrated by the in-vitro release study as a new drug delivery system for AD cytotoxicity of Mem HCl control ed with MTT test. Also new analytical method was successful y validated for Mem HCI. 5. REFERENCES 1. 2021 Alzheimer’s disease facts and figures. The Journal of Alzheimer’s Association, 2021. 17:327-406. 2. Tezel G., Timur S.S, et.al., A Snapshot on the Current Status of Alzheimer’s Disease, 3.2. In vitro Drug Release Treatment Perspectives, in-Vitro and in-Vivo Research Studies and Future Opportunities. The release profile of the Mem HCI loaded NP Chemical Pharmaceutical Bul etin, 2019. 67, (F11) was presented in Figure 1. 1030–1041 3. Sulochana P.S., Sharma K., et.al., Review of the validated HPLC and LC-MS/MS methods for determination of drugs used in clinical practice for Alzheimer’s disease. Biomedical Chromatography ,2014. 28: 1431–1490. Figure 1. In vitro release profile of Mem HCI loaded NP (F11) 122 OP32 AN ASSESSMENTS OF TITANATE NANOTUBES PERFORMANCE AS PROTEIN CARRIERS Tamás Sovány1, Yasmin Ranjous1, Natalie Deiringer2, Zoltán Kónya1, Géza Regdon jr.1, Wolfang Friess2 1Institute of Pharmaceutical Technology and Regulatory Affairs, University of Szeged, Hungary 2Chair of Pharmaceutical Technology and Biopharmaceutics, Ludwig Maximilians University, Germany 1. INTRODUCTION 2.2. Methods Biopharmaceuticals are of emerging interest for Zeta potential of H-TNTs and MgSt-TNTs was the treatment of various diseases such as cancer measured with Nano ZS Zetasizer (Malvern or autoimmune dieseases due to their potent Instruments, Herrenberg, Germany), using a therapeutic ability However, proteins have 4 mW He-Ne laser at 633 nm, in a filtrated certain limitations such as poor aqueous solution of 10 mM NaCl at 25°C. The pharmacokinetics, potential immunogenicity, pH was adjusted using NaOH or HCl. lack of stability, short circulation half-life, which necessitates the use of proper delivery Autosorb 1 (Quantachrome, Odelzhausen, systems. Knowledge on potential protein- Germany) apparatus was used to measure the carrier interactions and their effect on protein specific surface area of H-TNTs and MgSt- conformation is necessary for the selection of TNTs using nitrogen gas adsorption and suitable systems [1]. Brunauer-Emmet-Tel er isotherm analysis. Titanate nanotubes (TNTs) usual y have been To study the protein adsorption TNTs were utilized in immobilized form to modify the dispersed in respective formulation buffer in an surface of titanium implants to improve ultrasonic bath for 15 mins. After that, biocompatibility, but it is also possible to use lysozyme stock solution was added to the them as drug-delivery systems [19, 20]. TNTs Eppendorf tube which was placed in an may enhance the wettability and surface area of overhead shaker for 120 min. The samples were titanium, leading to a higher protein adsorption then centrifuged for 10 min at 10,000 rpm to [21], which indicates their potential as possible separate the free lysozyme in the supernatant delivery system for biopharmaceuticals. The from the TNT-Lysozyme complex in the aim of present study was to test the suitability sediment of hydrothermal y synthetized free TNTs as Lysozyme in the supernatant was quantified to possible carriers by evaluating their protein derive the amount of adsorbed protein on TNTs binding capacity. The adsorption proteins on (Calbiochem non-interfering CA protein naïve H-TNTs and Magnesium stearate (MgSt) assay;Merck , Darmstadt, Germany) . functionalized TNTs under different experimental conditions was investigated. The protein conformation was analysed by FT- IR (Bruker Optik GmbH, Et lingen, Germany), 2. MATERIALS AND METHODS using transmission mode, 4 cm-1 spatial 2.1. Materials resolution and scan number of 256. H-TNTs and MgSt-TNTs were prepared at the Lysozyme activity was measured by the Department of Applied and Environmental enzymatic assay from Sigma-Aldrich. Chemistry, University of Szeged fol owing the Micrococcus lysodeikticus bacterial suspension method of Ranjous et al [23], where the was prepared by using 10 mM pH=6.24 currently tested samples are referred as H-TNT potassium phosphate buffer. 100µl of TNT- and MgSt-TNT 47, respectively. Lysozyme bonded lysozyme sample prepared under (Ovobest Eiprodukte GmbH & Co. KG, different conditions was added to 2500 µl Neuenkirchen-Vörden, Germany), served as bacterial suspension and decrease in absorbance model protein during adsorption studies. was measured against blank of buffer by UV- 123 OP32 VIS spectrophotometry. TNT-free lysozyme HTNTs pH=4 64.00% stock solution was used as control. HTNTs pH=5 52.25% 3. RESULTS AND DISCUSSION HTNTs pH=6 57.85% Despite of the near identical size, a substantial y different was detected with 217 m2/g vs. 100 HTNTs pH=7 67.49% m2/g surface area in case of H-TNTs and MgSt- HTNTs pH=7 +140mm NaCl 84.21% TNTs, respectively. Interestingly, despite of the lower surface area the MgSt functionalized HTNTs/ PBS 78.42% samples bound almost double the amount of lysozyme per m2 as compared to H-TNTs (4,8 MgSt-TNTs/ PBS 87.13% mg vs. 2.2 mg, respectively) (Fig. 1), which may be due to the increased surface hydrophobicity of the functionalized samples 4. CONCLUSION which enables stronger binding and offers It may be concluded that TNTs may effectively proper binding sites for both polar and apolar bind proteins and may stabilize their regions of the protein The absorbed protein is conformation even under highly denaturising general y stable despite the low pH or high salt conditions and therefore may help to preserve concentrations necessary to facilitate protein- the activity of biopharmaceuticals. carrier interactions and increase the adsorbed amounts. 5. REFERENCES 6,0 2) 1. Dimitrov, D.S., Therapeutic proteins. Therapeutic Proteins, 2012. 899: 1-26. g/m 5,0 2. Liu, W., et al. Synthesis of TiO 2 nanotubes with ZnO nanoparticles to achieve antibacterial 4,0 properties and stem cell compatibility. ount (m Nanoscale, 2014. 6(15): 9050-9062. 3,0 3. Peng, L., et al. Long-term small molecule and protein elution from TiO2 nanotubes. Nano 2,0 Let ers, 2009. 9(5): 1932-1936. 4. Kulkarni, M., et al. Binding of plasma proteins 1,0 to titanium dioxide nanotubes with different diameters. International Journal of Adsorbed protein am 0,0 Nanomedicine, 2015. 10: 1359. M H H H H H H g -T -T -T -T -T -T 5. Ranjous, Y., et al. Evaluation of the p S h T NT NT NT NT N N b T T permeability and in vitro cytotoxicity of o - s T p N s p s p s p s p u s f p PB functionalized titanate nanotubes on Caco-2 a T h H H H H fe H p S t = = = = r = + cell line. 4 5 6 7 7 Acta Pharmaceutica Hungarica, 2021. e H b = b u 1 b u 1 b u 1 b u 1 1 4 1 u 4 f 0 f 0 f 0 f 0 0 0 91(1): 31-39. f 1 f fe m fe m fe m fe m m m e 0 r m r M r M r M r M M M M p h p p p N p o h h h h a s os os os o C s ACKNOWLEDGMENT ph p p p l p a h h h a t a a a h e te te te te Project no. TKP2021-EGA-32 has been implemented with the support provided by the Figure 1. Amount of absorbed lysozyme per m2 TNT Ministry of Innovation and Technology of from different buffers Hungary from the National Research, FT-IR results confirmed that binding may Development and Innovation Fund, financed induce moderate, but reversible changes in under the TKP2021-EGA funding scheme. protein structure, while activity assays revealed that activity was bet er preserved in case of higher bonded amounts. Table 1. Remaining enzymatic activity of various samples 124 OP33 IMMUNOMODULATORY PROPERTIES OF SIMVASTATIN EMBEDDED IN LIPOSOMES AND ELECTROSPUN NANOFIBERS FOR WOUND HEALING Anže Zidar1, Luca Casula2, Julijana Kristl1, Matjaž Jeras1, Špela Zupančič1 1University of Ljubljana, Faculty of Pharmacy, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia 2Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy 1. INTRODUCTION a nanofibrous scaffold as a delivery system for Wound can be described as a discontinuity of wound healing. the normal anatomic structure of the tissue due 2. EXPERIMENTAL METHODS to an exogenous laceration. Although the skin exhibits rapid self-regenerative capacity, 2.1. Delivery system preparation healing might fail, due to repeated infection, Firstly, phosphatidylcholine, SIM and BHA dysfunctional immune function and other were hydrated in water and immediately factors, which lead to formation of chronic sonicated using a high intensity ultrasonic wounds [1]. processor (Cole-Parmer, USA) to obtain Recent studies have shown the remarkable liposomes. Then the polymeric solutions were effects of simvastatin (SIM) as prepared by dissolving sodium alginate and immunomodulatory and antibacterial agent in polyethylene oxide (PEO) in water or liposomal chronic wound treatment. Its dispersions. After 10 h these solutions were immunomodulatory activity is mostly linked to transferred in a plastic syringe and fixed into the interference with the recruitment and activation electrospinning device (Fluidnatek LE100; of peripheral blood mononuclear cel s (PBMC) BioInicia SL, Valencia, Spain). The through suppression of their markers and electrospinning was carried out in a climatic cytokine expression. SIM, due to its chamber enabling control ed process antimicrobial activity also contributes to parameters. successful treatment of infected skin wounds 2.3. In vitro cellular tests [2]. Different formulations without and with SIM For efficient delivery of APIs to wounds many were tested on cel s to determine the effect of delivery systems have been studied and nano components and formulations. delivery systems showed great promise, The cytotoxicity of formulations was tested on especial y electrospun polymeric nanofibers human NCTC 2544 keratinocyte cel line cel s. with intrinsic wound healing and skin Keratinocytes were seeded in a 96-wel flat regeneration stimulating capacities. Nanofibers bot om tissue culture plate in DMEM medium mimic morphology of the extracel ular matrix, and incubated for 12 h. After that, tested which combined with their mechanical formulations were added with 0,1 % of dimethyl performance, physical properties, and sulfoxide (DMSO) to increase the solubility of flexibility, enables structural support, promotes SIM and 20 % of fetal bovine serum (FBS) for cel growth, and proliferation [3]. keratinocyte growth. Formulations contained Due to low aqueous solubility and stability of different SIM concentrations, i.e. 0, 0.4, 4, 40 SIM, the drug was first incorporated in and 400 µg/ml. Cultures were incubated for 72 liposomes with butylated hydroxyanisole h and then MTS assay was performed, with (BHA) to increase its stability. We expected that absorbances being measured at 490 nm, using a the combination of two nanocarrier microplate reader (Agilent BioTek Synergy H4, technologies would provide excel ent CA, USA). biocompatibility, control ed release, and Immunomodulatory effects were assessed as T mechanical properties of delivery systems. lymphocyte (main cel s in PBMCs) Therefore, the aim of this work was to design proliferation inhibition in a homeostatic and and evaluate the safety and immunomodulatory simulated hyper-inflamed microenvironments, efficacy of SIM-loaded liposomes embedded in fol owing the addition of phytohemagglutinin L (PHA-L). PBMCs were suspended in 125 OP33 BioTarget® medium and transferred in 96 - U reducing the harmful effects of over-activated wel microtiter plates, treated with different immune system. formulations and cultured for 72 h at 37 °C in a 5% CO2 incubator. Then the MTS assay was performed as described above. 3. RESULTS AND DISCUSSION 3.1. Effect of formulations on NCTC 2544 keratinocyte cell line cells viability We tested three groups of formulations: placebo, liposomal SIM and nanofiber formulations with liposomes and SIM. Figure 1. presents the dependence of keratinocyte viability on SIM concentration in each formulation tested. We found that nanofiber formulations had a slower release of liposomes Figure 2. PBMC metabolic activity assessed by MTS and SIM in comparison to liposomes with SIM. test in a non-inflammatory environment. As seen in Fig. 1. liposomal formulations 4. CONCLUSION showed no cytotoxicity up to 4 µg/ml of SIM, whereas nanofiber formulations were not Nanofiber formulations have superior safety cytotoxic up to 40 µg/ml. and efficacy properties in comparison to liposomal formulations containing SIM. They exert lower cytotoxicity on NCTC 2544 cel s, do not significantly influence PBMC functions in a non-inflammatory environment and stil hamper inflammation in a PHA-L induced inflammatory environment. The difference between liposomal and nanofiber formulations can be ascribed to multiple reasons: slower release of liposomes from nanofibers, different size and size distribution of liposomes after resuspension of nanofibers and/or the effect of nanofibers and polymers on the cells. Considering the results of al performed tests, nanofiber formulations proved to be an Figure 1. Effect of different formulations on NCTC 2544 excel ent option for incorporating SIM for cel s viability. LIPO – liposomes, NF – nanofibers. wound healing applications. 3.2. Immunomodulation of formulations 5. REFERENCES Immunomodulation of SIM containing 1. Raziyeva K, et al. Immunology of Acute and formulations was quantified by proliferation Chronic Wound Healing. Biomolecules. inhibition of lymphocytes T compared to 2021 May 8;11(5). controls (without SIM). SIM concentration of 2. Rego AC, et al. Simvastatin improves the 0.4 µg/ml was tested on lymphocytes. We show healing of infected skin wounds of rats. Acta in Figure 2 that the liposomal formulations Cir Bras. 2007 Mar-Apr;22 Suppl 1:57-63. inhibited homeostatic PBMC proliferation, 3. Zidar A, et al. Treatment chal enges and while nanofibers did not. This is bet er for the delivery systems in immunomodulation and safety profile, as the inhibition of basal cel probiotic therapies for periodontitis. Expert functions can lead to adverse effects. On the Opinion on Drug Delivery. 2021 other hand, al formulations containing SIM 2021/09/02;18(9):1229-1244. significantly inhibited PHA-L induced proliferation of T cel s (results not shown), thereby confirming their immunomodulatory properties. The immunomodulation in inflamed environment is especial y important for 126 OP34 THE EFFECT OF THE STRUCTURE – THE EVALUATION OF THE DISINTEGRATION AND THE DISSOLUTION PROCESSES OF 3D PRINTED ORODISPERSIBLE TABLETS Jolanta Pyteraf1, Witold Jamróz1, Mateusz Kurek1, Adam Pacławski1, Urszula Bąk1, and Renata Jachowicz1 1 Department of Pharmaceutical Technology and Biopharmaceutics, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland; jolanta.pyteraf@uj.edu.pl (J.P.) 1. INTRODUCTION Structures of tablets, presented in Fig. 1 were 3D printing technologies are considered as a selected based on the results of our previous potential way for the production of smal studies [2]. Printlets containing 50 mg of FLU batches of customized dosage forms [1]. were manufactured by FDM ZMorph® 2.0 S Printlets’ structure can be precisely designed (Poland) equipped with a 0.2 mm nozzle at and accurately reproduced during printing, 192°C and 10 mm/s of printing speed. which creates a great opportunity to fabricate a 2.4. Evaluation of tablets’ disintegration dosage form characterized by the targeted process and drug dissolution characteristics. quality profile. To evaluate the disintegration properties of The aim of the presented research was to printed ODTs the pharmacopoeial analyze the process of disintegration and drug disintegration test was performed in SAPO ED- dissolution from orodispersible tablets (ODTs) 2 apparatus (Electrolab, India) in 700 mL of printed with the fused deposition modeling purified heated water. Additionally, the tablets’ (FDM) method. height changes caused by disintegration were 2. MATERIALS AND METHODS registered using BJKSN-13 apparatus [3]. Surface dissolution imaging apparatus SDi2 2.1. Materials (Pion-Inc, UK) was applied to determine the Fluconazole (99.5%, Henan Tianfu Chemical disintegration and drug dissolution processes. Co. Ltd., China) served as a model active Tablets were placed in a custom-made holder ingredient (API), while poly(vinyl alcohol) and analyzed at λ1 = 255 nm and λ2 = 280 nm. (Parteck® MXP, Merck KGaA, Germany) was The drug dissolution profiles were analyzed in used as a filament-forming polymer. USP 4 apparatus at water (Erweka DFZ60, 2.2. Hot-Melt Extrusion Germany). For both above-mentioned tests, the Filament composed of 40% of FLU and 60% of amount of API released was assessed in-line PVA was prepared using a 12-mm corotating with UV-VIS Shimadzu 1800 twin-screw, hot-melt extruder (RES-2P/12A spectrophotometer (Japan) at λ = 261 nm. Explorer, Zamak Mercator®, Poland) equipped 3. RESULTS AND DISCUSSION with a 1.75 mm nozzle at 147°C. 3.1. 3D printed tablets 2.3. 3D printing of ODTs Filament was characterized by very good The tablets were designed using Blender® 2.90 printability which resulted in smal weight software (Blender Foundation, The variations (Tab. 1). Netherlands) and sliced using Voxelizer® (version 1.4.18, Zmorph S.A., Poland). Table 1. Tablets’ attributes. Basic Crown Infill 15% Average mass 118.8 118.9 121.1 (mg ± SD) ± 2.1 ± 1.5 ± 2.2 Dose 49.5 49.6 50.5 (mg ± SD) ± 0.9 ± 0.6 ± 0.9 Disintegration Figure 1. Tablets designs. time (s ± SD) 186 ± 11 112 ± 8 88 ± 3 127 OP34 3.2. Disintegration and dissolution over 80% of API was released within 15 minutes characteristics. of testing. The basic-shaped tablets had a ful outline and a porous infil , and their disintegration time slightly exceeded the limit of three minutes (Tab. 1). In the case of crown and infil 15% tablets, the porous structure facilitated the penetration of water, which resulted in a shorter disintegration time. The differences in the disintegration process are also visible in Fig. 2: in the case of basic-shaped tablets the gradual disintegration of the tablet’s outline is clearly marked. Figure 2. The disintegration profiles of 3D printed tablets obtained by BJKSN-13 apparatus. Figure 4. Drug dissolution profiles from 3DP tablets. 4. CONCLUSION The FDM technology allows for the production of tablets with a complicated internal structure, facilitating the penetration of water into the tablet, which results in its rapid disintegration. The applied methods al owed the analysis of the mechanisms of the tablets’ disintegration process and the release of the API. 5. REFERENCES Figure 3. UV views of the tablets in the initial phase 1. Zema, L., et al., Three-Dimensional Printing of of the study in SDi2 (time = 0 min, λ = 255 nm): basic Medicinal Products and the Challenge of (a), crown (b), and infill 15% (c). Personalized Therapy. J. Pharm. Sci. 2017, 106: 1697–1705. Based on the surface dissolution imaging 2. Tranová, T., et al., Fused Deposition Modeling analysis (Fig. 3), the outline of basic tablets as a Possible Approach for the Preparation of started to deform after 5 min. of the study. This Orodispersible Tablets. Pharmaceuticals 2022, effect was similar in the case of crown-shaped 15, 69. tablets, characterized by a porous outline. The 3. Brniak, W., et al., The Practical Approach to the disintegration of the infil 15% tablets was Evaluation of Methods Used to Determine the uniform throughout the entire tablet volume. Disintegration Time of Orally Disintegrating Tablets (ODTs). Saudi Pharm. J. 2015, 23: 437– The disintegration of the infil 15% tablets was 443. faster compared to the basic and crown printlets, resulting in a rapid release of API. However, ACKNOWLEDGMENT regardless of the tablet shape, the release profiles The authors acknowledge the National Science of the FLU presented at Fig. 4 were similar – Centre, Poland for financial support (grant OPUS 16 no 2018/31/B/ST8/01327). 128 OP35 EXPLORING THE POTENTIAL OF NEW BULKING AGENTS FOR LYOPHILISED BIOPHARMACEUTICAL FORMULATIONS INTENDED FOR SUBCUTANEOUS ADMINISTRATION Maja Bjelošević, Pegi Ahlin Grabnar University of Ljubljana, Faculty of Pharmacy, Chair of Pharmaceutical Technology, Aškerčeva c. 7, 1000 Ljubljana, Slovenia 1. INTRODUCTION 2. MATERIALS AND METHODS To date, intravenous (IV) injections and 2.1. Materials infusions are the most common dosage forms The model IgG mAb was obtained from Lek for administering biopharmaceuticals, although d.d. (Slovenia). Al amino acids and mannitol the current trend is oriented towards were from Merck (Germany). Polysorbate 20 subcutaneous (SC) administration, which was from J.T. Baker (USA). Ultra-pure water provides many advantages compared to the IV was from a Mil i-Q purification system route. On the negative side, the most limiting (Bedford, MA, USA). factor in SC application is the relatively smal 2.2. Methods: injection volume, i.e., ≤2 mL, and therefore highly concentrated (above 100 mg/mL) protein Sample preparation The starting lyophilised formulations were formulations are required. The development of reconstituted with ultra-pure water to obtain the such formulations is associated with many target mAb concentrations and the appropriate chal enges, with ensuring protein stability and amounts of mannitol or selected amino acid at formulation injectability being the most the different ratios to sucrose, were added. In al demanding. Proteins are general y susceptible formulations, the molar ratio between mAb and to chemical and physical instability. Stabilisers sucrose was 1:450. At the end, polysorbate 20 are essential to prevent protein aggregation, and was added (0.02% (w/v)) and 2.0 mL of the the lyophilised form of such a drug is often filtered solution was fil ed into vials. preferred. In addition, the increased viscosity in highly concentrated formulations is an obstacle Viscosity as it limits their development and Viscosity was measured on a viscometer administration [1, 2]. (RheoSense, USA) using microfluidic technology, at 25 °C. A chip with a 2 mm × 13 The research aims were to test the effects of mm rectangular slit and a 50-m-deep monoclonal antibody (mAb) concentration (30, microfluidic channel was used. 60, 90, 120 mg/mL) on viscosity, reconstitution Reconstitution time and visual appearance time, cake appearance and mAb stability. In The reconstitution time was determined by addition, the potential of amino acids dissolution of the lyophilised products and (phenylalanine, isoleucine, lysine, proline, visual evaluation was carried out after each arginine, methionine) as bulking agents on the lyophilisation cycle. critical quality at ributes of lyophilisate Dynamic light scattering (DLS) formulations was investigated at low and high The particle sizes were measured before and mAb concentrations compared to mannitol. after lyophilisation, to reveal any aggregates. Size exclusion chromatography (SEC) Measurements were performed at 210 nm, at a flow rate of 0.4 mL/min, and a column 129 OP35 temperature of 40 °C. The relative levels of acids resulted in prolonged reconstitution times aggregation products and monomers were (Figure 1). calculated. 3. RESULTS AND DISCUSSION 3.1. Effects of mAb concentration on viscosity, stability, and reconstitution time As hypothesized, the trend of increasing viscosity with increasing mAb concentration was confirmed, as none of the formulations exceeded the threshold of 50 mPa·s. However, during preparation of the formulation with 120 mg/mL mAb, there were some problems with filtration and fil ing. Because the amounts of sucrose in al samples were high enough to stabilise the mAb and the lyophilisation conditions were safe enough, neither the stability of the mAb nor the appearance of lyophilisates were compromised. In contrast, Figure 1. Reconstitution times of formulations with reconstitution times were greatly prolonged by 30 mg/mL (up) and 90 mg/mL (down) mAb. the increased mAb concentrations and were 4. CONCLUSION acceptable (<5 min) for formulations containing 30 mg/mL and 60 mg/mL mAb, whereas they The development of protein therapeutics for SC were >30 min for 120 mg/mL mAb (Table 1). administration raises several issues, some of which remain unresolved. The main conclusion Table 1. Viscosity and reconstitution time for formulations with no bulking agents. is that isoleucine for low mAb concentrations is a promising candidate to replace mannitol mAb concentration Viscosity Reconstitution time because it provides suitable reconstitution time, (mg/mL) (mPa·s) (s) cake appearance, and mAb stability, whereas 30 1.3 70 further strategies to shorten reconstitution times 60 2.6 170 of highly concentrated mAb formulations 90 6.5 360 should be discovered. 120 21.5 2070 5. REFERENCES 1. Bjelošević, M., et al., Effects of monoclonal 3.2. Impact of new bulking agents antibody concentration and type of bulking agent on Among al the amino acids tested, the most critical quality at ributes of lyophilisates. Drug appropriate appearance was found for Delivery Science and Technology, 2021. 63:102510. lyophilisates with isoleucine or phenylalanine 2. Bjelošević, M., et al., Excipients in freeze-dried biopharmaceuticals: Contributions toward in a sucrose ratio of 1:4, while for the other formulation stability and lyophilisation cycle amino acids shrunken cakes were obtained. It optimization. International Journal of was also found that both amino acids can Pharmaceutics, 2020. 576:119029. preserve the stability of mAb and thus represent ACKNOWLEDGMENT a potential for the replacement of mannitol and The authors acknowledge financial support glycine. However, we demonstrated that this from the Slovenian Research Agency (Research applies just for formulations with low mAb Core Funding, No. P1-0189), and analytical concentration, while for highly concentrated support from company Lek d.d. mAb formulations, the addition of both amino 130 OP36 CYCLODEXTRIN POLYMER-BASED siRNA DELIVERY SYSTEMS Ágnes Rusznyák1,2, Milo Malanga3, Judit Váradi1, Ildikó Bácskay1,2, Miklós Vecsernyés1, Ferenc Fenyvesi1 1Department of Pharmaceutical Technology, University of Debrecen, Debrecen, Hungary 2Institute of Healthcare Indsutry, University of Debrecen, Debrecen, Hungary 3CycloLab Ltd, Budapest, Hungary 1. INTRODUCTION were found along the cel membrane, even Cyclodextrin polymers are widely used polyplexes formulated with NHBCD polymer excipients mainly in the pharmaceutical were not taken up. industry, but are also increasingly used in the 4. CONCLUSION cosmetics and food industry today [1]. Cyclodextrin polymers, as wel as monomeric In summary, we have successful y formulated cyclodextrin molecules, are popular for siRNA-cyclodextrin polymer polyplexes, increasing the water solubility of lipophilic which did not affect cel proliferation and drugs and stabilization. siRNA is a double- QABCDP polyplex was taken up by cel s. stranded, non-coding RNA molecule containing 5. REFERENCES 20 to 27 base pairs and used for gene silencing. 1. Crini, G.; Fourmentin, S.; Fenyvesi, É.; Cyclodextrin-based systems are also used as Torri, G.; Fourmentin, M.; Morin-Crini, N. siRNA delivery agents [2], so we aimed to Cyclodextrins, from molecules to investigate the siRNA carrying capacity of two applications. Environ. Chem. Lett. 2018, 16, cyclodextrin polymers, quaternary amino beta-1361–1375, doi:10.1007/s10311-018-0763- cyclodextrin polymer (QABCDP) and amino 2. beta-cyclodextrin polymer (NHBCDP). 2. Liu, J.; Ding, X.; Fu, Y.; Xiang, C.; Yuan, Polyethyleneimine (PEI) was used as a control Y.; Zhang, Y.; Yu, P. Cyclodextrins based vehicle. Our aim was to investigate the delivery systems for macro biomolecules. properties and the cel ular internalization on Eur. J. Med. Chem. 2021, 212, 113105, Caco- 2 cel line. doi:10.1016/j.ejmech.2020.113105. 2. MATERIALS AND METHODS ACKNOWLEDGMENT The different polymer solutions effects on The work/publication is supported by the Caco-2 cel proliferation were measured by EFOP-3.6.1-16-2016-00022 and FK17 RTCA method. The properties of the (124634). Project no. TKP2021-EGA-18 has formulated polyplexes were investigated by been implemented with the support provided dynamic light scat ering technology (DLS) and from the National Research, Development and zeta potential measurements. The cel ular Innovation Fund of Hungary, financed under uptake of the polyplexes was investigated by the TKP2021-EGA funding scheme. confocal microscopy and flow cytometry. 3. RESULTS AND DISCUSSION Based on our RTCA studies, it can be stated that 50 and 100 nM polymer solutions did not affect cel proliferation. The complexation was successful, as the size and zeta-potential of both siRNA and polymer changed after complexation. Confocal microscopy and flow cytometry experiments revealed, that QABCDP polyplexes are taken up by cel s and localized in the cytoplasm. Complexes formed with PEI 131 OP37 OPEN LIQUID-SURFACE ULTRASOUND-ENHANCED ELECTROSPINNING FOR GENERATING MULTILAYERED NANOFIBER STRUCTURES Arle Kõrkjas1, Kaarel Laar1, Ari Salmi2, Joni Mäkinen2, Edward Hæggström2, Karin Kogermann1, Jyrki Heinämäki1, Ivo Laidmäe1,3,* 1Institute of Pharmacy, University of Tartu, Estonia 2Electronics Research Laboratory, Department of Physics, University of Helsinki, Finland 3Department of Immunology, Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia 1. INTRODUCTION measurements taken from a single sample, Chronic wounds are major chal enge for the using ImageJ software. health care system [1]. There’s a lack of chronic The presence of potential process induced wound specific dressings [2] and advanced transformations (PITs) of PEO in a needleless wound dressings could help with this issue [3]. USES process were studied by Fourier Conventional wound dressings face many transform infrared (FTIR) spectroscopy chal enges [3]. Advanced nanofiber dressings (IRPrestige 21, Shimadzu corporation) with a hold great potential in addressing these single reflection at enuated total reflection chal enges [4]. In this study we take a closer (ATR) crystal. look at specific process parameters of the Ultrasound-Enhanced electrospinning (USES, Figure 1) system and utilize them for creating multi-layered nanofiber structures which could be used for bet er chronic wound therapy [5]. 2. MATERIALS AND METHODS 2.1. Materials Polyethylene oxide, PEO (an average molecular weight of 900,000 Da) (Sigma-Aldrich Inc., U.S.A) was used as a mat forming polymer as a safe and established water-soluble synthetic Figure 1. Schematic of the USES system setup for polymer enabling the use of aqueous solution in electrospinning multi-layered nanofiber structures an electrospinning process. Purified water was used as a solvent for preparing the aqueous PEO 3. RESULTS AND DISCUSSION solutions (4% w/v and 3.5% w/v) applied in the 3.1. Study on USES parameters USES experiments. We investigated the impact of a stepwise 2.2. Fabrication of nanofibers change in two independent USES process An in-house USES method was used to generate parameters (ultrasound BR and BC) on the fiber nanofibers and multilayered nanofiber mats, diameter in the multilayered nanofibrous mats. describe in detail in prvious paper (Nieminen et The BR was increased for every subsequent al. 2018). Three types of tests were conducted – fiber layer that was generated, and the total 1) increase of burst rate (BR), 2) increase of number of nanofiber layers generated was eight. burst count (BC) and 3) increase of total relative The magnitude of other process parameters acoustic power (APrel) over multiple layers of were kept constant. Results from BR test are nanofibers. shown on Figure 2. 2.3. Characterization of nanofibers Scanning electron microscopy (SEM) was applied to study the size and morphology of the nanofibers with approximately 50 randomized 132 OP37 600 4. CONCLUSION ) 550 465 474 500 421 422 The fabrication of the multilayered nanofibrous 450 387 402 400 351 mats was further developed by stepwise altering 337 eter (nm 350 the critical process parameters in the USES 300 250 system to modify the nanofiber size in the 200 different fiber layers. In two sets of experiments Fiber diam 0 500 1000 1500 we showed great potential in modifying Burst rate (Hz) nanofiber thickness on fly to produce advanced nanofiber structures. Based on the results Figure 2. USES electrospun nanofiber diameters at obtained, we believe that USES could hold different BR values over eight separate layers. promise in fabricating multilayered (gradient) Similar investigation was conducted with BC nanofibrous structures for wound healing where parameter was increased over 11 applications. subsequent layers. BC test results are shown on 5. REFERENCES Figure 3. 1. Landén, N. X., Li, D. and Ståhle, M. ‘Transition 600 from inflammation to proliferation: a critical ) 488 step during wound healing’, Cel ular and 500 478 428429 455 Molecular Life Sciences 2016 73:20, 73(20), pp. 408 400 402 423 383 384 3861–3885. eter (nm 307 300 2. Gupta, S. et al. ‘Chronic wounds-Magnitude, 200 Socioeconomic Burden and Consequences’. 1000 3000 5000 7000 9000 11000 Wounds Asia 2021,Vol 4 Issue 1 Fiber diam Burst count (Cyc) 3. Zahedi, P. et al. ‘A review on wound dressings with an emphasis on electrospun nanofibrous polymeric bandages’, Polymers for Advanced Figure 3. USES electrospun nanofiber diameters at Technologies, 2021(2), pp. 77–95. different BC values over 11 separate layers. 4. Chen, S. et al. ‘Recent advances in electrospun 3.3. Discussion nanofibers for wound healing’, Nanomedicine As BR was increased stepwise from 150 Hz to (Lond). 2017 Jun;12(11):1335-1352. 1800 Hz, the average fiber size increased from 5. Ansari, S., Khorshidi, S. and Karkhaneh, A. 387 nm to 474 nm, respectively. In the present ‘Engineering of gradient osteochondral tissue: From nature to lab’, study, we used duty cycle (DC) levels in the Acta Biomaterialia, 2019 87, pp. 41–54. range of 2.2% and 26.2%, suggesting that there is stil room to increase the BR for generating ACKNOWLEDGMENT the nanofibers with higher fiber diameter, if the This work is supported by the Estonian ultrasonic fountain is stable enough. Research Council projects IUT 34-18, Increasing BC reduced the average diameter of PRG1507 and PRG712. The Estonian Ministry nanofibers in the fiber layers generated by of Education and Research is acknowledged for USES. As BC was increased from 1200 cycles a financial support. The present study was also to 10000 cycles, the average diameter of supported by the “Acouspin - Accelerated nanofibers decreased by 170 nm. Applying BC wound healing” project funded by the Business above 4500 cycles resulted in the average Finland. This presentation is funded by the diameter of nanofibers (fiber layers) ranging Doctoral School of Cinical Medicine, supported from 300 nm to 400 nm. Even though, there was by the European Union, European Regional a downward trend in the fiber thickness with Development Fund (University of Tartu’s increasing BC, trend was not monotoneus. ASTRA project PER ASPERA). Minor changes in other USES parameters were necessary per test to stabilize the ultrasonic cone on top of the solution in the vessel. Electrospinning was not possible with unstable ultrasonic cone. 133 OP38 DEVELOPMENT OF PEPTIDES AS AFFINITY LIGANDS FOR HUMAN ANTIBODY PURIFICATION Tomaž Bratkovič1, Krištof Bozovičar1, Anže Meden2, Barbara Jenko Bizjan3 1Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, Slovenia 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Slovenia 3Division of Paediatrics, University Medical Centre Ljubljana, Slovenia 1. INTRODUCTION 2.3. Structure-activity relationship (SAR) Peptides theoretical y overcome many analysis shortcomings of larger proteins as ligands for The lead peptide was subjected to on-phage N- affinity purification of biological molecules and C-terminal trimming and alanine scanning [1,2]. Firstly, they interact with cognate targets to identify residues involved in Fc binding [3]. with moderate affinity, al owing mild elution, 2.4. Design and screening of focused peptide thus preserving the target’s structural and library functional integrity. Secondly, lacking a higher- A phagemid-displayed focused library was order structure, peptides are resistant to harsh constructed based on the minimal peptide conditions of sanitization, thereby extending the sequence that retained Fc binding, in which the lifetime of the affinity column. Thirdly, they non-essential residues were randomized. The can be reproducibly and cost-efficiently library was screened against immobilized synthesized at large scale. Here, we report the human polyspecific IgG and eluted phagemid development of selective peptide ligands virions were subjected to NGS sequencing [4]. binding al 4 subclasses of human immunoglobulin G and an affinity matrix for 2.5. Affinity matrix production and antibody purification. characterization The peptide ligand exhibiting the highest affinity to human IgG was synthesized with a C- 2. MATERIALS AND METHODS terminal cysteine and coupled to bromohydrin- 2.1. Materials activated Workbeads resin (Bio-Works, PhD-12 random peptide phage display library Uppsala, Sweden) via tris(2-aminoethyl)amine kit was procured from New England Biolabs and bromoacetic acid [5]. Affinity matrix was (Ipswich, MA, USA). Phagemid pIT2, helper characterized with regard to selectivity, phage KM13, and E. coli strain TG1 were specificity, and dynamic binding capacity. obtained from Source BioScience (Not ingham, UK). Human Fc fragment was procured from 3. RESULTS AND DISCUSSION Athens Research & Technology (Athens, GA, USA). Leftover therapeutic polyspecific 3.1. Identification of lead peptide and SAR (Octagam) and monoclonal antibodies from analysis multidose vials were kindly donated by Profs. We identified 6 unique low-homology peptides Samo Zver and David Drobne (University as selective Fc binders by screening a random Medical Centre Ljubljana). dodecapeptide phage library. The peptide displaying the highest affinity 2.2. Screening of random peptide library (AGNGSYWYQVWF) was chosen as a lead for PhD-12 phage library was screened in solution further characterization. SAR analysis against a pool of biotinylated human IgG Fc identified the minimal sequence GSYWYQVWF regions [3]. Three selection rounds were required for binding with al 5 aromatic residues performed and affinities of peptide hits were seemingly making essential contacts to IgG Fc comparatively assessed by phage ELISA assay. region. 3.2. Development of improved peptide ligand We designed a focused peptide library based on the minimized affinity peptide by combining 134 OP38 soft and hard randomization of residues. 3.4. Outlook Peptides GX(Y/F)W(Y/F)XXW(Y/F) In addition to affinity chromatography, our (where X denotes any amino acid) were peptides might find use as selective ligands for displayed on phagemid virions at one copy per homogenous non-covalent antibody phage to prevent avidity effects. A single immobilization on immunoprecipitation beads panning round was performed and the entire and/or biosensor surfaces. We are currently eluted phagemid pool was sequenced. developing cyclized analogues of peptide A Enrichment was monitored by comparing clone with stil improved affinity towards the Fc frequency against that found in the pre-screened region. library. The highest affinity peptide was also most highly enriched and was termed peptide A 4. CONCLUSION (GSYWYNVWF). We have developed selective peptide binders of 3.3. Functionality demonstration of affinity human IgG Fc region and demonstrated their matrix use as ligands for affinity purification of We coupled synthetic peptide A to cross-linked antibodies. The peptide-based affinity matrix agarose beads via a short branched linker to displayed excel ent chemical stability and increase ligand density and prevent its steric dynamic binding capacity, and afforded high hindrance. The affinity matrix bound al 4 purity antibodies. human IgG subclasses. The determined dynamic binding capacity (DBC) for infliximab 5. REFERENCES (a chimeric IgG1) was ~44 mg/mL (Fig. 1), on par with that of a commercial protein A-based 1. Barredo-Vacchel i, G.R., et al., Peptide Affinity affinity column. Furthermore, our affinity Chromatography Applied to Therapeutic Antibodies Purification. International Journal matrix displayed high selectivity, enabling of Peptide Research and Therapeutics, 2021. enrichment of IgG from a mixture with bovine 27(4): 2905-2921. serum albumin (BSA) (Fig. 2). 2. Kruljec, N. & Bratkovič, T., Alternative Affinity Ligands for Immunoglobulins. Bioconjugate Chemistry, 2017. 28(8): 2009-2030. 3. Kruljec, N., et al., Development and Characterization of Peptide Ligands of Immunoglobulin G Fc Region. Bioconjugate Chemistry, 2018. 29(8): 2763-2775 Figure 1. A representative chromatogram of DBC 4. Bozovičar, K., et al., Focused Peptide Library determination for 1 mL peptide A affinity column. Screening as a Route to a Superior Affinity Ligand for Antibody Purification. Scientific Reports, 2021. 11(1): 11650. 5. Islam, T., et al., Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High- Capacity Affinity Adsorbents That Afford High IgG Product Quality. International Journal of Molecular Sciences, 2019. 20(1): 161. ACKNOWLEDGMENT We are grateful to former lab members Peter Molek, PhD and Nika Kruljec, PhD for Figure 2. SDS-PAGE analysis of eluted fractions for invaluable discussions and help with screening assessment of binding specificity. A 1:4 mixture phage libraries. This work was supported by the (mass ratio) of infliximab and BSA was loaded on the Slovenian Research Agency (program P4-0127) column and eluted with 20 mM glycine∙HCl buffer and the University of Ljubljana Innovation (pH 3.0). E1-E4 – sequential eluted fractions; FT – Fund (Grant 820-1/2020-33). flow-through. 135 OP39 ENGINEERING IL-6-BINDING LACTIC ACID BACTERIA FOR ALLEVIATION OF INFLAMMATORY BOWEL DISEASE Abida Zahirović1, Aleš Berlec1,2 1 Department of Biotechnology, Jožef Stefan Institute, Slovenia 2 Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION 2. MATERIALS AND METHODS Inflammatory bowel disease (IBD) is associated IL-6-binding affibody was previously selected with increased levels of interleukin (IL)-6 in by Yu et al. [2]. For L. lactis surface display, the serum and intestinal mucosa [1]. Its removal expression casset e consisted of Usp45 from the intestine can help reduce the secretion signal, IL-6-binding affibody inflammation in IBD patients. Systemic (denoted ZIL), and AcmA protein anchor. The administration of anti-cytokine agents can cause presence of affibody in the lysate of engineered side effects, including severe opportunistic bacteria was evaluated by Western blot ing. The infections and malignancies. extent of affibody surface display and its This can be avoided by local administration of functionality was assessed by confocal cytokine-binding proteins into the microscopy and flow cytometry. Removal of gastrointestinal tract using bacteria as an oral IL-6 from the surrounding medium by the delivery system. Food-grade lactic acid bacteria engineered L. lactis was evaluated using (LAB), such as Lactococcus lactis ( L. lactis), enzyme-linked immunosorbent assay (ELISA). represent a suitable host for that purpose. To determine the half-maximal effective This species is relatively resistant to gastric acid concentration (EC50), the percentage of IL-6 and bile salts, thrives in the intestinal removal was plot ed against various bacterial environment, but does not colonize the concentrations and fit ed to a four-parameter gastrointestinal tract and therefore has a low sigmoidal curve. potential to negatively affect the gut microbiota. 3. RESULTS AND DISCUSSION Microbial imbalance (dysbiosis) plays a crucial role in the pathology of IBD. LAB have been Anti-IL-6 affibody was expressed in L. lactis in shown to possess immunostimulatory and fusion with lactococcal secretion peptide and probiotic properties, which represents an anchoring protein. A high amount of affibody important advantage for IBD treatment. was detected on bacterial surface (Fig. 1a) and Probiotic administration has been shown to its functionality was validated by confirmation correct dysbiosis in IBD. of interaction with the target i.e. biotinylated Harnessing these beneficial properties of LAB, human IL-6 (Fig. 1b). we set out to develop L. lactis as a live carrier of the anti-IL-6 affibody that wil be able to decrease the content of free IL-6 in the intestine and thus block its detrimental effects in IBD. In this study, we displayed IL-6-binding affibody on the surface of L. lactis and demonstrated a high degree of IL-6 removal by the engineered bacteria in vitro. 136 OP39 patients. Affibody-displaying L. lactis reduced the content of IL-6 in the supernatants of both cel lines in a concentration-dependent manner by up to 94% (Fig. 2). Figure 2. Representative ELISA experiment showing concentrations of IL-6 that remained in the supernatant of differentiated THP-1 cel s fol owing incubation with affibody-displaying L. lactis (ZIL). Cont., L. lactis control cel s containing empty plasmid pNZ8148. Untr., untreated supernatant. Dose response analysis showed that bacterial Figure 1. (a) Representative confocal microscopy cel concentrations of 107 and 109 CFU/mL images visualizing ZIL-flag at the bacterial surface. were required for half-maximal removal of (b) Flow cytometric analysis showing binding of recombinant and macrophage-derived IL-6, ZIL-flag-displaying L. lactis to human biotin-respectively. conjugated IL-6 demonstrating that surface- displayed IL-6-binding affibody is functional. ZIL- flag, 4. CONCLUSION L. lactis harbouring pSD-ZIL-flag plasmid. Cont., L. lactis harbouring empty plasmid pNZ8148. Taken together, we developed Lactococcus Affibody-displaying L. lactis sequestered lactis with potent and selective IL-6 binding recombinant human IL-6 from the solution in a activity by displaying IL-6-specific affibody on concentration-dependent manner by up to 99%. its surface. Developed strain is suitable for The removal was equal y efficient across further development as an alternative IBD different IL-6 levels (150-1200 pg/mL) that treatment, which combines local neutralization were found to be clinical y relevant in IBD of IL-6 with beneficial effects of oral probiotics. patients, indicating high binding capacity. Engineered bacteria showed no binding to 5. REFERENCES mouse IL-6 or other pro-inflammatory 1. Neurath, MF. Cytokines in inflammatory bowel cytokines, thus proving to be highly species- disease. Nature Reviews Immunology, 2014. and antigen-specific. The ability of engineered 14:329-342. bacteria to capture IL-6 from cel culture 2. Yu, F., et al., An affibody-adalimumab hybrid supernatant was assessed using blocks combined IL-6 and TNF-triggered serum immunostimulated human monocytic cel lines amyloid A secretion in vivo. MAbs, 2014. 6:1598-1607. (THP-1 and U-937) differentiated into macrophage-like cel s. This model was established to mimic the inflammatory conditions in inflamed submucosa of IBD 137 OP40 EXPRESSION, PURIFICATION, AND NMR INVESTIGATION OF HUMAN KHSRP DNA BINDING PROTEIN USING E. COLI SYSTEM Pasquale Russomanno1, Diego Brancaccio1, Jussara Amato1, Antonio Randazzo1, Bruno Pagano1 1 Department of Pharmacy, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, Italy 1. INTRODUCTION Here, we present a new approach to express and Proteins that bind nucleic acids regulate many purify human KHSRP using the Escherichia cel ular processes, including transcription, Coli system. In addition, 1D 1H-NMR translation, gene silencing, microRNA experiments have been performed to detect its biogenesis and telomere maintenance. RNA- interaction with a G-quadruplex (G4) forming binding proteins are typical y considered DNA sequence from the promoter region of c-functional y distinct from DNA-binding myc gene. proteins and studied independently, but some of 2. MATERIALS AND METHODS these proteins, known as DNA- and RNA- 2.1. Materials binding proteins (DRBPs), have domains able The plasmids encoding KHSRP ful length and to bind both DNA and RNA with different KHSRP130-503 were cloned into pGEX-6P-1 specificity and manner. One of the known vector and al recombinant proteins were bivalent domains present in DRBPs is the K overexpressed in E. Coli BL21(DE3). The c- homology (KH) domain [1,2]. myc gene promoter DNA sequence was KHSRP (also known as KSRP or FBP2) is a chemical y synthesized at a 1-μmol scale on an multifunctional nucleic acid binding protein, ABI 394 DNA/RNA synthesizer (Applied present in the nucleus and cytoplasm [3]. It Biosystem, CA, USA). regulates transcription, mRNA translation, For 1D 1H-NMR assay, 200 μL of 20 μM miRNA biogenesis, and modulates diverse KHSRP or KHSRP130-503 and 20 μM c-myc in 25 cel ular functions, including cel mM Tris−HCl buffer (pH 7.4) supplemented differentiation/proliferation and innate with 150 mM NaCl and 10% D2O, were used. immunity, playing a key role in immune cel Al spectra were acquired on a BRUKER function and tumor progression [4-6]. AVANCE NEO 600 MHz spectrometer Particularly, the over-expression of KHSRP has equipped with a Z-gradient cryoprobe four- been shown to promote c-myc transcription, a channel equipped with a refrigerated SampleJet wel -known proto-oncogene involved in a broad autosampler at 298 K, using 256 scans per spectrum of human cancers [6,7]. spectrum with a recovery delay of 1.5 sec. KHSRP (1-711) is composed of a structured 2.2. Protein expression and purification central nucleic acid binding region that includes Cel s cultured in LB media were initial y grown four KH domains (130-503) and two N- (1-129) at 37 °C and subsequently cooled to 18 °C to and C-terminal (504-711) unfolded regions obtain the soluble protein. Then, after lysis by [3,8]. This particular structure and its ability to sonication, the solution was cyclical y loaded bind to nucleic acids have made its biochemical, onto a GST-column. Several elution steps, biophysical, and structural studies very using different buffers with increasing pH and difficult. To date, KHSRP expression and NaCl concentration, were performed to break purification have shown several limitations. the bond between KHSRP and the bacterial 138 OP40 DNA. Final y, GST-tag was removed, and the vitro studies are very difficult due to protein purified by size exclusion complicated protein expression and chromatography. Similar methodology was purification. Our results describe, for the first used to obtain KHSRP130-503 composed of only time, a new approach to express and purify the central nucleic acid binding region. human KHSRP, using Escherichia Coli system 2.3. NMR experiments and a different purification method without 1D 1H-NMR spectra confirmed the correct precipitation and refolding, to obtain the protein folding of KHSRP ful length and KHSRP in its native folding. Moreover, using 1D 1H- 130-503. 1D 1H-spectra of KHSRP NMR spectra we confirmed the correct folding 130-503 were recorded before and after the addition of increasing of KHSRP and thus validated our expression amounts of c-myc gene promoter sequence to and purification method. Final y, the solution containing the free protein. The macromolecule-based 1D 1H-NMR same macromolecule-based 1D 1H-NMR experiments were performed on KHSRP130-503 to spectra were performed on c-myc gene promoter detect and characterize its interaction with a G4- sequence before and after the addition of forming sequence from c-myc promoter. increasing amounts of KHSRP130-503. 5. REFERENCES 3. RESULTS AND DISCUSSION [1] Hudson WH, Ortlund EA. Nat Rev Mol Cel Analysis of 1D Biol. 2014, 15, 749-60. 1H-NMR spectra of KHSRP ful [2] García-Mauriño SM, Rivero-Rodríguez F, length and KHSRP130-503 indicates that both Velázquez-Cruz A, Hernández-Vel isca M, Díaz- proteins obtained with the expression and Quintana A, De la Rosa MA, Díaz-Moreno I. Front purification procedure described above are Mol Biosci. 2017, 4, 71. [3] Duncan R, Bazar L, Michelot i G, Tomonaga T, correctly folded: the methyl signal under 0 ppm Krutzsch H, Avigan M, Levens D. Genes Dev. 1994, indicates the presence of a hydrophobic region, 8, 465-80. while the good dispersion of NH signals at 9 [4] Briata P, Chen CY, Ramos A, Gherzi R. Biochim ppm indicates the presence of tertiary and Biophys Acta. 2013, 1829, 689-94. [5] Briata P, Bordo D, Puppo M, Gorlero F, Rossi M, secondary structures of the protein. Perrone-Bizzozero N, Gherzi R. Wiley Interdiscip In the first NMR experiment, when c- myc G4 is Rev RNA. 2016, 7, 227-40. [6] Palzer KA, Bolduan V, Käfer R, Kleinert H, Bros added to KHSRP130-503, a decrease in the M, Pautz A. Cells. 2022, 11, 1482. intensity of the signals in the aliphatic and [7] Ramdzan M. Zubaidah, Gek San Tan, Sandra B. aromatic regions of the free protein, as wel as E. Tan, Seng Gee Lim, Qingsong Lin and Maxey C. the appearance of new signals, are observed. M. Chung. Proteomics 2008, 8, 5086–5096 [8] Gherzi R, Chen CY, Ramos A, Briata P. Semin This confirms unequivocal y the formation of a Cell Dev Biol. 2014, 34, 2-8. DNA-protein complex. Comparable results were obtained upon the ACKNOWLEDGMENT addition of increasing amounts of KHSRP130-503 This work was supported by an AIRC grant (ID. to the solution of containing the free c-myc 24590 to B.P.) DNA. During the NMR titration specific aromatic signals of the free DNA decrease in intensity upon the addition of increasing concentrations of the protein. This indicates a specific binding between c-myc G4 and KHSRP130-503. 4. CONCLUSION KHSRP is a promising therapeutic target for the prevention and treatment of cancer, but its in 139 Poster Presentations 140 P01 Maja Bjelošević, Odon Planinšek, Pegi Ahlin Grabnar* ORAL LYOPHILISATES: A TYPE OF PATIENT-FRIENDLY DOSAGE FORM P02 Ivana Aleksić*, Slobodanka Ćirin-Varađan, Teodora Glišić, Mihal Đuriš, Jelena Đuriš, Jelena Parojčić EVALUATION OF DILUTION CAPACITY AND COMPACTION BEHAVIOUR OF THE EXCIPIENTS CO-PROCESSED BY IN SITU FLUIDIZED BED MELT GRANULATION P03 Fatima K. Al-Sulaiti, Fatma Kır, Selma Sahin* CRUSHING EFFECT ON DISSOLUTION PROFILES OF METOPROLOL SUCCINATE MODIFIED RELEASE TABLETS P04 Petra Arany*, Pálma Fehér, Zoltán Ujhelyi, Miklós Vecsernyés, Renátó Kovács, Mariann Zichar, Ildikó Papp, Géza Regdon, Jr., Mónika Béres, Melinda Szalóki, Ildikó Bácskay MANUFACTURING AND EXAMINATION OF 3D PRINTED VAGINAL DRUG DELIVERY SYSTEMS P05 Grega Bajc*, Gregor Ratek, Franc Vrečer, Klemen Korasa DEVELOPMENT OF A COLOURANT SELECTION MODEL IN FILM COATING FOR ACHIEVING TARGETED COLOUR OF FILM-COATED TABLETS P06 Hilke Lösing, Jonas Borregaard Eriksen, Regina Scherließ, Annet e Bauer-Brandl* INTRINSIC DISSOLUTION RATE: MEASUREMENT USING AN INEXPENSIVE ALTERNATIVE TO THE PHARMACOPOEIAL DIE HOLDER P07 Ana Baumgartner*, Odon Planinšek EFFECT OF PROCESS PARAMETERS IN HIGH SHEAR GRANULATION ON PARTICLE SIZE, COMPRESSIBILITY AND COMPACTIBILITY OF GRANULATED MESOPOROUS SILICA P08 Nevin Celebi*, Ayse Nur Oktay, Sibel Ilbasmis-Tamer, Sevtap Han, Orhan Uludag EVALUATION OF ANALGESIC AND ANTI-INFLAMMATORY EFFECTS OF CYCLODEXTRIN-BASED FLURBIPROFEN NANOGELS FOR DERMAL APPLICATION P09 Jeppe J. Christiansen*, Jonas B. Eriksen, Jarkko Rautio, Marika Ruponen, Annet e Bauer-Brandl, Martin Brandl COMBINED IN-VITRO DISSOLUTION-/ ENZYMATIC CONVERSION-/ PERMEATION-STUDIES OF THE PRODRUG FOSAMPRENAVIR P10 Mikolaj Czajkowski*, Paulina Skupin-Mrugalska, Bartłomiej Milanowski, Annette Bauer-Brandl, Martin Brandl THE INFLUENCE OF PHOSPHATIDYLCHOLINE CONTENT IN HYDROGENATED PHOSPHOLIPID/POLYMER-BASED SOLID DISPERSIONS ON THE DISSOLUTION PROFILE OF A POORLY SOLUBLE DRUG P11 Davide D’Angelo*, Eride Quarta, Stefania Glieca, Veronica Chierici, Giada Varacca, Martina Brandolini, Lisa Flammini, Simona Bertoni, Vit orio Sambri, Fabio Sonvico, Ruggero Bet ini, Francesca But ini DRY POWDER FORMULATION OF CYCLOSPORINE A FOR IMMUNOSUPPRESSIVE TREATMENT P12 Mine DİRİL*, K. Volkan Ozdokur, Yeliz Yıldırım, H. Yeşim Karasulu DEVELOPMENT AND IN VITRO-IN VIVO EVALUATION OF GLYCYRRETINIC ACID ACTIVE TARGETED PROLIPOSOMAL DRUG DELİVERY SYSTEMS FOR TREATMENT LIVER CANCER P13 Jelena Đoković*, Sanela Savić, Nebojša Cekić, Snežana Savić A PROPOSAL OF INNOVATIVE INJECTABILITY ASSESMENT METHOD FOR INTRAVENIOUS FORMULATIONS – CASE STUDY ON PEGYLATED NANOEMULSIONS P14 Črt Dragar, Sebastjan Nemec, Slavko Kralj, Petra Kocbek ELECTROSPINNING AS A DRYING METHOD FOR MAGNETIC NANOPARTICLE DISPERSIONS P15 Fanni Falusi*, Szilvia Berkó, Mária Budai-Szűcs, Anita Kovács 141 FORMULATION AND INVESTIGATION OF THE EFFECT OF POLYMERS ON DERMAL FOAM PROPERTIES USING THE QUALITY BY DESIGN (QbD) APPROACH P16 Pálma Fehér*, Marc Le Borgne, Florent Perret, Christelle Marminon, Zoltán Ujhelyi, Ágota Pető, Liza Józsa, Miklós Vecsernyés, Ildikó Bácskay FORMULATION OF NANOPARTICLES CONTAINING CASEIN KINASE 2 INHIBITOR FOR THE THERAPY OF GLIOBLASTOMA P17 Felicijan Tjaša*, Rede Katarina, Cof Greta, Bogataj Marija THE INFLUENCE OF SIMULATED WATER GASTRIC EMPTYING PROFILES ON DISSOLUTION OF MODEL DRUGS P18 Merve Geyik, Burcu Nacak, Melek Nur Bilal, Tugba Gulsun, Selma Sahin*, Levent Oner, Yagmur Akdag DRY POWDER INHALER FORMULATIONS CONTAINING GEFITINIB NANOPARTICLES P19 Teodora Glišić*, Ilija German Ilić, Jelena Parojčić, Ivana Aleksić COMPARATIVE COMPRESSION CHARACTERIZATION OF LIQUISOLID SYSTEMS PREPARED WITH MESOPOROUS CARRIERS P20 Filip Gorachinov*, Fatima Mraiche, Diala Alhaj Moustafa, Ola Hisari, Damjan Georgievski, Jensa Joseph, Katerina Goracinova DESIGN AND EVALUATION OF HYALURONIC ACID DECORATED MULTIFUNCTIONAL PCL-b-PEI NANOPARTICLES P21 Mirjam Gosenca Matjaž*, Katarina Bolko Seljak NANOCELLULOSE-BASED FILM-FORMING HYDROGELS CONTAINING BETAMETHASONE DIPROPIONATE: DEVELOPMENT AND PHYSICAL EVALUATION P22 Blaž Grilc*, Tjaša Felicijan, Timeja Planinšek Parfant, Odon Planinšek VALSARTAN BUCCAL FILMS FORMULATION DEVELOPMENT WITH THE IMAGE ANALYSIS P23 Adam Haimhoffer*, Licia Dossi, Ildikó Bácskai, Ferenc Fenyvesi NEW HIGHLIGHTS OF THE DRUG CARRIER PEG-Β-CYCLODEXTRIN POLYMER P24 Hana Hořavová*, Jan Gajdziok PREPARATION AND EVALUATION OF SPRAY-DRIED MICROPARTICLES CONTAINING N-ACETYLCYSTEINE FOR LUNG APPLICATION P25 Witold Jamróz*, Jolanta Pyteraf, Mateusz Kurek, Thao Tranová, Jan Loskot, Jitka Mužíková, Renata Jachowicz THE EFFECT OF THE HIGH DRUG LOAD – THE EVALUATION OF THE PROPERTIES OF 3D PRINTED ORODISPERSIBLE TABLETS (ODTS) WITH 70% OF DRUG CONTENT P26 Bisera Jurišić Dukovski*, Josip Ljubica, Petra Kocbek, Luka Bočkor, Jasmina Lovrić HCE-T CELL-BASED CORNEAL EPITHELIAL MODEL: SCALE-DOWN TO 96-WELL INSERT PLATES P27 Mohammed Kalayi*, Berna Uzun, Arkan Barbar, Buket Aksu FORMULATION AND CHARACTERIZATION OF MUCO-ADHESIVE ORAL FILMS CONTAINING LIDOCAINE HYDROCHLORIDE USING QUALITY BY DESIGN APPROACHES P28 Mateusz Kurek*, Jolanta Pyteraf, Witold Jamroz, Justyna Knapik-Kowalczuk, Marian Paluch, Renata Jachowicz STABILITY EVALUATION OF FLUCONAZOLE-LOADED FILAMENTS AND 3D PRINTED ORODISPERSIBLE TABLETS P29 Jakob T. Lynnerup*, Jonas B Eriksen, Ann Mari Holsæter, Martin Brandl THE EFFECT OF NANO-MILLING ON DISSOLUTION/PERMEATION OF POORLY SOLUBLE DRUG COMPOUNDS P30 Jelena Mitrović*, Maja Bjelošević, Daniel E. Knutson, Aleksandar Kremenović, Dominique Lunter, Pegi Ahlin Grabnar, James M. Cook, Miroslav M. Savić, Snežana D. Savić 142 FREEZE-DRIED NANOCRYSTAL DISPERSION OF NOVEL DEUTERATED PYRAZOLOQUINOLINONE LIGAND (DK-I-56-1): PROCESS PARAMETERS AND CRYOPROTECTANT SELECTION THROUGH STABILITY STUDY P31 Sebastjan Nemec*, Tanja Potrč, Petra Kocbek, Slavko Kralj PREPARATION OF ANISOTROPIC HOLLOW SILICA NANOSTRUCTURES P32 Zeliha Duygu Özdal*, Sevgi Takka PREPARATION AND IN VITRO CHARACTERIZATION OF ONDANSETRON HYDROCHLORIDE LOADED LIPOSOME FORMULATIONS P33 Petra Party*, Rita Ambrus DEVELOPMENT OF COMBINED INHALABLE FORMULATION OF IBUPROFEN AND MANNITOL FOR THE TREATMENT OF CYSTIC FIBROSIS P34 Nikola Pešić*, Mirjana Krkobabić, Ivana Adamov, Svetlana Ibrić, Branka Ivković, Đorđe Medarević ORAL DOSAGE FORMS WITH CARVEDILOL FABRICATED BY SELECTIVE LASER SINTERING (SLS) 3D PRINTING TECHNIQUE P35 Vladimir Petkov*, Zahari Vinarov, Slavka Tcholakova DISSOLUTION KINETICS OF GLIBENCLAMIDE AMORPHOUS SOLID DISPERSIONS IN BIORELEVANT MEDIA P36 Ágota Pető*, Dóra Kósa, Ádám Haimhoffer, Dániel Nemes, Pálma Fehér, Zoltán Ujhelyi, Judit Váradi, Ferenc Fenyvesi, Miklós Vecsernyés, Zoltán Tóth, Annamária Pallag, Tünde Jurca, Ildikó Bácskay TOPICAL FORMULATION OF LYOPHILIZED P. CORONARIUS FLOWER AND LEAF EXTRACTS, ANTIMICROBIAL ASSESSMENT OF THE PLANT P37 Odon Planinšek*, Ana Baumgartner, Blaž Grilc FENOFIBRATE ORODISPERSIBLE TABLET MADE WITH GRANULATED MESOPOROUS SILICA P38 Kaisa Põhako*, Kairi Lorenz, Marta Putrinš, Külli Kingo, Tanel Tenson, Karin Kogermann EX VIVO BIOFILM MODEL ON PIG SKIN TO TEST THE EFFICACY OF ELECTROSPUN ANTIMICROBIAL DRUG-LOADED FIBER MATERIALS AS WOUND DRESSING P39 Mitja Pohlen*, Jurij Aguiar Zdovc, Jurij Trontelj, Janez Mravljak, Mirjam Gosenca Matjaž, Iztok Grabnar, Tomaž Snoj, Rok Dreu RELATIVE BIOAVAILABILITY ENHANCEMENT OF SIMVASTATIN VIA DRY EMULSION SYSTEMS: COMPARISON OF SPRAY DRYING AND FLUID BED LAYERING TECHNOLOGY P40 Chrystalla Protopapa*, Marilena Vlachou, Angeliki Siamidi, Evi Christodoulou, Nikolaos D. Bikiaris COMPARISON OF THE RELEASE PROFILES OF MELATONIN FROM MATRIX TABLETS CONTAINING POLY(ε-CAPROLACTONE) AND COPOLYMERS P41 Zrinka Rajić*, Goran Poje, Lais Pessanha de Carvalho, Jana Held, Ivana Perković, Tana Tandarić, Robert Vianel o HARMIQUINS, NOVEL POTENT ANTIPLASMODIAL HITS P42 Dávid Sinka*, Enikő Doma, Mercédesz Varga, Pálma Fehér, Liza Józsa, Zoltán Ujhelyi, Ildikó Bácskay FORMULATION AND PERMEABILITY STUDIES OF FENUGREEK (TRIGONELLA FOENUM-GRAECUM) CONTAINING SEDDS P43 Bence Sipos*, Ildikó Csóka, Piroska Szabó-Révész, Gábor Katona SOLUBILITY ENHANCEMENT OF MEGESTROL-ACETATE VIA MICELLE AND POLYMERIC MICELLE FORMULATION P44 Barbara Sterle Zorec*, Hana Kokot, Stane Pajk, Janez Štrancar, Rok Dreu VISUALISATION OF SIMVASTATIN CORE-SHELL PARTICLES PREPARED BY ELECTROSPRAYING METHOD USING STIMULATED EMISSION DEPLETION MICROSCOPY P45 Spase Stojanov*, Tina Vida Plavec, Julijana Kristl, Špela Zupančič, Aleš Berlec ELECTROSPUN NANOFIBERS AS A DELIVERY SYSTEM FOR VAGINAL PROBIOTICS 143 P46 Boglárka Szalai*, Mária Budai-Szűcs, Orsolya Jójárt-Laczkovich EVALUATION OF DEXAMETHASONE CONTAINING IN SITU GELLING MUCOADHESIVE EYE DROPS P47 Eva Tavčar* SOLUBILITY ASSESSMENT OF CANNABIDIOL IN DIFFERENT SOLVENTS P48 Biljana Temova*, Betka Krampelj, Petra Kocbek DEVELOPMENT OF CHITOSAN AND ALGINATE BASED NANOFIBERS FOR WOUND HEALING APPLICATION P49 Erna Turković*, Ivana Vasiljević, Dragana Vasiljević, Svetlana Ibrić, Jelena Parojčić APPLICATION OF SUPPORT VECTOR MACHINE LEARNING FOR ORODISPERSIBLE FILMS DISINTEGRATION TIME PREDICTION P50 Luca Éva Uhljar*, Balázs Kürtösi, Rita Ambrus DEVELOPMENT OF A PREPARATION METHOD FOR NANOCAPSULES USING FACTORIAL DESIGN P51 Zoltán Ujhelyi*, Dóra Kósa, Ágota Pető,Thinh To Quoc, Ildikó Bácskay DEVELOPMENT AND EVALUATION OF FDM PRINTED NASAL DEVICE FOR SOLID NANOPARTICLES P52 Judit Váradi*, Lóránd Erdélyi, Ferenc Fenyvesi, Gábor Vasvári, Ádám Haimhoffer, Ilona Bereczki, György Vámosi, Miklós Vecsernyés, Ildikó Bácskay, Renátó Kovács INVESTIGATION OF THE EFFECTIVENESS OF CHITOSAN COATING ON PROBIOTIC MICROCAPSULES AND INTERACTION WITH LACTOBACILLUS PLANTARUM P53 Patrícia Varga*, Csil a Bartos,Rita Ambrus INVESTIGATION OF MELOXICAM POTASSIUM CONTAINING NANOPARTICLES FOR INTRANASAL ADMINISTRATION P54 Ivana Vasiljević*, Erna Turković, Jelena Parojčić HOW FORMULATION PARAMETERS AFFECT COMPRESSION BEHAVIOUR OF MULTIPARTICULATE UNITS PREPARED BY SELECTIVE LASER SINTERING? P55 Mercedes Vitek*, Alenka Zvonar Pobirk, Žiga Medoš, Mirjam Gosenca Matjaž FLAXSEED-OIL BASED LYOTROPIC LIQUID CRYSTALS: INFLUENCE OF MICROSTRUCTURE ON BETAMETHASONE DIPROPIONATE RELEASE PROFILE P56 Lamija Hindija, Jasmina Hadžiabdić, Merima Sirbubalo, Amina Tucak-Smajić, Ognjenka Rahić, Stanko Srčič, Edina Vranić* SOLUBILITY EHANCEMENT OF DIMENHYDINATE BY INCLUSION COMPLEXATION WITH SULFOBUTYL ETHER β-CYCLODEXTRIN P57 Alenka Zvonar Pobirk*, Mila Kovačević, Ilja German Ilić, Mirjana Gašperlin THE INFLUENCE OF POLYMERIC PRECIPITATION INHIBITORS ON SOLUBILITY CHARACTERISTICS OF CARVEDILOL-LOADED SMEDDS P58 Timeja Planinšek Parfant*, Anja Hrovat, Robert Roškar SIMULTANEOUS DETERMINATION OF FIVE NITROSAMINES IN SARTAN DRUG PRODUCTS BY A LC-MS/MS METHOD P60 Tin Takač*, Milena Jadrijević-Mladar Takač, Tomislav Jednačak PREDICTION OF ENVIRONMENTAL MICROBIAL DEGRADATION OF AZITHROMYCIN IN SOIL AND WATER AND METABOLISM IN HUMANS P61 Žane Temova Rakuša*, Robert Roškar A FAST HPLC-DAD METHOD FOR THE SIMULTANEOUS DETERMINATION OF ALL MAIN WATER-SOLUBLE VITAMINS IN FOODS AND SUPPLEMENTS P62 Ilona Bereczki*,Henrietta Papp, Veronika Nagy, Attila Agócs, Ferenc Jakab, Pál Herczegh, Anikó Borbás NATURAL APOCAROTENOIDS AND THEIR SEMISYNTHETIC GLYCOPEPTIDE CONJUGATES AGAINST SARS-COV-2 P63 Svit Ferjančič Benetik*, Boris Markoj, Matic Proj, Damijan Knez, Stanislav Gobec, Aleš Obreza, Urban Košak DESIGN, SYNTHESIS AND EVALUATION OF NOVEL BChE/p38α MAPK DUAL INHIBITORS FOR THE TREATMENT OF ALZHEIMER'S DISEASE 144 P64 Yavana Ganesh*, Jonathan E. Forman, Kabrena E. Rodda, John R. Cort, Ellen M. Wynkoop COUNTERING DIVERSION OF PHARMACEUTICAL-BASED COMPOUNDS ALONG THE CHEMICAL SUPPLY CHAIN: A WORKSHOP P65 Špela Gubič*, Louise Antonia Hendrickx , Xiaoyi Shi, Žan Toplak, Kenny M. Van Theemsche , Ernesto Lopes Pinheiro-Junior, Steve Peigneur, Alain J. Labro, Luis A. Pardo, Jan Tytgat, Tihomir Tomašič, Lucija Peterlin Mašič DESIGN OF NEW POTENT AND SELECTIVE THIOPHENE-BASED KV1.3 INHIBITORS AND THEIR POTENTIAL FOR ANTICANCER ACTIVITY P66 Bálint Lőrinczi*, Zsófia Sánta, István Szatmári SYNTHESIS AND TRANSFORMATION OF AZA KYNURENIC ACID DERIVATIVES P67 Marina Marinović, Zrinka Rajić SYNTHESIS OF NEW CARBAMATE-TYPE HARMICINES P68 Martina Piga*, Zoltán Varga, Ádám Fehér, Ferenc Papp, Eva Korpos Pintye-Gyuri, Tihomir Tomašič, Nace Zidar DISCOVERY OF INHIBITORS OF Hv1 PROTON CHANNELS AS POTENTIAL ANTICANCER DRUGS P69 Žan Toplak*, Louise Antonia Hendrickx, Špela Gubič, Jan Tytgat, Luis A. Pardo, Lucija Peterlin Mašič, Tihomir Tomašič IN SILICO DISCOVERY AND OPTIMISATION OF VOLTAGE-GATED POTASSIUM CHANNEL KV10.1 INHIBITORS WITH ANTIPROLIFERATIVE ACTIVITY P70 Selena Pavšič*, Janko Kos, Anja Pišlar DIFFERENTIATION OF SH-SY5Y CELLS INTO SPECIFIC NEURONAL PHENOTYPES: IN VITRO MODEL OF NEURODEGENERATION P71 Tina Vida Plavec*, Aleš Berlec ENGINEERING LACTOCOCCUS LACTIS TO INHIBIT INTESTINAL INFLAMMATION THROUGH SMALL PROTEIN BLOCKERS OF IL-23/IL-17 AXIS USING NOVEL BGLBRICK ASSEMBLY METHOD P72 Luka Hiti*, Tijana Markovič, Irena Mlinarič-Raščan UTILISATION OF LYMPHOBLASTOID CELL LINES AS IN VITRO MODELS OF OVER-ACTIVATED IMMUNE RESPONSE FOR DRUG REPURPOSING P73 Nataša Karas Kuželički*, Alenka Šmid, Tina Kek, Eberlinc Andreja, Irena Mlinarič-Raščan, Ksenija Geršak HIGHER INCIDENCE OF COMMON POLYMORPHISMS IN THE GENES OF FOLATE AND METHIONINE CYCLES IN CHILDREN WITH OROFACIAL CLEFS P74 Tijana Markovič*, Alenka Šmid, Helena Podgornik, Matevž Škerget, Irena Mlinarič-Raščan FACTORS INFLUENCING INTER-INIDIVIDUAL DIFFERENCES IN RESPONSE TO PROSTAGLANDIN EP4 RECEPTOR AGONIST IN CHRONIC LYMPHOCYTIC LEUKEMIA CELLS P75 Panagiotis-Dimitrios Mingas*, Jurij Zdovc, Iztok Grabnar, David Drobne, Tomaž Vovk DEVELOPMENT OF A METHOD FOR THE THERAPEUTIC DRUG MONITORING OF USTEKINUMAB IN DRIED BLOOD SPOTS P76 Armando Tratenšek*, Jurij Zdovc, Igor Locatelli, Iztok Grabnar, David Drobne, Tomaž Vovk ASSOCIATION OF OXIDATIVE STRESS-RELATED BIOMARKERS WITH DISEASE ACTIVITY IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE 145 P01 ORAL LYOPHILISATES: A TYPE OF PATIENT-FRIENDLY DOSAGE FORM Maja Bjelošević, Odon Planinšek, Pegi Ahlin Grabnar University of Ljubljana, Faculty of Pharmacy, Department of Pharmaceutical Technology, Aškerčeva c. 7, 1000 Ljubljana, Slovenia 1. INTRODUCTION in 10 mL beakers and kept at -20 °C for 24 Orodispersible drug formulations represent one hours. of the current trends in the pharmaceutical field, Lyophilisation process mostly with the purposes for pediatric and Lyophilisation was conducted in a laboratory geriatric patients. The main advantage of freeze-dryer (Epsilon 2-6D; Christ, Osterode orodispersible dosage forms is that they are am Harz, Germany). In freezing step, the shelf appropriate for patients with swal owing temperature was -45 °C. During primary drying, problems, leading to improvements in patients’ the shelf temperature was raised to 20 °C at 0.5 compliance. Oral lyophilisates are solid forms, °C/min, with chamber pressure of 0.10 mbar, obtained by lyophilisation, intended either to be while in secondary drying temperature was set placed in the mouth or to be dispersed (or to 40 °C. dissolved) in water before administration. Typical y, oral lyophilisates are composed of Product appearance binders and fil ers, taste modifiers, colorants, Oral lyophilisates were visual y evaluated after sweeteners, and preservatives. Binders, such as the completion of each lyophilisation cycle. gelatin, sodium alginate, polyvinylpyrrolidone, form an amorphous matrix of lyophilisate and Disintegration time provide appropriate mechanical strength. As Oral lyophilisates were placed in 200 mL of fil ers, sugars, and sugar alcohols, such as water (22 °C), and time needed to complete sucrose, mannitol or sorbitol are used [1, 2]. disintegration was determined. According to European Pharmacopoeia oral lyophilisates The objectives of the present work were: (i) to have to disintegrate within 3 min. develop and evaluate an optimised formulation for preparation of oral lyophilisates, based on 3. RESULTS AND DISCUSSION mannitol, gelatin, and polyvinylpyrrolidone 3.1. The effect of ratio between gelatin, PVP K25; and (i ) to examine the influence of K25, and mannitol glycine, and croscarmel ose on quality At the beginning, formulations with fixed at ributes of lyophilisates. gelatin to mannitol mass ratio 1:5 were tested, 2. MATERIALS AND METHODS while their total concentration in liquid formulations varied from 6 to 30 % (w/w). The 2.1. Materials obtained results showed that al these oral Gelatin, polyvinylpyrrolidone (PVP) K25 were lyophilisates with mannitol and gelatin express from Sigma-Aldrich, Germany, while glycine, disintegration time above 3 min, indicating that mannitol, and croscarmel ose were purchased gelatin formed rugged cake structure with no from Merck, Germany pores. On the contrary, lyophilisates which contain PVP K25 instead of gelatin, were friable 2.2. Methods and cracked. Therefore three-component Sample preparation lyophilisates formed from liquid formulations with 6 % (w/w) excipients (gelatin, PVP K25, First, the half amount of water was weighted in mannitol) were tested. Results in Table 1 beaker, and during constant mixing excipients indicate that higher content of gelatin resulted were added. At the end, the ful amount of water in longer disintegration time, while lower was added, and 2 mL of samples were aliquoted gelatin content results in unappropriate appearance of lyophilsates (Figure 1). By 146 P01 increasing PVP K25 content, the disintegration 1 2 0 0.5 4.5 15 ࿧ times were not significanlty different. Considering disintegration time and visual appearance, we determined gelatin: PVP K25: 4. CONCLUSION mannitol (1: 2: 5) formulation as the most suitable. The present study demonstrates that the selection of excipients has a great importance in obtaining oral lyophilisates with acceptable quality at ributes, among which disintegration time is one of the most important. To summarize, the most suitable formulations for further drug incorporation are liquid Figure 1. Appearance of oral lyophilisates with formulations containing 6 % (w/w) excipients ratio gelatin: PVP K25: mannitol = 0.5 :1 :5; 0.5: in the ratio 1: 2: 5 for gelatin: PVP K25: 2: 5; 1: 1: 5 and 1: 2: 5. ( from left to right). mannitol, and 0.5: 2: 0.5: 4.5 for gelatin: PVP 3.2. The effect of glycine and croscarmellose K25: glycine/ croscarmel ose: mannitol. Four-component formulations were prepared 5. REFERENCES from gelatin, mannitol, PVP K25, and glycine or croscarmel ose. As can be seen in Table 1, 1. Casian, T., et al., QbD for pediatric oral the disintegration time of the lyophilisates with lyophilisates development: risk assessment fol owed either glycine or croscarmel ose depended on by screening and optimization. Drug Development the gelatin concentration. While croscarmel ose and Industrial Pharmacy, 2017. 43(12):1932-1944. provided lyophilisates with shorter 2. Slavkova, M., et al., Orodispersible drug formulations for children and elderly. European disintegration time and also appropriate visual Journal of Pharmaceutical Sciences, 2015. 75:2-9. appearance, glycine only had a positive effect on the elegant appearance of lyophilisate cake ACKNOWLEDGMENT (Figure 2). The authors acknowledge financial support from the Slovenian Research Agency (Research Core Funding, No. P1-0189) and al col eagues contributed to the present research work. Figure 2. Appearance of lyophilisates with ratio gelatin: PVP K25: glycine/ croscarmel ose: mannitol = 0.5: 2: 0.5: 4.5 (glycine left, croscarmellose right). Table 1. The composition of tested formulations and their disintegration times and evaluated visual appearance. ratio of excipients disintegration time (s) appearance elose annitol gelatin PVP K25 glycine croscarm m 0.5 1 0 0 5 12 ػ 0.5 2 0 0 5 9 ػ 1 1 0 0 5 42 ࿧ 1 2 0 0 5 45 ࿧ 0 2 0.5 0 4.5 22 ࿧ 0.5 2 0.5 0 4.5 50 ࿧ 0 2 0 0.5 4.5 10 ࿧ 0.5 2 0 0.5 4.5 12 ࿧ 147 P02 EVALUATION OF DILUTION CAPACITY AND COMPACTION BEHAVIOUR OF THE EXCIPIENTS CO-PROCESSED BY IN SITU FLUIDIZED BED MELT GRANULATION Ivana Aleksić1, Slobodanka Ćirin-Varađan1, Teodora Glišić1, Mihal Đuriš2, Jelena Đuriš1, Jelena Parojčić1 1Department of Pharmaceutical Technology and Cosmetology, University of Belgrade – Faculty of Pharmacy, Serbia 2Department of Catalysis and Chemical Engineering, Institute of Chemistry, Technology and Metallurgy—National Institute of the Republic of Serbia, Serbia 1. INTRODUCTION circularity (C) were calculated for granule shape evaluation. Co-processing has emerged as a suitable approach to meet the increasing demands for 2.3. Determination of the Carr index excipients with improved tableting The bulk and tapped (1250 taps) densities of co- performance. Apart from the most commonly processed excipients and their mixtures with 30, used energy-consuming co-processing methods 40 or 50% paracetamol were determined using (e.g. spray-drying and wet granulation), melt tap density tester STAV 2003 (J. Engelsmann granulation as a solvent-free and more AG, Germany), and Carr index was calculated. environmental y friendly technique has recently 2.4. Dynamic compaction analysis gained more at ention [1]. Co-processed excipients and their mixtures with The aim of the present study was to investigate paracetamol were compressed on a single punch the influence of meltable binder particle size instrumented tablet press (GTP D series, and compaction parameters on dilution capacity Gamlen Tableting Ltd, UK). Compacts (100 and compaction behaviour of lactose-based co- mg) were compressed under compression load processed excipients. of 200 kg (≈ 70 MPa) or 500 kg (≈ 173MPa), and compression speed of 60 or 120 mm/min. 6 2. MATERIALS AND METHODS mm flat faced punches were used. The obtained 2.1. Materials force-displacement curves were used to calculate: net work of compression (NW), Paracetamol (Acros Organics, Belgium) was detachment stress (DS), ejection stress (ES). used as the model drug. Lactose monohydrate Tablet crushing force was determined using (Carlo Erba Reagents, Italy) was used as fil er tablet hardness tester Erweka TBH 125D and glyceryl palmitostearate (Precirol® ATO 5 (Erweka GmbH, Germany), and the values Gattefossé S.A.S, France) as meltable binder. obtained were used to calculate tensile strength 2.2. Preparation of co-processed excipients (TS). Elastic recovery (24 h after compression) Co-processed excipients were prepared by in was calculated, as well. situ melt granulation in Mycrolab fluid bed 2.4. Experimental Design processor (OYSTAR Hüt lin, Germany). In order to investigate the influence of binder Precirol® particles (15%) from the 125–180 μm particle size, paracetamol content and (≈ 150 µm) or 600–710 μm sieve fraction (≈ 655 compression speed on the abovementioned µm) were used for granulation of lactose (85%). compaction properties, compacts were The inlet air flow rate was 30 m3/h, and product prepared, at compression load of 500 kg, temperature during agglomeration was 65 °C. according to 23 ful factorial design. 2.3. Particle size and shape analysis 3. RESULTS AND DISCUSSION Granule size distribution was evaluated by sieve analysis, and median particle diameter (d50) was 3.1. Particle size and shape calculated by linear interpolation of the Larger initial binder particle size led to cumulative percentage frequency curve. formation of larger and more spherical granules Granule shape was examined by 2D scanned (Table 1). image (4800 dpi resolution) analysis using ImageJ software. The aspect ratio (AR) and 148 P02 Table 1. The size and shape of the co-processed Relatively low values of detachment and excipients’ particles. ejection stress (< 3.5 MPa) indicate good antiadhesive and lubricating properties of the Binder PS (µm) d50 AR C investigated excipients. Lower values of both 150 564.9 1.33 0.81 parameters were observed in the case of P655 655 846.2 1.14 0.86 which could be related to different 3.2. Flowability agglomeration mechanisms involved. Besides The Carr index values obtained indicated binder particle size, compression speed and considerably bet er flowability of the co- paracetamol content were found to significantly processed excipient prepared by using larger influence these properties. binder particles (P655) in comparison with the Elastic recovery values of the investigated excipient prepared with smal er binder particles (P150). This might be ascribed to more samples ranged between 12 and 28%. In the case of both excipients, higher elastic recovery spherical and larger particles of P655. However, values were obtained at higher compression the addition of paracetamol led to an increase in Carr index values and less pronounced pressure. ER values of the compacts prepared at higher compression pressure were significantly differences between two excipients (Fig. 1). affected by compression speed and interactions P655 of the investigated variables. ) 20 P150 4. CONCLUSION 15 The results obtained show that meltable binder 10 particle size affects granule size and shape, and 5 Carr index (% consequently may influence flowability and 0 compaction behaviour of the co-processed 0 30 40 50 excipients. Interactions between binder particle Paracetamol loading (%) size and compaction variables were also found to affect compaction properties of the Figure 1. The influence of paracetamol loading investigated excipients. on flowability of co-processed excipients. 5. REFERENCES 3.3. Compaction behaviour The results obtained revealed bet er mechanical 1. Ćirin-Varađan, S., et al., Comparative properties of P150 in comparison with P655 evaluation of mechanical properties of lactose- compacts, irrespective of the compression based excipients co-processed with lipophilic pressure applied. The addition of paracetamol, glycerides as meltable binders. Journal of Drug as the model API with poor compaction Delivery Science and Technology, 2022. 67: properties, led to decrease in tensile strength of 102981. the compacts prepared with both excipients, and ACKNOWLEDGMENT paracetamol content showed statistical y significant influence on TS (p < 0.0001). This research was funded by the Ministry of Acceptable tensile strength (> 1 MPa) could be Education, Science and Technological achieved for compacts with 30% paracetamol Development, Republic of Serbia through compressed at higher compression pressure (≈ Grant Agreement with University of Belgrade- 173 MPa). Faculty of Pharmacy No: 451-03-68/2022- 14/200161. Paracetamol content, compression speed and interaction between binder particle size and paracetamol content were found to significantly affect NW. The influence of binder particle size was more pronounced at higher paracetamol content, with lower NW observed in the case of P655. Higher compression speed led to higher NW. 149 P03 CRUSHING EFFECT ON DISSOLUTION PROFILES OF METOPROLOL SUCCINATE MODIFIED RELEASE TABLETS Fatima K. Al-Sulaiti1; Fatma Kır1; Selma Sahin1 1Department of Pharmaceutical Technology, Hacettepe University Faculty of Pharmacy, Ankara, Turkey Correspondence: sahin.selma@gmail.com 1. INTRODUCTION 2.2. Method for Tablet Crushing Technique Oral dosage forms are one of the most used Whole MS-MR tablets were manual y crushed formulations in clinical practice, due to easy and to powder state using mortar and pestle practical administration and lower costs. For technique to mimic the hospital practices. patients with swal owing difficulties or enteral 2.3. Dissolution Studies feeding tubes, solid dosage forms are modified to facilitate the administration by crushing. Dissolution studies were conducted in According to the dosage form specifications compliance with USP monograph (USP30) and clinical condition of the patient, immediate conditions (Table 1) using Sotax dissolution release (IR) tablets can be crushed and testing instrument (Basel, Switzerland). Each dispersed in water. However, other oral dosage sample (5 mL) was withdrawn and then forms (i.e. modified-release (MR), enteric- replaced with an equal volume of fresh medium. coated tablets, film-coated tablets) should not Samples were filtered through a 0.45-μm be crushed due to potential y unpredictable membrane filter and then analysed (Shimadzu release properties, which may result in UV-1800) at 227 nm using UV- supratherapeutic or subtherapeutic in vivo drug Spectrophotometer. concentrations. Table 1. Conditions of dissolution studies. On the other hand, the absence of an IR- Apparatus Apparatus 2 (paddle) alternative makes it sometimes necessary for Speed 50 rpm clinicians to apply this practice, although the evidence based-guidelines clearly state that Medium pH 6.8 phosphate buffer non-IR oral solid dosage forms cannot be Medium crushed. Therefore, in the real clinical practice, Volume 500 mL many non-IR dosage forms are crushed and Temperature 37 ± 0.5°C administered to patients with swal owing Sampling Time 1, 4, 8, 20, 24 h difficulties or feeding tubes (i.e. dysphagia, enteral-feeding tubes) [1, 2]. 3. RESULTS AND DISCUSSION The effect of crushing on dissolution profiles of No standard tablet-crushing protocol exists to commercial y available metoprolol succinate date. Hospital practices include the use of a MR (MS-MR) tablets available in Turkish mortar and pestle or pil crusher device, mixing market was evaluated in this study. with food or thickened fluid vehicles, or 2. MATERIALS AND METHODS flushing the powder through the enteral feeding tubes with water. In our study, we crushed MS- 2.1. Materials MR tablets using a mortar and pestle to mimic MS-MR (100 mg, AstraZeneca) tablets were the crushing process of tablets applied in purchased from a community pharmacy in hospitals. Turkey. Monobasic potassium phosphate (≥ 99%: Sigma-Aldrich, St. Louis, MO USA), and The sampling time continued for up to 24 hours sodium hydroxide (≥ 97%; Merck, Germany) as it was stated in the medication package insert were used as received. that MS-MR tablets maintain stable concentration in blood for 24 hours. 150 P03 The dissolution profiles of MS whole tablets concentration profiles in the feeding tube and crushed tablets in pH 6.8 phosphate buffer patient population. Therefore, future research is are shown in Figure 1. needed to determine specific dosing recommendations, achieve therapeutic efficacy Whole Crushed 100 and prevent possible toxicity when crushing MS-MR tablets. ) 80 S (% 60 5. REFERENCES 40 1. Demirkan, K., et al., Assessment of drug issolved MD administration via feeding tube and the 20 knowledge of health-care professionals in a 0 university hospital. European Journal of 0 5 10 15 20 25 30 Clinical Nutrition, 2017. 71(2): 164-8. Time (h) 2. Wasylewicz, ATM., et al., Clinical decision Figure 1. Dissolution profiles of whole and support system-assisted pharmacy intervention crushed MS-MR tablets (mean ± standard reduces feeding tube-related medication errors deviation; n=6). in hospitalized patients: A focus on medication suitable for feeding-tube administration. The dissolution profile obtained for the whole Journal of Parenteral and Enteral Nutrition, tablet is in accordance with the reference values 2021. 45(3): 625-32. in the USP (not more than 25% in 1 hour, between 20 and 40% in 4 hours, between 40 and ACKNOWLEDGMENT 60% in 8 hours, not less than 80% in 20 hours). According to the dissolution profile of crushed This study was supported by Hacet epe tablets, dissolution was greater than 50% within University Scientific Research Projects 1 hour but then approached approximately 80% Coordination Unit with TSA-2022-19747 fund. within 24 hours. Therefore, the results for the crushed tablet were not found to meet the USP criteria. The f2 similarity factor used to compare the similarities of the two dissolution profiles was calculated as 28.02 (<50) indicating that the two dissolution profiles were not similar. The f1 difference factor was calculated as 40.02 (≥15) conforming the difference between the dissolution profiles. The results suggest the possibility that release profile has changed as a result of the deformation of the polymeric membrane structure that controls the drug release rate from microencapsulated cores containing MS. Since the deformation of polymeric membrane structures after tablets were crushed could be heterogeneous, the standard deviation of six replicates was found higher than the whole tablets. 4. CONCLUSION In Turkey, MS-MR tablets are crushed in critical y il patients using feeding tubes. Our study demonstrated that crushing changes the dissolution profile of MS-MR tablets. This suggests the clinical impact on blood- 151 P04 MANUFACTURING AND EXAMINATION OF 3D PRINTED VAGINAL DRUG DELIVERY SYSTEMS Petra Arany1, Pálma Fehér1; Zoltán Ujhelyi1; Miklós Vecsernyés1; Renátó Kovács2; Mariann Zichar3; Ildikó Papp3, Géza Regdon, Jr. 4, Mónika Béres 5, Melinda Szalóki 6, Ildikó Bácskay*1 1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Debrecen, Hungary 2Department of Medical Microbiology, Faculty of Medicine and Faculty of Pharmacy, University of Debrecen, Hungary 3Department of Computer Graphics and Image Processing, Faculty of Informatics, University of Debrecen, Hungary 4Institute of Pharmaceutical Technology and Regulatory Affairs, University of Szeged, Hungary 5Department of Medical Imaging, Faculty of Medicine, University of Debrecen, Debrecen, Hungary 6Department of Biomaterials and Prosthetic Dentistry, Faculty of Dentistry, University of Debrecen, Hungary 1. INTRODUCTION metronidazole and chloramphenicol separately. Vaginal drug delivery systems can provide a This gels were manual y fil ed to the pre-printed long-term and constant liberation of the active vaginal rings. pharmaceutical ingredient even for months. [1] 2.3. Method – Material structure For our experiment, FDM 3D printing was used Sample characterisation by thermogravimetric to manufacture the vaginal ring samples from (TG) and heatflow (DSC) analysis, contact thermoplastic polyurethane filament, which angle measurement and microcomputed enables fast manufacturing of complex, tomography (MicroCT). personalized medications. 3D printing can be an excel ent alternative instead of industrial 2.4. Method – Dissolution test manufacturing, which is complicated and time- The dissolution test was carried out by a USP consuming. [2] In our work, the 3D printed Type I Erweka DT 800 dissolution apparatus. vaginal rings were fil ed manual y with jel ified Dissolution medium of 200 ml of simulated metronidazole or chloramphenicol for the vaginal fluid at pH 4.2 was used at 37°c. treatment of bacterial vaginosis. The samples material structure and biocompatibility 2.5. Method – Biocompatibility test properties were determined. [3] A long-term MTT cel viability assay was performed to determine if any kind of 2. MATERIALS AND METHODS xenobiotic is dissolved from the samples. Microbiological test was performed to 2.1.Materials determine the effectiveness of the samples. For the 3D printing process, polylactic acid (PLA), PLA Gypsum, PLA Foam and 3. RESULTS AND DISCUSSION thermoplastic polyurethane (TPU) was used. As a jel ifying agent Carbopol 934, medium 3.1. Sample manufacturing molecular weight chitosan, hydroxyl ethyl Our research group had successful y printed the cel ulose and agar-agar was used. TPU based vaginal rings by FDM 3D printing As an active pharmaceutical ingredient with the adequate printing parameters. metronidazole and chloramphenicol were used. Successful y manufactured API containing gels in three different formulations and successful y 2.2.Method – Sample manufacturing fil ed the samples manual y. The digital design of the samples were designed using SolidWorks. The shape was based on a 3.2.Material structure commercial y available vaginal ring but the The TG/DSC curves confirmed that the API’s edges were chamfered. Each sample was 5 mm should be fil ed manual y to eliminate the tal and printed in two different diameters degradation due to high printing temperature. (39.19 and 41.19 mm) to provide the perfect (Fig. 1) fit ing. Four different formulations were prepared based on the jel ifying agent and al four formulation was homogenized with 152 P04 4. CONCLUSION In conclusion, vaginal rings from thermoplastic polyurethane were successful y 3D printed by FDM technology. The pre-printed samples were fil ed with chloramphenicol or metronidazole and jel ified with chitosan/HEC or agar-agar. Based on the dissolution curves, the used API and jel ifying agent can modify the dissolved Figure 1. Thermogravimetric (I) and heatflow API amount. Based on the MTT assay results, (II) analysis of the chloramphenicol (a) and the TPU polymer can be considered metronidazole (b). cytocompatible. 3.3. Dissolution test The microbiological evaluation confirmed that Based on the dissolution test results and the f1- metronidazole and chitosan have a synergistic f2 analysis al samples can be described with effect against E. coli. Based on the overal different dissolution test results. (Fig. 2) project, TPU polymer fil ed with metronidazole was suggested for further investigations. 1 0 0 2 n d f o r m u la ti o n ) 3 rd f o r m u la ti o n 5. REFERENCES 8 0 t (%n 4 th f o r m u la ti o n uo 60 I am 1. Pel e P. et al., Preparation and clinical use of P A 40 combined broad spectrum vaginal suppositories. edlv ORV Hetil, 1980, 17: 1015-7. isso 2 0 D 0 2. Chia H. N. et al., Recent advances in 3D printing 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 of biomaterials. J. Biol. Eng., 2015, 9: 1–14 T i m e ( h ) Figure 2. Dissolved API amount (%) versus time 3. Arany et al., Manufacturing and examination of (h) for samples jel ified with metronidazole. vaginal drug delivery system by FDM 3D printing. Pharmaceutics, 2021, 13, 1714 3.4. Biocompatibility test ACKNOWLEDGMENT The vaginal ring showed no cytotoxicity based on the ISO 10993-5:2009(E) A.2.4 standard. Supported by the ÚNKP-20-3 New National Based on the microbiological experiment the Excel ence Program of the Ministry for metronidazole and chitosan containing sample Innovation and Technology from the source of showed bactericid effect within 24 hour against the National Research, Development and the just metronidazole or just chitosan Innovation Fund. The project was supported by containing samples. (Fig. 3) the Gedeon Richter’s Talentum Foundation (1103 Budapest, Gyömrői út 19-21, Hungary). This work was supported by the European 1 0 10 a ) 1 0 9 Union and the European Regional Development b ) 1 0 8 c ) Fund [GINOP-2.3.2.-15-2016-00011]. The 1 0 7 l) d ) /m 1 0 6 publication is supported by the GINOP-2.3.4- UF (C 1 0 5 g 15-2020-00008 project. The project is co- lo 1 0 4 financed by the European Union and the 1 0 3 B a c te r ic id a l lim it 1 0 2 European Regional Development Fund. The 1 0 1 research was supported by the Thematic 0 2 4 6 8 10 12 14 16 18 20 22 24 T im e ( h ) Excel ence Programme (TKP2020-IKA-04) of Figure 3. The logarithmical colony-forming unit the Ministry for Innovation and Technology in (CFU)/mL in case of E. coli versus time (h) Hungary”. where (a) empty; (b) metronidazole containing (c) 3 w/w% chitosan and 4 w/w% hydroxyethyl cel ulose-containing; (d) 3 w/w% chitosan and metronidazole containing vaginal ring. 153 P05 DEVELOPMENT OF A COLOURANT SELECTION MODEL IN FILM COATING FOR ACHIEVING TARGETED COLOUR OF FILM-COATED TABLETS Grega Bajc1, Gregor Ratek1, Franc Vrečer1,2, Klemen Korasa1 1R&D, Krka d.d., Šmarješka cesta 6, 8501 Novo mesto, Slovenia 2University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia 1. INTRODUCTION Colour is a very important characteristic of film-coated tablets. It ensures easier recognition, harder forgery and it often leads to bet er patient compliance. The desired colouring of a film-coated tablet is achieved by using an abundance of pigments [1]. In recent years, though, this is becoming increasingly more difficult due to new toxicological studies suggesting some of those pigments are unsafe Figure 1. Model development plan [2]. New EMA recommendations in a reflection paper on the pharmaceutical development of Experiments were planned using the Design of medicines for use in older population are experiments method (Design Expert software, suggesting innovator and generic products version 12 (StatEase, USA)). The quantitative should have the same key visual appearance limits of components included were set based (i.e. colour, size etc.) to the degree (legal y) on previous experiences and are described in possible [3]. When developing a product, the Table 1. obstacle how to reproduce reliably the desired Table 1. Design space limits shade of colour is often encountered. Parameter Lower limit Upper limit In the present study, a model was created that Opadry® 97.0 % 100.0 % converts the L*, a*, and b* colour Red iron oxide 0.0 % 3.0 % Yel ow iron oxide 0.0 % 3.0 % coordinates/values of a reference film-coated tablet into the colourant composition, resulting Based on the proposed limits, an experimental in the targeted colour. space with 19 experiments was created. Two experiments had the same composition as the 2. MATERIALS AND METHODS test of colour reproducibility. 2.1. Materials Tablets were coated in GCII coating machine (Glat , Germany). Colours of tablets were Film coatings of different colours, using red and measured with HunterLabUltraScan VIS colour yel ow pigments were prepared. The materials measurement spectrophotometer (Hunter Lab, needed were placebo tablet cores and USA), which defines the colour in CIEL*a*b* components of film coating including yel ow colour space with L*, a* and b* colour values or/and red iron oxide pigment (Venator on the basis of which the model was set. Pigments S.p.A., Italy) and commercial y It was used to predict colourants for film coating available mixture Opadry® (Colorcon, USA) of the independent test batches, which were consisting of HPMC polymer, plasticizer later compared with reference products. (PEG), lubricant (talcum) and titanium dioxide. 3. RESULTS AND DISCUSSION 2.2. Method 3.1. L*, a* and b* models Colours were defined with L*, a* and b* values. L* value describes the brightness on a scale between 0 and 100. a* value indicates how green to red the colour is on scale between -127 and +127 and b* indicates how blue to yel ow it is on scale between -128 and +127. 154 P05 With the tested film coatings we achieved ߂ܧכ = ඥሺ߂ܮכሻଶ ൅ ሺ߂ܽכሻଶ ൅ ሺ߂ܾכሻଶ colour coordinates in the range: L* between ௔௕ 68.24 and 103.33, a* between -0.18 and 28.25 ߂E reveals the largest colour difference for the and b* between 4.87 and 41.94. For designing brightest tablets and the lowest for the light the experimental space, special quartic design pink. Even though the ߂E was more than 2, model was used. From results we gained there was only a slight difference in colour polynomial (regression) equations for L*, a*, observed by visual inspection. However, for and b* which enable calculation of colour inexperienced observers al the test tablets are values out of colourants selection, and, in virtually identical to the reference tablets. reverse colourant selection, out of colour Table 3. Comparison of reference film-coated tablet and test values. The fol owing contour graphs show the film-coated tablet based on proposed colourant selection. best visualization of those three equations. 1 2 3 Reference film- coated tablet Test film-coated tablet Colour 7.32 3.50 1.81 difference (߂E) 4. CONCLUSIONS Figure 2. Contour graphs for L*, a* and b*. In the left corners of graphs is 3% of red FeO, in the right corners is In our study, a colourant selection model was 3% of yel ow FeO and in the upper corners is 100% of developed by applying DoE principle and Opadry®. Red shows the highest value, while blue shows colourimeter. The colours of reference tablets the lowest value of L*, a* and b*. were measured, and a designed model was used The first graph shows that the highest value of to predict the optimal selection of two pigments L* is reached in an area with the highest amount needed to achieve the desired colour of film- of Opadry® and the lowest in the area with the coated tablets. The model successful y highest amount of red iron oxide. The a* graph predicted the optimal colourant composition. shows that the highest a* is achieved in the area Further research is necessary to improve with the highest amount of red iron oxide. The accuracy and expand the measuring range of b* graph shows the highest b* is within the area similar predictive models. of the highest yellow oxide amount. 5. REFERENCES The suitability of the model can be assessed 1. M. Mishar, P. Biswal, Bhadouriya Anupam, and with the value of adjusted R2. It represents the Yadav Vivek, “An updated review on colorants as proportion of variability in our results explained the pharmaceutical excipients,” International by our model. The adjusted R2 values of al journal of pharmaceutical, chemical and biological three models are presented in Table 2. These sciences, vol. 5(4), pp. 1004–1017, 2015. values indicate a good fit of the results. 2. F. Aguilar et al. , “Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids Table 2. Adjusted coefficient of determination of L*, a*, and b* models and Food Contact Materials (AFC) PANEL MEMBERS *,” 2008. L*model a*model b*model 3. E. Medicines Agency, “Reflection paper on the adjusted R2 0.9915 0.9787 0.9797 pharmaceutical development of medicines for use 3.2. Validation of a colour selection model in the older population,” 2020. The best comparison between the reference ACKNOWLEDGMENT tablets and test tablets is made by ߂E value (i.e. Authors would like to thank Krka, d. d., Novo colour difference). The human eye does not mesto for providing support for the study. perceive the difference between two colours when ߂E is below 2. 155 P06 INTRINSIC DISSOLUTION RATE: MEASUREMENT USING AN INEXPENSIVE ALTERNATIVE TO THE PHARMACOPOEIAL DIE HOLDER Hilke Lösing1,2, Jonas Borregaard Eriksen1, Regina Scherließ2, Annette Bauer-Brandl1 1Department of Physics Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark 2Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany 1. INTRODUCTION Tack-ALL" was purchased from Hamelin The dissolution rate is in many cases rate- (Denmark). determining for absorption of poorly water- 2.2.Preparation of substance and tablet soluble active pharmaceutical ingredients. Co-crystals were prepared in a bal mil model Intrinsic dissolution rate (IDR) is dissolution Retsch MM2000 in a molar ratio of 1:1. were rate normalized to the surface area and is bal mil ed for 4 h at an amplitude of 20 mm. measured of compacted substances with Flat faced tablets (approx. 60 mg of substance) minimal porosity in a special die hoplder to were compressed on a EK0 tablet press by hand keep the surface area constant. and kept under ful pressure for 30 sec. Monograph 2.9.29 in the European Pharmacopoeia (Ph. Eur.) describes testing the 2.3. Preparation of the device IDR of a substance in the dissolution apparatus, The device was made by kneading approx. 1.5 either top-down or at the bot om with the paddle g of RAG thoroughly and shaping it into a bal , rotating above [European Pharmacopoeia, placing the bal on a coin, and then pushing a 2021]. The standard apparatus needs 150 - 700 tablet of the compound of interest into the RAG. mg, which is not always available in drug The RAG was then shaped around the tablet by development [Teleki et al., 2020; Tseng et al., hand to ensure a constant surface area. We 2014]. It appears to be a common problem that confirmed no visible grooves in the RAG. APIs may not at ach to the holders [Bartolomei Wearing gloves proved best to ensure that the et al., 2006]. RAG did not get contaminated. The coin Removable adhesive gum (RAG) used in ensured that the tablet was heavy enough to sink households and offices consists of the polymer to the bot om, and the stirring motion did not polyisobutylene with inorganic fil ers such as move it from its position. If the coin did not sink titanium-(IV)-oxide and contains no added to the centre of the vessel, it was careful y organic solvents,. In the present study we tested pushed in place with a bar. We used a € 10-cent whether removable adhesive gum (RAG) can coin, but any other coin or disc would do the replace the die holder when evaluating the IDR. same. Co-crystals of acyclovir and glutaric acid were used as a model substance of tricky properties 2.4. Intrinsic dissolution testing in terms of sit ing in the die holder. 900 ml of degassed 0.1 M phosphate buffer pH 7.4 were utilized as the medium at 37 °C, and 2. MATERIALS AND METHODS the stirring speed was 50 rpm. Each tablet had a mass of approx. 60 mg and approx. 1.5 g of the 2.1.Materials RAG were utilized. the exposed area was 0.22 Acyclovir and co-crystals of acyclovir and cm2 . Every five minutes, a sample of 5 ml was glutaric acid in a molar ratio of 1:1 were used as taken with a syringe and filtered with a 0.45 µm model compounds. Acyclovir was from Abcr filter. In total, the intrinsic dissolution was GmbH (Karlsruhe, Germany) and glutaric acid determined for half an hour, and HPLC was from Sigma Aldrich (Brøndby, Denmark). A used for quantification. The IDR of acyclovir 0.1 M phosphate buffer pH 7.4 was prepared as in mg/h/cm2 was calculated from the slope of described in Ph. Eur. monograph 4.1.3. the regression line. At each time point, the mean [European Pharmacopoeia, 2021]. The and standard deviations were calculated (n = 3). removable adhesive gum RAG named "Linex 156 P06 3. RESULTS AND DISCUSSION The IDR was found to be 12.76 mg/h/cm2 for acyclovir, and 15.46 mg/h/cm2 for Co GA. This 3.1.Validation of the use of RAG means that the co-crystals had a 1.2-fold higher The RAG did not release any detectable substances into the media. No adsorption nor IDR. permeation of the tested substances through the 4. CONCLUSION RAG was detected. These values confirm that the RAG may be a 3.2. Intrinsic Dissolution Rates good alternative for investigating the IDR. For both Acyclovir and the co-crystal, the dissolution process of the substance was RAG does not interfere with the media or fol owed and plot ed cumulative vs. time substances tested Linear and reproducible (Figure 1). values for the IDR were obtained with the model substances acyclovir and acyclovir: glutaric acid co-crystals. It can therefore be argued that the RAG is a good alternative for determining the IDR of these substances. The RAG is widely available and not expensive. It is easy to shape and can therefore be used for different tablet sizes including minitablets. 5. REFERENCES 1. European Pharmacopoeia, 2.9.29. Intrinsic dissolution, 10th edition, Council of Europe, Strasbourg, 2021. Figure 1. Cumulative amount of acyclovir in mg against the time in hours, for acyclovir and ACKNOWLEDGMENT Co GA (from intrinsic dissolution testing). Data The authors are grateful for a mobility grant to are represented as mean +/- SD (n = 3).. HL, and col aborative work between the two Plot ing the cumulative amount of acyclovir in institutions enabled by funding from Nordforsk mg vs. time (5, 10, 15, 20, 25, 30 min time Program Nordic University Hub (Project points) produced a linear relation with number 85352 “Nordic PoP”). correlation coefficients larger than 0.998. The coefficient of variation (% CV) (n = 6) of the IDR was less than 3.5 %, indicating good reproducibility. Figure 2 shows the intrinsic dissolution rate values: Figure 2. Intrinsic dissolution values f in mg/h/cm2 or acyclovir and Co GA. Data are represented as mean +/- SD (n = 3). 157 P07 EFFECT OF PROCESS PARAMETERS IN HIGH SHEAR GRANULATION ON PARTICLE SIZE, COMPRESSIBILITY AND COMPACTIBILITY OF GRANULATED MESOPOROUS SILICA Ana Baumgartner1, Odon Planinšek1 1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION were isomalt amount and water amount, water Mesoporous silica is investigated as a addition rate and granulator impel er speed. For promising carrier for amorphous solid statistical analysis and model setting, α=0.10 dispersions, but its major limitations are poor was chosen to determine significant variables flow and compression properties, which are for the fol owing observed responses: particle related to smal particle size [1]. Hence, a novel size parameters (d10, d50 d90, span) and co-processed material was developed consisting compressibility and compactibility parameters of mesoporous silica and a sugar alcohol (Heckel constant and compactibility slope [cp; isomalt acting as a binder which connects smal tensile strength vs. compression pressure]). silica particles into granules. Besides bigger 2.4. Particle size determination particle size, such material should have bet er Particle size parameters d10, d50 and d90 were flow and compression properties as wel as high measured by laser diffraction method with a dry enough specific surface area for impregnation powder feeder (Mastersizer 3000, Malvern, with poorly water-soluble drug. The purpose of UK). Span as a measure of particle size this study was to examine the effect of process distribution was calculated according to the parameters in high shear granulation on particle fol owing equation: span = (d90 – d10)/d50. size in relation to compressibility and compactibility of such material by Design of 2.5. Compactibility and compressibility Experiments (DoE) study. evaluation For each granulate, 10–15 500 mg tablets were 2. MATERIALS AND METHODS compressed at different compaction pressures 2.1. Materials using single-punch tablet press (Kilian SP300, Isomalt (GalenIQ 800) was kindly donated by IMA, Germany) with round flat-faced punches. Beneo (Germany). Mesoporous silica (Syloid The fol owing tablet characteristics were 244 FP) was obtained from Grace Davison, evaluated: tablet mass (Met ler Toledo, Grace GmbH & Co. KG (Worms, Germany). Switzerland), thickness and diameter Water was purified by reverse osmosis. (micrometer, Mitutoyo, Japan), crushing force (hardness tester VK200, Varian Inc., USA), true 2.2. Preparation of granulates density (helium pycnometer AccuPyc 1330, Co-processed excipients were made by wet Micromeritics, USA). Heckel constant and cp granulation in a high-shear mixer (ProCept were determined by linear regression. 4M8-TriX, 1L vessel, Belgium). Distil ed water was added as granulation liquid to the blend of 3. RESULTS AND DISCUSSION isomalt and silica dropwise at a specific flow 3.1. Effect of process parameters on particle rate. Wet granulate was sieved through a 2 mm size sieve and dried on trays at 60 °C until moisture Out of 27 performed experiments, 7 ended in an content was below 2%. Dried granulate was over-wet granulate and were therefore not sieved through 710 µm sieve, and particles that further evaluated. After eliminating passed 80 µm sieve were discharged. insignificant factors, good models for d10, d50, 2.3. Experimental design d90 and span were obtained (Table 1). A central composite orthogonal design was Table 1. Model characteristics for d10, d50 and used for DoE study. Experimental design and d90 analysis were performed by Modde Software d10 d50 d90 span (Sartorius, Germany). Independent variables R2 0.92 0.88 0.93 0.88 158 P07 R2 (adj) 0.88 0.86 0.82 0.82 which means more liquid on the surface and Q2 0.78 0.78 0.57 0.66 thus granule growth by coalescence [2]. Model validity 0.79 0.78 0.85 0.44 However, if both factors are set on high levels, Reproducibility 0.93 0.92 0.89 0.98 there are high chances of overgranulation, as was the case in the experiments that were It was observed that amount of added water and discarded. On the other hand, water addition impel er speed have a positive effect on particle rate has a negative effect on particle size. The size, meaning that the higher those values are, reason for this could be that slower addition rate the larger particles are formed (Figure 1). This means longer process time and thus more time can be explained by the fact that increasing for the granules to form. Slower water addition these factors contributes to higher liquid rate also results in narrower particle size saturation of the granulate during the process, distribution, which is a desired characteristic as wel . Figure 1. Visualisation of effect of water amount and addition rate on d50 and d90. Impel er speed was 200 rpm and isomalt amount was 17.5 g in both cases. 3.2. Effect of process parameters on 5. REFERENCES compressibility and compactibility The models acquired to explain the effects of 1. Baumgartner A, Planinšek O. Application of process parameters on compressibility (Heckel commercially available mesoporous silica for drug constant) and compactibility (c dissolution enhancement in oral drug delivery. p) were less good EJPS, 2021. 167:106015. (R2 (adj) = 0.57 and 0.66, respectively; Q2 = 0.49 and 0.49, respectively). However, it is stil 2. Badawy SI, et al. Effect of process parameters on possible to distinguish the main effects. Water compressibility of granulation manufactured in a addition rate has a positive effect in both cases, high-shear mixer. International Journal of meaning that faster rate results in more Pharmaceutics, 2000. 198(1):51-61. compressible and compact granulate. This is in ACKNOWLEDGMENT contrast to its effect on particle size, which is somewhat surprising. Also, higher amount of The authors acknowledge the financial support binder seems to increase granule provided by the Slovenian Research Agency compressibility. (Program Code P1-0189). 4. CONCLUSION High shear granulation of mesoporous silica with isomalt is a complex process with many process parameters in interplay that govern product characteristics. Although some trends can be seen from the models acquired by a DoE study, more detailed studies are needed to predict the characteristics studied in this research. 159 P08 EVALUATION OF ANALGESIC AND ANTI-INFLAMMATORY EFFECTS OF CYCLODEXTRIN-BASED FLURBIPROFEN NANOGELS FOR DERMAL APPLICATION Nevin Celebi1,2, Ayse Nur Oktay1,3, Sibel Ilbasmis-Tamer1, Sevtap Han4, Orhan Uludag4 1Department of Pharmaceutical Technology, Gazi University-Faculty of Pharmacy, Ankara, Türkiye, 2Department of Pharmaceutical Technology, Başkent University-Faculty of Pharmacy, Ankara, Türkiye 3Department of Pharmaceutical Technology, University of Health Sciences- Gulhane Faculty of Pharmacy, Ankara, Türkiye 4Department of Pharmacology, Gazi University-Faculty of Pharmacy, Ankara, Türkiye 1. INTRODUCTION (FB-free or FB-loaded) and then stirred at 500 Flurbiprofen (FB) is an effective nonsteroidal rpm under a magnetic stirrer (IKAט Eurostar anti-inflammatory drug utilized in the 20) for 60 min at room temperature until a treatments of chronic and acute inflammatory homogeneous gel was obtained. diseases such as soft tissue injures, 2.4. Evaluation of Analgesic and Anti- osteoarthritis, rheumatic arthritis and sunburn. Inflammatory Effects of FB-loaded But its poor water solubility limits its Cyclodextrin Based Nanogels absorption, thus its dermal bioavailability [1]. Al the animal protocols were approved by the Cyclodextrin-based nanogels could be ethical commit ee of Gazi University (148677). promising for the dermal application of drugs Formulations were applied at the dosage of 20 with low water solubility. The main aim of this mg/kg (FB) to al animal groups (Table 1). study was to investigate the in vivo analgesic and anti-inflammatory effects of the optimized Table 1. Animal groups flurbiprofen cyclodextrin-based nanogel formulation in rats. 2. MATERIALS AND METHODS 2.1. Materials Flurbiprofen (FB) was kindly provided by n1: Number of rats used to evaluate the anti-inflammatory effect Sanovel Drug Company (Istanbul, Turkey). n2: Number of rats used to evaluate the analgesic effect A1-A4: Groups for evaluations of anti-inflammatory effect Hydroxypropyl beta cyclodextrin (HPβCD) and B1-B4: Groups for evaluation of analgesic effect Span 80 were purchased from Sigma- Aldrich®. 2.4.1. Evaluation of the anti-inflammatory Al the other chemicals were analytical grade. effect 2.2. Preparation of FB-loaded cyclodextrin- Carrageenan induced rat hind paw edema based nanogel formulation method was used to examine anti-inflammatory effect. Al formulations were applied topical y FB-loaded cyclodextrin-based nanogel to the plantar surface of the hind paw of rats, formulations were prepared via and 100 μl of 1% w/v carrageenan was injected emulsification/solvent evaporation process and into the right hind paw of each rat, 30 min after optimized with Design of Experiment(DoE) administration. Physiologic saline (100 μl/paw) with factorial design. To prepare the FB-loaded was injected into the subplantar tissue of the left cyclodextrin based nanogels, the FB: HPβCD hind paw of each rat. Paw volumes were ratio was determined as 1:1 molar ratio with measured by a plethysmograph at 0, 60, 180, phase solubility studies [2]. The FB content in 240, 360 and 480. minutes and 24 hours after nanogel formulation was adjusted 4% w/w. carrageenan injection. The percentage inhibition of edema was calculated by the 2.3. Preparation of HPMC gel containing fol owing formula: cyclodextrin-based nanogel Inhibition of edema % = [1 (Vt/Vc)]x100 The weighed HPMC K100 LV powder (5% Vt: Rat paw edema volume of the treated groups w/v) was dispersed in the CD-based nanogel Vc: Mean edema volume of the control group 160 P08 2.4.2. Evaluation of the analgesic effect The tail flick method was used to determine of analgesic effect. The time between placing the rat’s tail on the radiant heat source (IR intensity: 75, Cut off time: 10 s) and sharp withdrawal of the tail was noted as reaction time. Tail-flick test was performed before, and at 30, 60 and 180 minutes after drug administration. The percentage analgesic effect was calculated according to the fol owing formula: Figure 2. Latency time in the tail-flick test in the rats. % Analgesic activity = (Observed - Basal reaction time) x 100 *p<0.05 different from control. Values were expressed as Cut off - Basal reaction time Mean±SEM (n=6). 3. RESULTS AND DISCUSSION 4. CONCLUSION PS, PDI and ZP values of optimum nanogel This study demonstrated that the anti- formulation were found 295.5±7.5 nm, inflammatory and analgesic effects of 0.336±0.128, -31.9±0.5 mV; respectively. Al flurbiprofen were increased by FB nanogel in characterization studies of gel formulations HPMC gel formulation compare with FB in (visual examination, pH, drug content analysis, HPMC gel. viscosity measurement, dynamic rheological properties, stability and permeability studies) 5. REFERENCES showed the FB-loaded nanogel in HPMC gel 1. Oktay, A.N., Karakucuk, A., Ilbasmis-Tamer, S., was found bet er than coarse FB in HPMC gel Celebi, N. Dermal flurbiprofen nanosuspensions: (Fig.1). Optimization with design of experiment approach and in vitro evaluation. European Journal of Anti-inflammatory effect studies showed the Pharmaceutical Sciences, 2018. 122: 254-263. lower paw volume was observed in the groups 2. Oktay A.N. Insights to the phase solubility applied with FB-loaded nanogel in HPMC gel diagrams of flurbiprofen with inclusion complex, than those of the coarse powder of FB in HPMC Journal of Research in Pharmacy, 2021. 25(2): 196- gel. FB-loaded nanogel in HPMC gel (20 208. mg/kg) inhibited the development of edema from the 1st hour and its effect proceeded ACKNOWLEDGMENT during the 24 hours with the lowest paw edema This study was supported by Tubitak (Project volume. No:117S149). Figure 1. Effect of different formulations on paw volume in carrageenan-induced paw edema in rats. Values were expressed as mean±SEM (n=6). As results of analgesic effect studies; latency times in the al groups were similar before and 30 min after gel administrations. In the 60 min and 180. min of FB-loaded nanogel in HPMC gel treatment, latency time was found to be significantly prolonged compared to the control group (p<0.05) (Fig.2). 161 P09 COMBINED IN-VITRO DISSOLUTION-/ ENZYMATIC CONVERSION-/ PERMEATION-STUDIES OF THE PRODRUG FOSAMPRENAVIR Jeppe J. Christiansen1, Jonas B. Eriksen1, Jarkko Rautio2, Marika Ruponen2, Annette Bauer-Brandl1, Martin Brandl1 1Department of Physics, Chemistry & Pharmacy, University of Southern Denmark, Odense, Denmark 2School of Pharmacy, University of Eastern Finland, Kuopio, Finland 1. INTRODUCTION 3. RESULTS AND DISCUSSION Prodrugs are pharmacological y inactive 3.1. MDCK II permeability study substances that are converted in the body (e.g. The more water-soluble prodrug fosamprenavir by enzymatic action) into a pharmacological y has the potential of causing a supersaturation of active drugs. amprenavir, when enzymatical y cleaved by In recent years a tendency towards more alkaline phosphatase. The bioconversion of lipophilic, but poorly water-soluble new drug fosamprenavir to amprenavir in the MDCK II molecules has been observed. Various cel s was poor, and a supersaturation of candidate-enabling strategies have been tested amprenavir was not achieved. This resulted in in order to overcome this chal enge. A probate amprenavir solutions permeating more than the strategy used to overcome poor water solubility bioconverted amprenavir from the concentrated is to make a phosphate ester prodrug. Phosphate fosamprenavir solutions (Figure 1). Another esters are charged moieties of enhanced issue was the loss of sink conditions during the solubility. They are hypothesized to be readily experiments, likely due to too leaky MDCK II absorbed upon cleavage by alkaline cel s. phosphatase found in the intestine. 3.2. PermeaPad® permeability study The current study aimed to conduct combined The permeability experiments with artificial in vitro dissolution / enzymatic conversion / set-up showed great potential to fol ow the permeability studies of the prodrug bioconversion rate combined with the fosamprenavir, which by cleavage, turns into permeability study. The system was modulated the parent drug amprenavir, a potent HIV by the amount of alkaline phosphatase added to protease inhibitor. the donor side. A supersaturation of amprenavir was observed in al experiments and caused an 2. MATERIALS AND METHODS increased flux compared to an amprenavir suspension. A human dose dissolution 2.1. MDCK II permeability study permeation experiment was performed with The Permeability studies with amprenavir and fosamprenavir, where the microdialysis showed fosamprenavir were conducted with MDCK II two replicates, where the supersaturation cel s that natural y express the alkaline remained stable over 3 hours, and one col apsed phosphatase. after 2 hours (Figure 2). 2.3. PermeaPad® permeability study Figure 1. The permeability studies were made on an artificial set-up in side-by-side cells with added bovine alkaline phosphatase. In the artificial set-up, the donor and acceptor compartment was separated by the biomimetic barrier PermeaPad®. A microdialysis probe was added to the donor to fol ow the conversion of fosamprenavir to amprenavir. 162 P09 Figure 1. Permeability experiments of amprenavir and 2. Brouwers J, Tack J, Augustijns Patrick. In vitro fosamprenavir converted to amprenavir over MDCK II behavior of a phosphate ester prodrug of amprenavir cells (n=6). in human intestinal fluids and in the caco-2 system: Figure 2. Il ustration of intraluminal supersaturation. Int j pharm. 2007;336: 302–9 ACKNOWLEDGMENT The authors are grateful for a mobility grant to JJC and col aborative work between the two institutions enabled by funding from Nordforsk program Nordic University Hub (project #85352) “NordicPOP”. Figure 2. Human dose in vitro dissolution / enzymatic conversion / permeability study of fosamprenavir with 30 U/mL alkaline phosphatase. The measurements were done with a microdialysis setup. Three replicates are individual y plot ed. Figure 3. Figure 3. Amprenavir suspension measured with a microdialysis setup (n=3). 4. CONCLUSION The side-by-side cel set-up with microdialysis and PermeaPad® showed great potential for permeability studies on prodrugs. The microdialysis system made it easy to follow the bioconversion rate during the permeability studies. The MDCK II permeability studies were not as optimal as the bioconversion rate of fosamprenavir was slower than expected. 5. REFERENCES 1. Stel a VJ, Nti-addae KW. Prodrug strategies to overcome poor water solubility. Advanced drug delivery reviews. 2007;59(7): 677–94. 163 P10 THE INFLUENCE OF PHOSPHATIDYLCHOLINE CONTENT IN HYDROGENATED PHOSPHOLIPID/POLYMER-BASED SOLID DISPERSIONS ON THE DISSOLUTION PROFILE OF A POORLY SOLUBLE DRUG Mikolaj Czajkowski1, Paulina Skupin-Mrugalska1, Bartłomiej Milanowski2, Annette Bauer-Brandl3, Martin Brandl3 1Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Poland 2Department of Pharmaceutical Technology, Poznan University of Medical Sciences, Poland 3Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Denmark 1. INTRODUCTION 2.2. Freeze-drying of binary and ternary Oral formulations remain the preferable way of dispersions administering drugs due to patient compliance, Solid dispersions were prepared by freeze- non-invasiveness, ease of use and safety. drying from water/alcohol solutions. Currently, more than 90% of novel drug entities Formulations were frozen overnight at -82°C are poorly soluble [1]. One strategy employed and placed on a pre-cooled (-60°C) freeze-dryer to overcome this problem is using amorphous shelf. The freeze-drying cycle compromised the solid dispersions (ASD), usual y polymer-based main drying (+25°C, 0.1 mbar) for 24 h and formulations. Novel excipients are stil needed final drying (+50°C, 0.01 mbar) for 3 h. to improve the stability and performance of 2.3. Powder X-ray diffraction (PXRD) ASD. Due to their potential solubilizing ability PXRD (D2 PHASER, Bruker, Bil erica, MA, and stabilizing effect in ASD, phospholipids USA) was performed to characterize the solid- (PLs) seem valuable materials as co-formers of state of obtained binary and ternary dispersions. ASD. Hydrogenated PLs are of interest to be Samples were exposed to Cu Kα radiation and applied. measured within the angular range of 5°-45° The aim of this study was to establish the with 0.02 step size mode and scanning speed of content of saturated PL in ternary solid 10°/min. dispersions of fenofibrate (FEN) in mixed PL/polymer matrices. Additional y, we 2.4. Dissolution test in USP 4 apparatus evaluated whether phosphatidylcholine content (flow-through cell) in the saturated PL may affect the dissolution Dissolution testing was performed in a closed- rate of FEN. loop configuration of a semi-automated flow- through cel dissolution system comprising a 2. MATERIALS AND METHODS SOTAX CE 7 smart unit with a set of seven cel s with 12 mm diameter, a SOTAX CP 7–35 2.1. Materials piston pump and a SOTAX C-613 7 channel Reagents used in this study were of analytical fraction col ector. Each cel was prepared by grade unless stated otherwise. Kol idon VA64 placing a 5-mm ruby bead in the cone's apex, (VA64) was kindly donated by BASF SE and the cone was fil ed with 1-mm glass beads. (Ludwigshafen, Germany). FEN, trifluoracetic The cel was closed with a prepared insert and acid (TFA), sodium hydroxide, sodium filter head containing a microfibre glass filter chloride, sodium phosphate monobasic and a hydrophilic PTFE membrane with a pore monohydrate, sodium phosphate dibasic size of 2.7 and 0.2 µm (Munktel -Munskjö, dihydrate, and tert-butanol were purchased Helsinki, Finland and Omnipore, Sigma- from Sigma-Aldrich (St. Louis, MI, USA). Aldrich, St. Louis, MI), respectively. The Hydrogenated soybean phosphatidylcholine, Phosphate Saline Buffer pH 7.4 was used as i.e., PHOSPHOLIPON P90H (containing >90% dissolution medium, pumped at the rate of 16 hydrogenated soybean phosphatidyl-choline) mL/min. Samples were withdrawn at 5, 10, 15, and LIPOID P 75-3 (P75-3) (with 30, 60, 120, 240, 480, 720, and 1440 min. with 70% phosphatidylcholine) were kindly donated diluted with a twofold volume of methanol, and by Lipoid GmbH (Ludwigshafen, Germany). FEN was quantified by HPLC. 164 P10 3. RESULTS AND DISCUSSION 3.1. PXRD Al binary and ternary formulations obtained by the freeze-drying method were amorphous, with no Bragg peaks present at the X-ray diffractograms. 3.2. Dissolution testing The dissolution profiles of FEN in various formulations were determined in phosphate buffer saline pH 7.4 are presented in Figure 1. A fixed drug loading of 20% (w/w) was used, and the saturated PL (P90H or P75-3) was applied at variable content ranging from 0.5 to 20% (w/w). The dissolution testing showed a synergistic effect of polymer and saturated PL when ternary PL/polymer/FEN dispersions were compared to binary PL/FEN or polymer/FEN dispersions. Also, it was demonstrated that the phosphatidylcholine content in the saturated PLs influences the ability to induce supersaturation. The higher supersaturation concentrations were observed Figure 1. Phospholipid dissolution profiles of FEN when saturated PL with lower from PL/VA64/FEN, VA64/FEN and PL/FEN dispersions with a) P75-3 and b) P90H, obtained using phosphatidylcholine content was used. the USP 4 apparatus. Noteworthy, supersaturation increased up to a critical amount of saturated PL of 2.5% (P75-3) 4. CONCLUSION and 1% (P90H) and subsequently dropped down with the increasing PL content (Figure 1). FEN's amorphous solid dispersions can be We assumed that at higher PL content, polymer obtained by freeze-drying. Ternary matrices and PL molecules form large multilamel ar composed of PL/polymer/FEN have a higher micel es that precipitate and entrap FEN. While ability to induce and maintain supersaturation at lower PL content, upon hydration in the than binary ones. The ability to generate and dissolution media, VA64/PL creates smal er sustain supersaturation increases up to the col oidal species that can pass through the critical content of saturated PL (2.5% for applied filter and be observed as a P75-3, 1% for P90H). The content of supersaturation. phosphatidylcholine in hydrogenated PL influences the supersaturation maintenance ability. 5. REFERENCES 1. Gigliobianco M. R. et al. Pharmaceutics (2018), 10(3), 134 ACKNOWLEDGMENT The authors acknowledge the financial support from the Phospholipid Research Center under grant PSM-2020-085/1-1. 165 P11 DRY POWDER FORMULATION OF CYCLOSPORINE A FOR IMMUNOSUPPRESSIVE TREATMENT Davide D’Angelo1, Eride Quarta1, Stefania Glieca1, Veronica Chierici1, Giada Varacca1, Martina Brandolini2, Lisa Flammini1, Simona Bertoni1, Vittorio Sambri2, Fabio Sonvico1, Ruggero Bettini1, Francesca Buttini1 1Department of Pharmacy, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy 2Microbiology Unit, The Great Romagna Area Hub Laboratory, 47522 Pievesestina, Cesena, Italy 1. INTRODUCTION 2.2.Methods Cyclosporine A (CsA) is a calcineurin inhibitor drug, widely used in the treatment of The inhalation powder was produced by spray autoimmune conditions and in the drying a solution of water and ethanol at 45% immunosuppressive treatment after organ v/v with 1% w/v of solids concentration. The transplantation [1]. This requires the use of high dissolution of the powder was studied by using dosages between 5 and 15 mg/kg/day which can a vertical diffusion cel apparatus cal ed lead to nephrotoxicity and hepatotoxicity. Respicell®. Respirability was studied using Moreover, it is reported that the drug NGI. Each HPMC capsule was fil ed with 20 bioavailability upon oral administration is only mg of CsA and loaded into a dry powder inhaler about 40%. RS01 activated at a flow rate of 60 L/min. Cel viability on A549 and THP-1 was assessed. The The aim of this work was the formulation of a anti-inflammatory effect as reduction of IL-6 dry powder for inhalation containing expression on A549 and THP-1 in co-culture cyclosporine. This formulation was designed was evaluated through the IL-6 ELISA test. The for the prevention of rejection after lung antiviral effect was studied on Vero E6 cel s transplantation and potential y to treat the viral infected with SARS-CoV-2 variant omicron infection Covid-19 [2]. The advantage of this BA.1 at a concentration of 0.005 m.o.i. Cel s formulation is the possibility of reducing the were treated with CsA one hour before dose currently administered oral y, and the infection, two hours post-infection or achievement of a greater local concentration of simultaneously with a viral infection. drug in the lung. 3. RESULTS AND DISCUSSION 2. MATERIALS AND METHODS 3.1.Results and Discussion 2.1.Materials The powder containing 80% CsA and 20% CsA (CAS Number 59865-13-3) was purchased mannitol was efficiently produced through the from Metapharmaceutical (Barcel ona, Spain). spray-drying method and this powder has been HPMC extra-dry capsules for use in dry powder shown to have a higher dissolution rate than raw inhalers, Quali-V®-I size #3, were provided by material. Moreover, this powder presented a Qualicaps (Madrid, ES) and a single dose dry FPF of 83.28± 0.69 and an MMAD of 2.5 µm. powder inhaler RS01 high resistant was gifted No significant cytotoxic effect was observed by Plastiape (Lecco, IT). from in vitro studies, moreover, a powerful anti- Mannitol was purchased from Roquet e anti-inflammatory effect was observed on cel (Lestrem, France), glycine was purchased from co-culture. In this study, it was also Sigma Aldrich (St Louis, MO, USA). Al other demonstrated a significant antiviral effect chemicals used were obtained from commercial against the omicron BA-1 variant of SARS- suppliers and were of analytical grade. The human lung adenocarcinoma cel line A549, monocytic cel line THP-1 and VERO E6 cel s were purchased from the American Type Culture Col ection. 166 P11 CoV-2, in particular, during pretreatment and simultaneous treatment to infection (Figure 1). Pretreatment Post-treatment Simultaneous treatment Pretreatment Post-treatment Simultaneous treatment ٓ ٓ ٓ ٓ ٓ ٓ ٓٓ 100 eduction ٓ R 50 0 iralInfectivityV% 64μM 32μM 16μM 8μM 64μM 32μM 16μM 8μM 64μM 32μM 16μM 8μM M20 CsA raw Mannitol Figure 1. Viral concentration 0.005 m.o.i.; Cel line: Vero E6; Pretreatment: one hour before infection; Post-treatment: two hours post infection; Samples suspended in PBS. *P <0.05 vs CsA raw. 4. CONCLUSION Due to its technological characteristics and encouraging in vitro results, this new inhalable CsA dry powder formulation could represent an innovative strategy, not only in the prevention of transplant rejection but potential y also in the treatment of patients with covid-19. 5. REFERENCES 1. Fahr A Cyclosporin Clinical Pharmacokinetics. Clinical Pharmacokinet 1993. 24:472–495 2. Molyvdas A, Matalon S,Cyclosporine: an old weapon in the fight against coronaviruses. European Respiratory Journal 2020. 56:2002484 167 P12 DEVELOPMENT AND IN VITRO-IN VIVO EVALUATION OF GLYCYRRETINIC ACID ACTIVE TARGETED PROLIPOSOMAL DRUG DELİVERY SYSTEMS FOR TREATMENT LIVER CANCER Mine DİRİL1, K. Volkan Ozdokur 2 , Yeliz Yıldırım 3 , H. Yeşim Karasulu 1 1Department of Pharmaceutical Technology, Faculty of Pharmacy, Ege University, Izmir, TURKEY 2Department of Chemistry, Faculty of Science and Art, Erzincan Binali Yıldırım University ,Erzincan, TURKEY 3Department of Physicochemistry, Faculty of Science, Ege University, Izmir, TURKEY 1. INTRODUCTION rate: 1 mL/min; Colon temperature: 25°C; Liver cancer is the fifth most common cancer in Wavelenght: 254 nm. men and the ninth most common cancer in 2.3. DSPE-PEG 2000- Glycerretinic acid Synthesis women, and the survival rate in the last five GA and DSPE-PEG2,000-NHS at a molar ratio years has been reported to be less than 9% (1). of 3:1 was dissolved in dimethyl sulfoxide in the Doxorubicin Hydrochloride (Dox-HCl) , one of presence of 1-Ethyl-3-(3- the active ingredients used in cancer treatment, dimethylaminopropyl) carbodi mide (1.58 mg) is an anthracycline antibiotic that acts on the cel and 20 μL triethylamine was added to the by interfering between DNA base pairs ant it reaction system to regulate pH. has many side effects (2). For this reason, it is very important to develop a drug delivery 2.4. Development Pegylated Active-Targeted system that can eliminate these side effects. Proliposome Formulation Studies have shown that Glycyrretinic acid Lipid hydration method was used for liposome (GA), which is an active targeting molecule, formulation. Different drug:lipid ratio was tried stops cel cycle against hepatocel ular for develop liposomal drug delivery systems. carcinoma (3). The primary goal of this study is For proliposome formulation, different amounts to reduce the toxic effect of Doks-HCl in of mannitol was used lyophilization step. healthy tissues by providing active targeting by 2.5. In vitro Studies of Pegylated Active- conjugation of glycyrrhetinic acid to the PEG Targeted Proliposome Formulation molecule. Liposomal drug delivery systems are In vitro characterization studies (pH, particle composed of phospholipids, which are the main size, zeta potential, viscosity, encapsulation, components of the body, however, it is drug loading, humidity, time to disperse), in relatively unstable col oidal systems(4). vitro release studies, cytotoxicity studies, Another goal of this study is to prevent the stability studies, drug permeability studies were stability problems of lipids in liposomal drug performed. delivery systems. For this reason develop proliposomal drug delivery systems by 2.6. In vivo studies lyophilizing liposomal formulation. Liver cancer was developed on CD-1 nude mice with Hep G2 cel lines for the efficiency studies. 2. MATERIALS AND METHODS Comparative IVIS images were taken in the 2.1. Materials negative control group, Caleyx-administered Doxorubicin Hydrochloride supplied by Deva group and pegylated active targeted İlaç. All other excipient are pharm grade. proliposome formulation-administered group. Caelyx® pegylated solution for infusion 3. RESULTS AND DISCUSSION (Schering plough /ABD) was used as the reference drug. 3.1 HPLC validation of Doxorubicin Al results were found to comply with the 2.2. HPLC validation of Doxorubicin criteria given in the ICH guide. The fol owing system was used for the validation of Dox-HCl; Dedector: UV; Mobile 3.2. DSPE-PEG 2000- Glycerretinic acid phase: Acetonitrile- 0.01 M (sodium lauryl Synthesis sulfate pH adjusted to 2.5) (50:50); Colon: GA and DSPE-PEG2,000-NHS conjugation Eclipse Plus C18, 4.6 x 250mm, 5um; Flow was carried out successful y. 168 P12 3.3. Development Pegylated Active-Targeted Table 1. In vitro characterisation results Proliposome Formulation The highest encapsulation capacity was found Pegylated active- in the formulation with a drug:lipid ratio of 0.4. targeted, proliposome The use of 7% mannitol was provided effective formulation drying and cake formation. pH 6.5 3.4. In vitro Studies of Pegylated Active- Particle size (nm) 120 Targeted Proliposome Formulation Zeta potential (mV) 9.57 Pegylated active-targeted proliposome formulation characterisation results were Polydispersity index 0.36 shown Viscosity (cp) 11.3 Encapsulation (%) 90.0 . In vitro release studies were shown Figure 4. IC50 values of Hep G2, Mahlavu and Huh 7 cel Drug Loading(%) 100.0 lines, Dox-HCl and pegylated active-targeted Humidity (%) 0.0019 proliposome formulation were found 6.5 µM. Time to disperse (sec) 10 3.5. In vivo studies Liver cancer model has been successful y developed. Efficiency studies were shown Error! Reference source not found. IVIS images were taken at the beginning, after the first dose treatment and after the second dose treatment. After the first dose treatment of pegylated active-targeted proliposome formulation, a significant reduction in tumor was observed. Figure 4. In vitro release studies 4. CONCLUSION Obtained results indicated that; pegylated active-targeted proliposome formulation containing Dox-HCl has been shown to be promising in the treatment of liver cancer in future studies. Negative control group Caleyx®- group Active targeted proliposome formulation-group Figure 5. Efficiency studies-IVIS 5. REFERENCES 1. Schlachterman, A., Craft, W., et al . World J Gastroenterol, 2015, 21 (28): 8478-8491 2. Hanna, A., Lam, A., at al . Adverse Effects of Doxorubicin and Its Metabolic Product on Cardiac RyR2 and SERCA2A, 2014, Mol Pharmacol 86: 438–449. 3. Cao, M., Gao, Y., et al . Glycyrrhizin Acid and Glycyrrhetinic Acid Modified Polyethyleneimine for Targeted DNA Delivery to Hepatocel ular Carcinoma, 2019, Int. J. Mol. Sci, 20, 5074. 4. Hiral, M., Bijal, P., Rakesh, P. Review of Preliposomes as novel drug delivery system. 2015, Pha In J, 4(3), 61-67. 169 P13 A PROPOSAL OF INNOVATIVE INJECTABILITY ASSESMENT METHOD FOR INTRAVENIOUS FORMULATIONS – CASE STUDY ON PEGYLATED NANOEMULSIONS Jelena Đoković1, Sanela Savić2, Nebojša Cekić2,3, Snežana Savić1 1Department of Pharmaceutical Technology and Cosmetology, Faculty of pharmacy, University of Belgrade, Serbia 2DCP Hemigal, Serbia 3Department of Pharmaceutical Technology and Cosmetology, Faculty of Technology, University of Niš, Serbia 1. INTRODUCTION the PEGylated ones were marked S1, S3 and S6, Syringeability and injectability are recognised referring to the PEG2000-DSPE concentration. as fundamental performance parameters / 2.2. Physicochemical characterization critical quality at ributes of any parenteral The NEs droplet size (Z-Ave) and droplet size dosage form. Syringeability refers to the ability distribution (PDI) were determined with of an injectable preparation to transfer from a Zetasizer Nano ZS90 (Malvern Instruments vial through a hypodermic needle prior an Ltd., Worcestershire). Rheological analysis was injection, while injectability is defined as the performed using MCR 302 air-bearing force, or pressure, required to inject the rheometer (Anton Paar, Graz, Austria) equipped formulation from a syringe-needle system into with coaxial cylinders system (CC27 measuring the tissue [1]. When developing drug delivery bob with C-PTD 180/Air) with sheer rate range systems, the priority is usual y the release of 0.1-100 s-1 at 20°C. kinetics, biocompatibility or other factors that may come in conflict with the optimal 2.3. Injectabilty assesment parameters for the applicability of those The injectability of the NEs was expressed as systems [2]. The aim of this research was to force (N) needed to extrude the NE in the develop a method that could be used for function of the extruded volume (ml). About 10 injectability assessment of the intravenous ml of the NE was loaded into the 10 ml syringe formulations and the application of this method and extruded through the 25 G scalp vein on curcumin-loaded PEGylated nanoemulsions infusion set (Romed, Wilnis, Netherlands) into (NEs) in order to gage the impact of PEGylation the blood mimicking solution, circulating on NEs injectability. through pump at 4 ml/min, in order to assess the NEs’ performance in the prospective 2. MATERIALS AND METHODS intravenous administration. The NEs were extruded at 1 mm/s croshead speed of the 2.1. Nanoemulsion preparation loading cel of the texure analyzer (EZ-LX Nanoemulsions were prepared using high Compact Table-Top Testing Machine, pressure homogenization method. The aqueous Shimadzu, Japan) with the Trapezium software phase (glycerol, polysorbate 80, sodium oleate version 1.5 used for data col ection and analysis and highly purified water) was added into the oil phase (soybean oil, soybean lecithin, 3. RESULTS AND DISCUSSION medium chain triglycerides, butylhydroxytoluene, benzyl alcohol, curcumin 3.1. Physicochemical characterization and PEGylated phospholipid – PEG2000-DSPE The NEs have average size of about 100 nm, in 0.1 %, 0.3 % or 0.6 % concentrations) and with the PDI values below 0.20, indicating mixed using rotor-stator homogenizer (IKA suitability for intravenous application. It could Ultra-Turrax® T25 digital, IKA®-Werke be observed from Fig. 1 that the addition of GmbH and Co. KG, Staufen, Germany), and PEGylated phospholipids caused an increase in further processed on high pressure homogenizer NE viscosity, as could be expected given that (EmulsiFlex-C3, Avestin Inc., Canada) at 800 the polyethylene glycols are used in parenteral bar for 10 discontinued cycles. The non suspensions as stabilizing - rheology modifying PEGylated formulation was marked as CS, and agents [3]. 170 P13 4. CONCLUSION The injectability method used in this research proved as a useful tool in screening formulations adequate for prospective intravenous use. 5. REFERENCES 1. Cilurzo, F., et al. Injectability Evaluation: An Open Issue. AAPS PharmSciTech, 2011. 12(2): 604-609. 2. Sarmadi, M., et al. Modeling, design, and machine learning-based framework for optimal injectability of microparticle-based drug Figure 1. NE viscosity formulations. Science advances, 2020. 6: eabb6594. 3.2. Injectability assessment 3. Gul apal i, R. P., Mazzitel i, C. L. Polyethylene The injectability assessment was performed glycols in oral and parenteral formulations—A with syringe-needle system used in our critical review. International Journal of laboratory for intravenous administration in in Pharmaceutics, 2015. 496(2): 219-239. vivo animal studies. As blood-mimicking 4. Yousif, M. Y., et al. Deriving a blood- solution, 36.6 %, v/v, glycerol solution was mimicking fluid for particle image velocimetry used [4]. It could be observed from Fig. 2 that in Sylgard-184 vascular models. In Annual the injectability of NEs depended on their International Conference of the IEEE Engineering in Medicine and Biology Society, viscosity, with the higher pressure needed to 2009 (pp. 1412-1415 extrude the formulations with the higher 5. Wat , R. P., et al. (2019). Injectability as a PEG2000-DSPE concentration. Even though, to function of viscosity and dosing materials for the best of our knowledge, there are no studies subcutaneous administration. International investigating the injectability of the intravenous Journal of Pharmaceutics, 2019:554, 376-386. preparations, based on some previous research ACKNOWLEDGMENT on subcutaneous model [5], it is recommended the maximum force used to inject the This research was funded by the MESDT, formulations should be kept about 20 N, which Republic of Serbia through Grant Agreement would eliminate S3 and S6 from further with University of Belgrade-Faculty of investigation (Fig. 2). Pharmacy No: 451-03-68/2022-14/200161 and supported by the Science Fund of the Republic of Serbia, GRANT No 7749108 , Neuroimmune aspects of mood, anxiety and cognitive effects of leads/drug candidates acting at GABAA and/or sigma-2 receptors: In vitro/in vivo delineation by nano- and hiPSC-based platform - NanoCellEmоCog. Figure 2. NE injectability 171 P14 ELECTROSPINNING AS A DRYING METHOD FOR MAGNETIC NANOPARTICLE DISPERSIONS Črt Dragar1, Sebastjan Nemec1,2, Slavko Kralj1,2,3, Petra Kocbek1 1University of Ljubljana, Faculty of Pharmacy, Department of Pharmaceutical Technology, Slovenia 2Jožef Stefan Institute, Department for Materials Synthesis, Slovenia 3Nanos SCI, Nanos Scientificae d.o.o., Slovenia 1. INTRODUCTION polymers, namely poloxamer 188 and With their unique chemical, physical, and polyethylene oxide in weight ratio 1:1, in the biological properties iron-oxide-based magnetic initial ethanol-based MNP dispersion. nanoparticles (MNPs) are believed to be one of Table 1. Composition of MNP dispersions for the most promising novel drug delivery systems drying. [1]. However, their long-term physical stability Formulatio MNP Polymer Ethanol in dispersions stil represents an important n s [mg] s [mg] * [g] technological chal enge [2]. To improve the F0 0 240 8 physical stability of nanoparticulate F1 60 240 8 dispersions, the particle surface modifications F2 120 240 8 or the addition of stabilizers have been F3 180 240 8 investigated, but it was shown that such F4 240 240 8 approaches may strongly affect the properties of F5 300 240 8 MNPs [2]. Therefore, the methods for the F6 360 240 8 transformation of MNP dispersions into stable, redispersible dry products have been intensively *The ethanol used was 96% (v/v). investigated in the last two decades. However, 2.3. Characterization of Electrospun the currently available methods usual y require Products relatively large amounts of excipients to The electrospun products were characterized preserve MNP size and result in fluffy regarding the morphology (scanning electron powdered products, which can provoke adverse microscopy, SEM), the internal structure health effects in humans, if inhaled (transmission electron microscopy, TEM), and unintentional y [3]. The aim of our study was MNP content (thermogravimetric analysis, thus to adopt the electrospinning method for the TGA). The electrospun products were transformation of MNP dispersions into a dry reconstituted in 0.9% (w/v) NaCl (~1 mg/mL) non-powdered product, which would enable the by vortex mixing for 3 min. The MNP rapid and simple reconstitution of MNPs. hydrodynamic size and particle size distribution were measured by photon correlation 2. MATERIALS AND METHODS spectroscopy, whereas MNP zeta potential was 2.1. Materials measured by laser Doppler anemometry. Al materials used were of reagent grade and 3. RESULTS AND DISCUSSION from commercial sources. The initial ethanol- based MNP dispersions (iNANOvative™) were MNPs used in our study were composed of self- kindly provided by Nanos Scientificae Ltd. assembled multiple 10 nm superparamagnetic (Nanos SCI, Ljubljana, Slovenia). iron oxide nanocrystals. The nanocrystal clusters were coated with a 10 nm thick silica 2.2. Drying of Magnetic Nanoparticle shel as previously reported [4]. Their mean Dispersions by Electrospinning hydrodynamic size in the initial dispersion was MNP dispersions with different amounts of (356 ± 9) nm. MNPs (Table 1) were dried using the electrospinning device in the horizontal set ing The electrospinning enabled the formation of (temperature, 25 °C; relative humidity, ≤ 35%; the dry products with the nanofiber morphology voltage, 15 kV; flow rate, 1.77 mL/h; col ector- as revealed by the SEM analyses (Fig. 1). The needle distance, 15 cm). The MNP dispersions TEM analyses confirmed that MNPs have been for drying were prepared by dissolution of 172 P14 successful y incorporated into the polymer matrix of nanofibers (Fig. 2). Figure 3. Hydrodynamic size of MNPs after the reconstitution in 0.9% (w/v) NaCl from formulations F1-F6 and the hydrodynamic size of MNPs in the initial dispersion (MNPs/e) and initial dispersion diluted with 0.9% (w/v) NaCl (MNPs/s). 4. CONCLUSION Figure 1. Representative SEM images of the We have established electrospinning as an electrospun product with 50% (w/w) of MNPs efficient method for the transformation of MNP (formulation F4) at lower (top) and higher dispersions into redispersible dry products, (bot om) magnification. which can contain up to 50% (w/w) of MNPs. The electrospun product in a form of a nanofiber mat prevents unintentional MNP aerosolization and thus ambient exposure to MNPs. The excipients used in the formulation, i.e., hydrophilic polymers, enable simple and rapid reconstitution of MNPs just prior to application, insignificantly contribute to the tonicity of the obtained dispersion, and improve MNP stability Figure 2. Representative TEM image of the in 0.9% (w/v) NaCl. electrospun product with 50% (w/w) of MNPs (formulation F4). 5. REFERENCES Additional y, the TGA revealed no losses of 1. Dragar, Č., et al., Bioevaluation methods for MNPs during electrospinning and homogenous iron-oxide-based magnetic nanoparticles. distribution of MNPs in the electrospun International Journal of Pharmaceutics, 2021. products. Based on the hydrodynamic size of 597: 120348. MNPs after the reconstitution, we can conclude 2. Ong, H.T., et al., Fatty acid coated iron oxide nanoparticle: Effect on stability, particle size that the maximal MNP loading in the and magnetic properties. Col oids and Surfaces electrospun product, which stil enables A: Physicochemical and Engineering Aspects, efficient reconstitution and preserved size of 2020. 606: 125371. MNPs, is 50% (w/w) (Fig. 3, formulation F4). 3. Valdiglesias, V., et al., Are iron oxide The hydrodynamic size of MNPs after the nanoparticles safe? Current knowledge and reconstitution from formulations F1-F4 in 0.9% future perspectives. Journal of Trace Elements (w/v) NaCl changed only slightly compared to in Medicine and Biology, 2016. 38: 52-63. the MNPs in the initial dispersion (MNPs/e), 4. Kralj, S., Makovec, D., Magnetic assembly of indicating the suitability of the technological superparamagnetic iron oxide nanoparticle procedure to preserve the particle size also in clusters into nanochains and nanobundles. ACS Nano, 2015. 9: 9700-9707. the presence of salts in the dispersion medium. ACKNOWLEDGMENT The authors grateful y acknowledge the financial support provided by the Slovenian Research Agency (Program Codes P1-0189, P1-0420, and P2-0089 and Projects J2-3043, J2- 3040, J2-3046, J3-3079, and J1-7302). 173 P15 FORMULATION AND INVESTIGATION OF THE EFFECT OF POLYMERS ON DERMAL FOAM PROPERTIES USING THE QUALITY BY DESIGN (QbD) APPROACH Fanni Falusi1, Szilvia Berkó1, Mária Budai-Szűcs1, Anita Kovács1 1University of Szeged, Institute of Pharmaceutical Technology and Regulatory Affairs, Hungary 1. INTRODUCTION Dermal foams are promising drug delivery systems due to their advantages and ease of 2.2. The QbD methodology application. In particular, they are beneficial for The QbD is a science-based and risk-managed the treatment of skin conditions when patients approach to drug development that starts with have highly inflamed and sensitive skin, as the pre-defined goals. The CQAs were defined with application of the foam minimizes the need for the consideration of the at ributes of the liquid skin contact [1]. system and the foam system. After the Foams provide an innovative, easy to apply, determination of CQAs, the fol owing step is to novel alternative for creams and ointments, determine the critical material at ributes and the hence there are stil few test methods in the critical process parameters of the foams with literature. For this reason, our research aimed to risk assessment. develop stable foam formulations containing different types of polymers, determine the 2.3. Preparation of foams proper methods to investigate their Based on the results of the initial risk physicochemical and structural properties and assessment, the polymer concentration and type compare the results of different methods. To were critical material at ributes. Therefore, the ensure quality-based development, the QbD effect of five different polymers on the foam approach was applied. The QbD concept structure has been investigated. The involves identifying the quality target product formulations contain 2 different concentrations profile (QTTP), the critical material at ributes of xanthan gum, hydroxyethyl cel ulose (HEC), (CMAs), and critical process parameters (CPPs) low molecular weight hyaluronic acid (LMW- into the critical quality at ributes (CQAs) of HA), high molecular weight hyaluronic acid dermal foam at the beginning of the (HMW-HA), and cross-linked hyaluronic acid development [2]. (CL-(Phase B). The foaming agents are mainly surfactants (Phase A). Al formulations contain 2. MATERIALS AND METHODS the same amount of surfactants (Phase B). Phase 2.1. Materials C contains the microbiological preservative. The bulk liquid is prepared by mixing Phase A, Kol iphor RH 40 was obtained from BASF SE B, and C. To produce the foam, the bulk liquid Chemtrade GmbH (Ludwigshafen, Germany). is stirred at 2000 rpm for 5 minutes. Labrasol ALF was from Gat efossé (Saint- Priest Cedex, France), Xantural® 180 was 2.4 Investigation of foam properties provided by CP Kelco A Huber Company (Atlanta, GA, USA). Verstatil PC was Through the determination of macroscopic purchased from Biesterfeld GmbH (Hamburg, properties, information on the stability of foam Germany). Hydroxyethylcel ulose (Ph. Eur. 9.) formulations can be acquired. With a light was supplied by Molar Chemicals Ltd. microscope, the stability of foams, as wel as the (Budapest, Hungary). Purified and deionized kinetics of the destabilization mechanism was water was used (Mil i-Q system, Mil ipore, analyzed. Rheological measurements could Milford, MA, USA). HyaCare50, HyaCare detect deformations in the structure of the foam Fil er CL and HyaCare Tremel a were product due to different forces. The dermal application samples from Evonik Industries AG (Essen, of foam could be modeled with a texture North Rhine-Westphalia, Germany). analyzer. 174 P15 3. RESULTS AND DISCUSSION is higher than the viscous one, and the moduli are constant up to higher strain values, 3.1. Initial risk assessment indicating a more stable coherent structure. The The QTPP of the foams includes the route of administration, dosage form, site of action, the other type of rheological behavior of the foam is when G” dominates over G’ in the entire appearance of the drug delivery system, strain range, and a crossover point cannot be stability of the liquid system and the foam detected. These preparations did not form a real system, appearance of the liquid and the foam system, and the polymer content of the foam for coherent foam structure. stability. The CQAs were defined with the Spreadability consideration of the at ributes of the liquid system and the foam formed, too. In the case of On the basis of the results, in general, the risk assessment quality management tools have polymer content improved the firmness of the been used (e.g. Ishikawa diagram, Pareto foam, which would prevent the formulation analysis, risk estimate matrix, etc.). from flowing off the skin. Our results indicate that xanthan gum-containing and HMW-HA- 3.2. Investigation of foam properties containing foams met the pre-established Macroscopic properties requirement and correlated with the results of The fol owing parameters can be determined the macroscopic investigations. through the macroscopic investigations: foam 4. CONCLUSION expansion (FE, %), foam volume stability (FVS, %), relative foam density (RFD). In the In summary, the polymer content has a great tested formulations, foams containing xanthan effect on the properties of the foams. The gum and HMW-HA had the highest stability but chosen methods reinforce each other and help to the lowest foam expansion values. Among the select a formula for dermal application. Based tested formulations, the wel -foaming on our results, formulations containing xanthan composition were foams with an FE-value gum and HMW-HA had good foam properties above 100 % and the most stable formulations and wil be appropriate delivery systems for an were the formulations with FVS-value above active pharmaceutical ingredient. The results of 70 %. the different methods showed good correlation and can be used in preformulation studies to Microscopic properties select the optimal formulation. Al foam systems were polydisperse. The 5. REFERENCES kinetics of foam destabilization was also observed with microscopic examinations (Fig. 1. Parsa et al., Foam in pharmaceutical and 1.). The microscopic pictures showed that the medical applications, Current Opinion in increasing size of the bubbles leads to the Col oid&Interface Science 44 (2019) 2. F.Falusi, et al., Investigation of the effect of destabilization of the foam over time. Foams polymers on dermal foam properties using the with an initial bubble count above 100 were QbD approach. European Journal of microscopical y stable. Pharmaceutical Sciences 173 (2022). ACKNOWLEDGMENT The research was supported by the Gedeon Richter Plc. Centennial Foundation (Gyömrői 19-21, Budapest, H-1103, HU). Project no. TKP2021-EGA-32 has been implemented with the support provided by the Ministry of Figure 1. The analysis of foam kinetics through Innovation and Technology of Hungary from microscopic images. the National Research, Development and Innovation Fund, financed under the TKP2021- Rheology EGA funding scheme. Two typical amplitude sweep curves can be distinguished: one which represents a wider linear viscoelastic envelope, the elastic modulus 175 P16 FORMULATION OF NANOPARTICLES CONTAINING CASEIN KINASE 2 INHIBITOR FOR THE THERAPY OF GLIOBLASTOMA Pálma Fehér1, Marc Le Borgne2, Florent Perret2, Christelle Marminon2, Zoltán Ujhelyi1, Ágota Pető1, Liza Józsa1, Miklós Vecsernyés1, Ildikó Bácskay1,3 1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Debrecen 2Chemistry Department, Faculty of Pharmacy, University of Claude Bernard Lyon 3Institute of Healthcare Industry, University of Debrecen 1. INTRODUCTION method using a Büchi Encapsulator B-395 Pro apparatus. CK 2 inhibitor was finely distributed Glioblastoma (GBM) is the most common in 40 mL of 1.50% sodium-alginate solution. primary central nervous system tumour in The polymer–pharmacon mixture was forced adults, represents about 15% of al primary into the pulsation chamber by a syringe pump at brain tumours. The limited and less-effective speed 5.00 mL/min. The alginate beads were treatment options for these highly aggressive left to harden for 15 min in calcium-chloride GBMs cal for the development of new solution. The finely divided particles were techniques and the improvement of existing washed with distil ed water and filtered on a 0.4 technologies [1,2]. Casein kinase 2 (CK2) is µm pore size membrane by a vacuum pump and overexpressed in GBM samples. Recent freeze dried for 24 h at −110 ◦C [4]. evidence suggests CK2 inhibitor is a promising therapeutic target for GBM. CK2 inhibition was 2.3. Encapsulation efficiency shown to decrease cel migration and adhesion, To determine the encapsulated drug content in increase cel ular apoptosis and inhibit tumour the beads, a 1 mL sample was measured from growth in GBM cel s [3]. 100 mM of the calcium-chloride hardening The aim of research was the formulation and solution right after formulation. Drug investigation of microcapsules containing a CK concentration was determined by 2 inhibitor. spectrophotometer. 2. MATERIALS AND METHODS 2.4. Determination of Mean particle size and Zeta potential 2.1. Materials The nanoparticle size and Zeta potential of the CK 2 inhibitor was synthesized by the Faculty formulations were determined by Malvern of Pharmacy, University of Claude Bernard Zetasizer Nano S instrument. Lyon. Sodium alginate was obtained from BÜCHI Labortechnik AG (Flawil, 2.5. In vitro dissolution test Switzerland). Calcium chloride dehydrate, Erweka DT800 equipment was used for the ninety-six-wel cel plates was purchased from experiment. Rotating paddle method was VWR International (Debrecen, Hungary). chosen, (75 rpm and 900 ml dissolution media) Caco-2 cel line was originated from the Samples were taken at predetermined intervals. European Col ection of Cel Cultures (ECACC, Duration: 24 h. Absorbance of dissolved CK2- Public Health England, Salisbury, UK). inhibitor was measured at 420 nm (UV-VIS, 2.2. Preparation of CK2 inhibitor- loaded Shimadzu UV-1601). alginate beads The CK2 inhibitor-loaded alginate nanobeads 2.6. Cell Viability Assay were prepared by a control ed polymerization Caco-2 cel viability was evaluated using the MTT method. Cel s were harvested and seeded 176 P16 at a density of 104 Cel s/wel on flat-bottom 96- wel tissue culture plates. Cel s were al owed to grow for 7 days. Mitochondrial activity of the cel s was determined after 3 h incubation with MTT at the concentration of 0.50 mg/ml. Dark blue formazan crystals were dissolved in acidic isopropanol. Absorbance was measured with a FLUOstar OPTIMA micro-plate reader at 570 nm against a 690 nm reference. Figure 2. Results of MTT test 3. RESULTS AND DISCUSSION 4. CONCLUSION 3.1. Encapsulation efficiency Microencapsulation of CK2 inhibitor offers the possibility of releasing therapeutic agents to The encapsulation efficiency measurements targeted areas of the brain, which could provide resulted in at least 64±0.72 %. new directions for the treatment of neurological diseases such as GBM. 3.2. Mean Particle Size and Zeta potential 5. REFERENCES Table 1. Particle size and zeta potential of CK2 1. Marc Fakhoury, Drug delivery approaches for the inhibitor beads treatment of glioblastoma multiforme Artificial Mean Particle size 345.8± 6.02 Cel s, Nanomedicine, and Biotechnology, 2016, (±SD) nm 44(6):1365 –1373. Zeta potential -23.6 ±5.04 mV 2. Justin S. Michael Et Al, Nanotechnology for treatment of glioblastoma multiforme, Journal Of Translational Internal Medicine, 2018, 6 (3):128-3.3. Results of in vitro dissolution test 133. 3. Haitao Ji et al, The Role of Protein Kinase CK2 in Glioblastoma Development, 2013, 19(23):6335-6337 4. Yu-Tsai Yang et al., Preparation of alginate beads containing a prodrug of Diethylenetriaminepentaacetic Acid, Carbohydrate Polymers, 2013, 92(2):1915-1920 ACKNOWLEDGMENT Figure 1. In vitro dissolution profile of CK2 2019-2.1.11-TÉT-2020-00098 „Formulation of inhibitor beads Casein Kinase 2 inhibitor nanoparticles in glioblastoma brain tumor therapy” 3.4. MTT test “Project no. TKP2021-EGA-18 has been According to the results of MTT test the implemented with the support provided from samples were safe in the applied concentrations. the National Research, Development and Cel viability is demonstrated as the percentage Innovation Fund of Hungary, financed under of the untreated control. the TKP2021-EGA funding scheme.” 177 P17 THE INFLUENCE OF SIMULATED WATER GASTRIC EMPTYING PROFILES ON DISSOLUTION OF MODEL DRUGS Felicijan Tjaša1, Rede Katarina1, Cof Greta1, Bogataj Marija1 1Department of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION basal medium volume in the working vessel (i.e. Different and highly variable physiological 35 mL) was reached. factors can affect formulation / drug 2.2. Tablet manufacture performance in the gastrointestinal tract and 400 mg tablets were manual y compressed at influence drug absorption in the smal intestine. the University of Ljubljana, Faculty of Especial y of great importance are properties of Pharmacy, using 12 mm flat-faced punches and the fluids throughout the GI tract: their were composed of: drug (25 %; paracetamol composition (buffer species and concentrations, (PAR) or diclofenac sodium (DF-Na) or presence of solubilizing components), buffer dipyridamole (DPL)), lactose monohydrate capacity, volumes, pH values, viscosity, and (66.5 %), HPMC SB-4 (7.5 %) and Mg-stearate hydrodynamics, especial y gastric emptying (1 %). (GE) [1]. 2.3. Dissolution experiments The study aimed to simulate different fasted Dissolution experiments were performed using water GE profiles in an in vitro dissolution the glass-bead flow-through dissolution system system and assess their influence on dissolution under the conditions of the selected simulated of three model drugs. GE profiles. The experiments began by placing a weighed tablet into the working vessel 2. MATERIALS AND METHODS containing a mixture of HCl and water. 2.1. Selection of the GE profiles Samples, pumped out of the vessel, were Two distinct individual in vivo GE profiles after col ected in different time intervals, filtered ingestion of 240 mL of water were selected through 0.45 µm RC filters, diluted and the from the literature [2], that represented slow GE concentration of dissolved drug was measured (subject 8) and fast GE kinetics (subject 13). using the UV-Vis spectrophotometer. 2.2 Simulation of GE profiles using the glass- 2.4. pH measurements in the vessel bead flow-through system In separate experiments, pH in the working Using the in vitro glass-bead flow-through vessel during simulation of GE profiles with system [3], the selected in vivo GE profiles were and without tablet addition was also measured. simulated. In the beginning, 35 mL of 0.001 M 3. RESULTS AND DISCUSSION HCl and 240 mL of water were combined and added to the working vessel. Fresh 0.001 M HCl 3.1. In vitro simulation of the selected GE was constantly delivered into the vessel with a profiles flow rate of 2 mL/min. 25 g of glass beads With some adjustments compared to in vivo (diameter 1 mm) and a magnetic stirring bar values of gastric volumes, both GE profiles were added to the vessel. The medium in the were successful y and reproducibly simulated in working vessel was heated to 36.5 °C and the in vitro flow-through system. In vivo and in stirred at 50 rpm. The volume of the medium in vitro simulated fast and slow GE profiles are the working vessel was changing throughout the presented in Figure 1. experiment to resemble the in vivo gastric volumes. The flow rates through the tube, that pumped the medium out of the vessel, were accordingly altered to cause suitable change in the medium volume in the working vessel. Simulation of GE profiles proceeded until a 178 P17 not alter pH in the vessel. Both, DPL and DF- Na dissolution increased medium pH for a certain time. Figure 1. The individual in vivo gastric fluid volumes after ingestion of 240 mL of water (red lines, [2]) and simulated in vitro GE profiles in the flow-through dissolution system (black lines). Figure 3. pH profiles during simulation of slow (ful 3.2. Dissolution results lines) and fast (dashed lines) GE profiles without or Dissolution experiments were performed with with tablets (PAR, DF-Na or DPL). tablets containing PAR, DF-Na or DPL. Differences in the amount of dissolved drug can pH changes were the greatest in the case of DF- be seen for tablets containing DF-Na or PAR Na and affected its dissolution. High pH value when two different GE profiles were simulated at the beginning increased dissolution, whereas (Figure 2). decreasing the pH back to pH 3 prevented the dissolution of the whole dose due to lower solubility of diclofenac at pH 3. 4. CONCLUSION Simulation of GE profiles after ingestion of 240 mL of water was performed in the in vitro glass-bead flow-through system. The effect of different GE rates on dissolution was observed for tablets with PAR and DF-Na, but not for DPL. The results show, that the effect of water ingestion and its GE on dissolution could depend on drug physico-chemical properties, Figure 2. Dissolved drug (%) from tablets containing especial y its solubility and dissolution rate. PAR (blue lines), DF-Na (red lines) or DPL (green lines) in experiments with simulated slow GE profile 5. REFERENCES (full lines) or fast GE profile (dashed lines). 1. Mudie D.M., et al., Physiological parameters for The simulation of a fast GE profile resulted in a oral delivery and in vitro testing. Molecular faster dissolution and/or higher percent of the Pharmaceutics, 2010. 7(5): 1388-1405 dissolved drug for DF-Na and PAR tablets 2. Mudie D.M., et al., Quantification of gastrointestinal liquid volumes and distribution compared to a slow GE profile. For tablets fol owing a 240 mL dose of water in the fasted containing DPL, the GE profile did not affect state. Molecular Pharmaceutics, 2014. 11(9): the amount of dissolved drug, probably due to 3039-3047 the drug’s poor solubility and low dissolution 3. Bogataj M., et al., Development of a glass-bead rate in 0.001 M HCl, additional y diluted by device for dissolution testing. Dissolution water. In Figure 3, pH profiles during Technologies, 2015. 22(3): 18-24 experiments with or without tablet are ACKNOWLEDGMENT presented. The pH of the medium at the beginning of the experiment was approximately The authors acknowledge dr. Luca Marciani 3.9 and it gradual y decreased back to pH 3 due (University of Not ingham) for providing raw to the delivery of fresh 0.001 M HCl into the data of individual in vivo GE profiles and vessel. The decrease in pH was faster when fast Slovenian Research Agency for the financial GE profile was simulated. PAR dissolution did support (grant number P1-0189). 179 P18 DRY POWDER INHALER FORMULATIONS CONTAINING GEFITINIB NANOPARTICLES Merve Geyik1, Burcu Nacak2, Melek Nur Bilal2, Tugba Gulsun1, Selma Sahin1, Levent Oner1, Yagmur Akdag1 1Department of Pharmaceutical Technology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey 2Faculty of Pharmacy, Hacettepe University, Ankara, Turkey 1. INTRODUCTION 2.2. Preparation of HSA-gefitinib Lung cancer is one of the deadliest types of nanoparticles cancer, and chemotherapy treatment is at the HSA-gefitinib nanoparticles are prepared with forefront of treatment methods. However, the NAB™ technology method with minor severe side effects resulting from conventional modification [2]. The gefitinib solution slowly chemotherapeutic applications have led injected to aqueous solution (0.2% HSA(w/v)) researchers to search for new drug delivery under 11,000 rpm. Then, the crude emulsion systems in chemotherapy. Nanoparticulate was homogenized in high pressure homogenizer systems enable to accumulate at the tumor site (20,000 psi, 20 cycles). Afterward, the at lower doses thanks to active and passive nanoemulsion was rotary evaporated to remove targeting methods. the organic solvent. Inhalation therapy is a method that has been 2.3. Preparation of dry powder inhaler used for years and can provide a local effect on formulations the lung, and at the same time, a systemic effect Gefitinib-HSA nanoparticle suspensions (220 can be easily achieved thanks to the capil aries nm) are covered by %50 (w/w) with four in the alveoli. Dry-powder inhalers (DPIs) are different carriers (mannitol, lactose, sucrose, advantageous inhaler products due to their good and trehalose) and frozen at -20ºC for 24 hours. formulation stability, environment friendliness, Then, they were lyophilized for 72 hours at a easy storage conditions and least requirement pressure of 0.01 mmHg at -80ºC. Obtained on actuation-inhalation coordination [1]. In this powders were weighed into size 3 HPMC study a nanoparticulate system which contains capsules and were placed in RS01 devices. human serum albumin (HSA)-gefitinib is covered with different carriers to produce a dry 2.4. Andersen Cascade Impactor analysis powder inhaler with suitable aerodynamic parameters for pulmonary delivery. The aerodynamic parameters of the formulations were calculated using the 2. MATERIALS AND METHODS Andersen Cascade Impactor (ACI) taking into account the criteria of European and American 2.1. Materials Pharmacopoeias.ACI plates were coated with a Gefitinib was kindly provided by Nobel 2% (w/v) solution of Tween 20 in ethanol to (Istanbul, Turkey). HSA solution of Grifols (1 ensure that the particles adhere to the plates at mL of the aqueous solution containing 0.2 g each stage of the device. The device was human albumin, 0.016 mmol sodium caprylate, activated for 4 seconds, with an air flow rate of 0.016 mmol sodium N-acetyltryptophanate) 60 L/min, to provide 4 L of air flow from the was used as an HSA source. D-Mannitol and inhaler. Inhaler device, capsule, mouthpiece, trehalose were purchased from Merck and induction port, stages and filter were washed Goldbio, respectively. Sucrose was purchased separately with acetonitrile. from Isolab, lactose monohydrate was supplied The emit ed fraction (EF %) is calculated by by BASF Pharma. RS01 device was donated by dividing the emit ed dose by the recovered Plastiape SpA (Italy). Al other chemicals were active substance from the ACI system. Mass- of analytical grade. median aerodynamic diameter (MMAD) is obtained from the cumulative percent graph of active substance content (log-probability graph) 180 P18 as a function of aerodynamic diameter. Fine DP 219.9±3. 0.358±0 34.0±1.51 252.5±3.33 0.448±0. 24.6±0 particle dose (FPD) was calculated as the I-2 758 .019 0 2 008 .265 amount of active substance in the formulation DP 219.9±3. 0.358±0 34.0±1.51 26.0±0 I-3 758 .019 0 195.2±5.62 0.452±0. 017 .600 with an aerodynamic diameter of less than 5 μm. DP 219.9±3. 0.358±0 34.0±1.51 167.9±5.05 0.372±0. 26.2±0 The fine particle fraction (FPF) was calculated I-4 758 .019 0 2 018 .404 DP by dividing the FPD by ED. 219.9±3. 0.358±0 34.0±1.51 160.1±343. 0.361±0. 20.9±2 I-5 758 .019 0 897 027 .370 2.5. HPLC method 3.3. Aerodynamic parameters The aerodynamic parameters of the listed Active substance at every stage of ACI device formulations have shown in Table 2. DPI-2 and was quantified by a HPLC method. The mobile DPI-5 have the most desired MMAD values phase was acetonitrile:130 mM ammonium which are smal er than 5 µm. Al the acetate buffer pH 5; 37: 63 (v/v %). Active formulations have a high EF % shows that the substance was observed at 30ᵒC column formulations have good emit ing capacity. The temperature at 260 nm [3]. FPF % higher than 40 % has been achieved with 2.6. Measurement of particle size the mannitol as a carrier. distributions Table 2. Aerodynamic parameters of DPI The particle size distributions and zeta potential formulations. of obtained nanoparticle suspension were MMAD FPD measured with Malvern Zetasizer Nano ZS Formulation EF % (µm) (µg) FPF % (Malvern Instruments, UK). DPI-1 99.6 5.11 89.1 36.1 DPI-2 97.9 4.34 13.0 38.3 3. RESULTS AND DISCUSSION DPI-3 99.9 5.75 15.9 13.5 DPI-4 98.8 5.18 41.5 36.5 3.1. Redispersibility in water after DPI-5 98.9 4.27 27.2 41.4 4. CONCLUSION lyophilization Redispersibility can be explained as ability of DPI formulations, which contain positively nanoaggregates, nanocomposites or charged nanoparticles of approximately 220 nm microcarrier systems to release nanoparticles in size combined with different carriers, have good similar characteristics to the primary water redispersion. Formulations that have FPF nanoparticles upon exposure to humidity in % of more than 35 % show that they can vitro or to lung fluids in vivo [4]. Obtained dry penetrate deep enough into the lungs for drug powder inhaler formulations with no carrier delivery. (DPI-1), with lactose (DPI-2), with sucrose (DPI-3), with trehalose (DPI-4) and with 5. REFERENCES mannitol (DPI-5) were dispersed in distil ed 1. Zhong, Q, Co-Spray Dried water and particle size distributions and zeta Mannitol/Poly(amidoamine)-Doxorubicin Dry- potential were measured (Table 1). Powder Inhaler Formulations for Lung Adenocarcinoma: Morphology, In Vitro DPI-1 showed that lyophilization process did Evaluation, and Aerodynamic Performance, not cause growth of particles, but rather reduced AAPS PharmSciTech, 2018. 19(2): 531-540 the size of them. Also, al the carriers except 2. Desai et al., (2010). US20100226996. lactose caused a decrease in particle size. The 3. Chandrashekara, K.A., et al., Separation and high positive charge may prevent the particles estimation of process-related impurities of from aggregation during the lyophilization gefitinib by reverse-phase high-performance process. liquid chromatography. Journal of Chromatographig Science, 2014. 52(8): 799- Table 1. Particle size distribution and zeta 805. potential of nanosuspensions and DPIs (n=3, 4. Al -Hal ak, M. K., et al. Pulmonary delivery of mean±SD) inhalable nanoparticles: drypowder inhalers. Therapeutic Delivery, 2011. 2(10): 1313–1324. Nanoparticle suspension (before lyophilization) Lyophilized powder ACKNOWLEDGMENT For Zeta mul Particle Zeta Particle potent This study was supported by a grant from the atio size PDI potential size (nm) PDI ial n (nm) (Mv) (mV) Scientific and Technological Research Council DP 256.3±1 0.368±0 33.3±0.02 233.5±11.4 0.432±0, 28.1±0 of Turkey (TÜBİTAK; SBAG-120S895). I-1 3.12 .020 0 017 .551 181 P19 COMPARATIVE COMPRESSION CHARACTERIZATION OF LIQUISOLID SYSTEMS PREPARED WITH MESOPOROUS CARRIERS Teodora Glišić1, Ilija German Ilić2, Jelena Parojčić1, Ivana Aleksić1 1Department of Pharmaceutical Technology and Cosmetology, University of Belgrade, Faculty of Pharmacy, Serbia 2Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION Table 1. Composition of prepared LS Maintaining good compaction properties of admixtures liquisolid systems (LSS) is particularly Liquisolid PEG chal enging in the case of high-dose drugs [1]. admixturesa Rb Liquid load 400 (%) High amount of liquid phase within LSS, S1 10 0.7 38.9 required to dissolve/suspend higher amount of S2 30 0.7 40.4 drug substance, while necessary for S3 10 0.6 35.3 improvement of bioavailability, can cause S4 30 0.6 36.7 difficulties during the tableting process, N1 10 1.1 49.8 resulting in low tablet hardness or even inability N2 30 1.2 54.7 of admixtures to be directly compressed. This a type of carrier used: S1, S2 - Syloid® XDP has led to the development of new highly porous 3050, S3, S4 - Syloid® XDP 3150, N1, N2 - carriers, specifical y designed for LSS, that can Neusilin® US2; bcarrier to coating material ratio adsorb/absorb very high amount of liquid phase. 2.3. Powder density The aim of this study was to investigate the LS admixtures’ true densities were determined compaction properties of LSS, prepared with by helium pycnometer (AccuPyc 1330, three types of novel silica-based mesoporous Micromeritics, GA) while bulk and tapped carriers, using dynamic compaction analysis as densities were measured using a graduated a tool, with the focus on compressibility, cylinder and a volumeter (STAV 2003, J. compactibility and tabletability of these systems Engelsmann AG, Germany). [2]. 2.4. Powder morphology 2. MATERIALS AND METHODS The morphology of LS particles was examined using a scanning electron microscope (SEM, 2.1. Materials Supra 35VP, Carl Zeiss, Germany). Amorphous magnesium aluminometasilicate (Neusilin® US2, Fuji Chemical Industry Co, 2.5. Dynamic compaction analysis Ltd., Japan) and two types of amorphous Dynamic compaction analysis was performed mesoporous silicon dioxide (Syloid® XDP 3050 on an instrumented tablet press (GTP D series, and Syloid® XDP 3150, Grace GmbH, Gamlen Tableting Ltd, UK). 6 mm flat faced Germany) were used as carriers. Col oidal punches were used at a compaction speed of 60 silicon dioxide (Aerosil 200, Evonik Industries mm/min, with compression load ranging from AG, Germany) was used as coating material and 250 to 500 kg, with a 50 kg increment. polyethylene glycol 400 (PEG 400, Fagron, Netherlands) was used as liquid phase. 3. RESULTS AND DISCUSSION 2.2. Liquisolid Admixture Preparation 3.1. Compressibility of LS admixtures LS admixtures (Table 1) were prepared using Regardless of the compaction pressure applied Mycrolab fluid bed processor (OYSTAR and differences in liquid load, very high values Hüt lin, Germany), with the operating of solid fraction were observed in LS compacts temperature of 30°C, inlet air flow rate of 20 with Neusilin® US2 (0.90-0.94). On the other m3/h, and liquid feed rate of 12 g/min. hand, LS compacts with both Syloid® XDP carriers exhibited lower relative density (0.59- 0.89) that was affected by changes in the 182 P19 applied compaction pressure. Compressibility compact tensile strength lower than 1 MPa at al profiles suggest that carrier particle size and the but highest compaction pressure applied. amount of coating material used, had an effect on relative density. An increase in the amount 4. CONCLUSION of coating material used had a negative impact Out of the three investigated carriers, Neusilin® on compressibility and lower values of solid US2 showed the best compaction properties fraction were achieved. despite its high liquid load. LS admixtures with 3.2. Compactibility of LS admixtures this carrier exhibited the highest values of Admixtures N1 and N2 could be considered as tensile strength and solid fraction at relatively having good compactibility [3]. Compacts with low compression pressures. Pronounced Neusilin® US2 achieved higher tensile strength differences have been noticed between the two values compared to compacts with Syloid® Syloid carriers, which indicates the effect of XDP, even at low compaction pressures. carrier particle size on compaction properties of Particle geometry and shape (Fig. 1) can affect LS admixtures. the way particles interact during tableting and 5. REFERENCES therefore may affect their mechanical characteristics. Differences in particle size 1. Lu, M., et al., Liquisolid technique and its could be a reason for lower values of solid applications in pharmaceutics. Asian Journal of fraction and tensile strength observed in Pharmaceutical Sciences, 2017.12(2):115-123. compacts prepared with Syloid® XDP 3150 2. United States Pharmacopeia and National compared to compacts with Syloid® XDP 3050 Formulary (USP 44 - NF 39). United States as carrier. Pharmacopeial Convention; 2021. 3. Pit , KG., et al., Compression prediction accuracy from small scale compaction studies to production presses. Powder Technology, 2015, 270: 490-493. ACKNOWLEDGMENT This research was funded by the Ministry of Education, Science and Technological Figure 1. SEM micrographies of LS particles: Development, Republic of Serbia through Grant admixture N1 (left) and S1 (right) Agreement with University of Belgrade Faculty 3.3. Tabletability of LS admixtures of Pharmacy No: 451-03-68/2022-14/200161. Despite the significantly higher liquid load, bet er tabletability was observed in LSS with Neusilin® US2 as carrier with tensile strength ranging from 1,68 to 2,55 and 1,61 to 2,11 for formulations N1 and N2, respectively. Although relatively similar values of tensile strength were achieved, tabletability profiles indicate that there are differences in compaction behavior between formulations N1 and N2. Higher values of tensile strength observed at the same compression pressure indicate bet er tabletability of LS admixtures with Syloid® XDP 3050 compared to those with Syloid® XDP 3150 as carrier. Interestingly, formulations with Syloid® XDP 3050 had higher liquid load which implies that this formulation factor had lesser influence on tabletability compared to the properties of the carrier itself (such as particle size and specific surface area). The lowest tabletability was observed in LS admixtures S3 and S4 with 183 P20 DESIGN AND EVALUATION OF HYALURONIC ACID DECORATED MULTIFUNCTIONAL PCL-b-PEI NANOPARTICLES Filip Gorachinov1, Fatima Mraiche2, 3, Diala Alhaj Moustafa2, 3, Ola Hisari2, 3, Damjan Georgievski1, Jensa Joseph2, 3, Katerina Goracinova1 1Faculty of Pharmacy, Ss. Cyril and Methodius University in Skopje, Skopje, N.Macedonia 2College of Pharmacy, Qatar University, 3Biomedical and Pharmaceutical Research Unit, QU Health Doha, Qatar University, Doha, Qatar 1. INTRODUCTION quadratic regression model) where each factor Paclitaxel (PTX) loaded hyaluronic acid (HA) was varied at five different levels in the D- decorated polycaprolactone-b-polyethyleneimi- optimal design algorithm was developed ne (PCL-b-PEI) multifunctional nanocarriers (MODDE experimental design software, were developed with the goal of increasing Umetrics, Sartorius, Stedim Biotech) and used payload delivery in NSCLC cel s positive for to evaluate the influence of copolymer, CD 44. Hyaluronic acid forms a bioresponsive Lutrol®F127, and drug concentration upon the shel with a dual role, improving tumor particle size (PS), drug content (DC) and localization by the EPR effect and targeting the efficacy of encapsulation (EE). Optimization overexpressed CD 44 receptor. Further, in the procedure was carried out using the desirability tumor microenvironment, hyaluronidase function. The goal was set to minimize the overexpression may catalyze the degradation of particle size, maximize the efficacy of loading hyaluronic acid, expose polyethyleneimine and drug content. chains and reverse the nanoparticle (NP) charge Ultrasound assisted nanoprecipitation was used which wil further influence cel ular trafficking for preparation of the PCL-b-PEI NPs and endosomal escape of the nanoparticles (1). (Ultrasound homogenizer 300VT, Biologics The aim of this study was to develop a Inc) according to the D-optimal design matrix. reproducible technological approach for PCL-PEI-HA conjugation was performed by preparation of HA decorated PCL-b-PEI amidation of HA carboxylates with amino- nanoparticles using D-optimal experimental groups from PEI using EDC as a coupling design and to evaluate NPs properties. agent. PCL-b-PEI-HA conjugates were further subjected to polyelectrolyte complexation of 2. MATERIALS AND METHODS unreacted amino groups from PEI with HA. 2.1.Materials PS, size distribution and zeta potential of the Poly- -caprolactone-branched-polyethylene polymeric NPs were determined by Zetasizer imine (PCL-b-PEI, Mw ~ 40.000-800 Da) was Nano ZS (Malvern Instruments, UK). purchased from Akina, Inc. IN, USA and poly Previously validated HPLC method was used (ethylenoxide)-b-poly(propyleneoxide)-b-poly for quantification of encapsulated Paclitaxel as (ethyleneoxide) (PEO-PPO-PEO, Lutrol® wel as calculation of the EE and DC (Waters F127) was purchased from BASF, Germany. N- HPLC system, mobile phase ammonium acetate Hydroxysulfosuccinimide sodium salt and N- pH=5.0/ acetonitrile/ methanol; 50:40:10; (3-Dimethyl-aminopropyl)-N'-ethylcarbodiimi- column Symmetry® C18, 4.6x100mm, 3.5 m de hydrochloride were acquired from Sigma- (Waters, USA), flow rate 1.4 ml/min; Agilent Aldrich, USA. Low molecular hyaluronic acid 1100 DAD detector at a wavelength of 230 nm). sodium salt HA5K (Mw less than 10.000 Da) In-vitro drug release studies of optimized was purchased from Lifecore Biomedical; and formulation, FTIR analysis, SEM studies as al other chemicals were analytical grade wel as cytotoxicity and efficacy evaluation purchased from Sigma-Aldrich, USA. using MTT and LDH assay on H322 and A549 cel lines were also performed. Results from the 2.2.Experimental design, preparation and D-optimal design study and characterization of physicochemical evaluation of NPs design matrix samples wil be presented further. D-optimal design matrix (19 experiments, 184 P20 3. RESULTS AND DISCUSSION 3.1.D-optimal design results The design was fit ed with multiple regression analysis and the statistical results pointed to high correlation (R2) among the formulation parameters and the factors as wel as high pre- dictivity (Q2) of the values for the evaluated parameters (PS R2 = 0.9502 and Q2 = 0.6218; Figure 3. Design space and % of failure EE R2 = 0.9481 and Q2 = 0.5710, DC R2 = 3.2.Characterization of the design matrix 0.9845 and Q2 = 0.9020). samples and HA decorated NPs The factors influencing the particle size, efficacy of encapsulation and the drug content PCL-b-PEI self-assembled into nanoparticles are presented in Fig 1. with size in a range of 80±1.2 - 120±2.1 nm and displayed zeta-potential of +36.2±1.4 mV (average value, n=19, nonsignificant difference among matrix samples). Conjugated PTX(PCL- PEI-HA) NPs showed zeta potential of -1.2±0.4 mV and particle size of 103±2.2 nm (PDI = 0.11±0.02). After additional HA electrostatic adsorption, the NPs showed –15±0.56 mv zeta potential and the average size of 111.3±1.4 nm (PDI = 0.18±0.02). Conjugation of HA and the presence of stable HA layer was confirmed Figure 1. Factors and factor coefficients using FTIR and DSC studies. MTT and LDH influencing PS, EE and DC test of the blank HA decorated NPs pointed to The influence of polymer and drug high viability of treated A549 and H322 cel s concentration on the drug content, efficacy of (2 4 and 48 h). encapsulation and particle size at fixed value of Pluronic F127 is presented in Fig. 2. 4. CONCLUSION D-optimal design was successful y used to optimize the PS, EE and DC of the PCL-b-PEI NPs. HA decoration improved biocompatibility and EPR effect by reversing the positive PEI charge to almost neutral charge or negative charge. HA is specific for overexpressed CD44 receptor but may also enable multimodality feature leading to exposure of PEI chains in tumor microenvironment due to enzyme hydrolysis leading to improved internalization and endosomal escape. Figure 2. Response surface plot for EE, DC, PS (Pluronic F125 value 5 mg) 5. REFERENCES The desirability function gave several 1. Maiolino S., et al., Biodegradable nanoparticles optimized formulations. The design space for sequentially decorated with Polyethyleneimine DCmax, EEmax and PSmin is presented in and Hyaluronan for the targeted delivery of Figure 3. docetaxel to airway cancer cells., Journal of Nanobiotechnology (2015) 13:29 ACKNOWLEDGMENT This project has received support from UREP24-057-3-022 award from the Qatar National Research Fund. The statements made herein are solely the responsibility of the author[s]. 185 P21 NANOCELLULOSE-BASED FILM-FORMING HYDROGELS CONTAINING BETAMETHASONE DIPROPIONATE: DEVELOPMENT AND PHYSICAL EVALUATION Mirjam Gosenca Matjaž1* and Katarina Bolko Seljak1 *mirjam.gosenca.matjaz@ffa.uni-lj.si 1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION 2. MATERIALS AND METHODS Atopic dermatitis (AD) is a chronic 2.1.Materials inflammatory skin disease with the lifetime prevalence of up to 30 % in children and 10 % For preparation of film-forming hydrogels gel in adults. Dysfunction of skin barrier, typical for NCC ((gNCC)Nanocrystacel , Slovenia) or AD, results in characteristic features of atopic powder NCC ((pNCC) Cel uforce, Canada), skin being dry, sensitive, accompanied by sodium alginate ((ALG)Protanal® LF 10/60, intense itch. Daily skin care routine has an FMC BioPolymer, USA), pectin ((PEC) Sigma- important role in AD management, whereas Aldrich, USA), glycerol (Pharmachem, treatment options span from local anti- Slovenia) and purified water were used. inflammatory therapy to systemic therapy in CapryolTM 90 (Gat efosse, France) and cases of severe AD [1]. Cremophor® EL (BASF, Germany) were used for formation of self-microemulsifying drug Various dermal formulations are being delivery system (SMEDDS) for dissolving BDP investigated for AD treatment. One of advanced (Sicor, Italy). approaches is use of hydrogels as film forming systems upon dermal application. Namely, 2.2.Hydrogel preparation and DSC analysis hydrogel is applied directly to the skin forming For NCC-ALG or NCC-PEC hydrogels, BDP- a thin, transparent film in situ upon solvent loaded or unloaded SMEDDS diluted with evaporation [2]. While primarily suggested for water was added to NCC, fol owed by alginate prolonged delivery, in the case of AD treatment or pectin plus glycerol. The NCC/natural the additional value of film-forming hydrogels polymer ratio was 1/2, while SMEDDSS and is providing a physical barrier on the skin glycerol content was 3 and 5 %, respectively. surface that is lacking in atopic skin. Drug content in BDP-loaded hydrogels was 0,64 mg/1 g. In drug delivery, biocompatible and biodegradable polymers are used. One such For DSC measurements (Met ler Toledo, polymer, nanocrystal ine cel ulose (NCC), is Switzerland) samples were heated from 25 to gaining interest due to its excel ent and 250 °C with heating rate 10 K/min at nitrogen at ractive mechanical and chemical flow of 50 ml/min. characteristics in addition to renewability, 2.3.Film preparation and evaluation although its properties may differ based on Hydrogels were evenly distributed across glass source and manufacturing procedure [3]. plate using ZUA applicator and dried at 32 °C In this study, novel film-forming hydrogels to form films that were cut into even pieces. based on two different NCCs, combined with Films were evaluated for residual moister one of the natural polymers (alginate or pectin)) content and thickness. I n vitro ability to retain plus glycerol were developed for incorporation water evaporation was evaluated using of betamethasone dipropionate (BDP) and Tewameter®TM 300 (Courage&Khazaka evaluated as innovative delivery system for AD GmbH, Germany) with films placed in Franz treatment. diffusion cells. 186 P21 3. RESULTS AND DISCUSSION 3.1.Hydrogel preparation and evaluation BDP is practical y insoluble in water, therefore a chal enge to prepare BDP-loaded hydrogels was resolved by prior dissolution in SMEDDS. The mixture of Cremophor® EL/CapryolTM 90 in 8/2 ratio was chosen on the merit of its high BDP saturated solubility and ability to form microemulsions upon water dilution without BDP precipitation (data not shown). Regarding DSC analysis, broad endothermic peak can be seen for al hydrogels, ranging between 104-108 °C representing water Figure 2. Water evaporation (g/m2/h) for evaporation. No additional endothermic peak cel ulose acetate membrane (basal value) was observed in case of BDP-loaded hydrogels, compared to cel ulose acetate membrane with confirming that BDP remains dissolved upon film (the water evaporation was measured prior incorporation into hydrogels (example of NCC- wet ing of films). Data are expressed as mean ± ALG hydrogels in Fig. 1). S.D. (n = 3). 4. CONCLUSION Both types of NCC were proven to be suitable for formation of film-forming hydrogels in combination with alginate or pectin. BDP was successful y incorporated and remained dissolved using SMEDDS. Using solvent evaporation method flexible films were formed at 32 °C and significantly reduced in vitro water evaporation was observed. Figure 1. DSC heating curves of BDP-loaded NCC-based film forming hydrogels therefore and unloaded NCC-ALG hydrogels. present novel and perspective delivery system for AD treatment. 3.3.Film preparation and evaluation Films were prepared from hydrogels using 5. REFERENCES solvent evaporation method at 32 °C, i.e. skin surface temperature. The prepared films 1. Weidinger S., et al., Atopic dermatitis. Nature showed a low residual moisture content (up to Reviews Disease Primers, 2018. 4(1): 1. 2. Kathe K., et al., Film forming systems for 2,15 % for NCC-PEC films and up to 1,5 % for topical and transdermal drug delivery. Asian NCC-ALG films, being higher in case of gNCC Journal of Pharmaceutical Sciences, 2017. or/and incorporated BDP). Incorporation of 12(6): 487-497. BDP had also an impact on film thickness, 3. Trache D, et. al. Nanocellulose: From being the maximum in case of BDP-loaded Fundamentals to Advanced Applications. pNCC-ALG films, i.e. 0,078 ± 0,008 mm. Frontiers in Chemistry, 2020. 8: 392. 4. Montero-Vilchez T., et al., Skin Barrier Increased transepidermal water loss due to Function in Psoriasis and Atopic Dermatitis: impaired skin barrier function is distinctive for Transepidermal Water Loss and Temperature atopic skin [4], therefore formulations that as Useful Tools to Assess Disease Severity. enable water retention in skin are especial y Journal of Clinical Medicine, 2021. 10(2): 359. beneficial in AD treatment. Prepared films ACKNOWLEDGMENT decreased in vitro water evaporation (Fig. 2) with statistical y significant difference (p < The authors acknowledge financial support 0,05) compared to cel ulose acetate membrane from the Slovenian Research Agency (research that was used as carrier for films between donor core funding, No. P1-0189). and acceptor compartment of Franz diffusion cel s. 187 P22 VALSARTAN BUCCAL FILMS FORMULATION DEVELOPMENT WITH THE IMAGE ANALYSIS Blaž Grilc1, Tjaša Felicijan2, Timeja Planinšek Parfant2, Odon Planinšek1 1Department of Pharmaceutical Technology, 2Department of Biopharmaceutics and Pharmacokinetics, University of Ljubljana, Faculty of Pharmacy, Slovenia 1. INTRODUCTION (Matrix Fine Chemicals, Switzerland), ethanol Buccal administration of drugs is a promising (Merck, Germany). alternative for patients who are less responsive 2.2. Preparation of films to conventional oral administration, such as Films were prepared by applying the wet film pediatric patients. Buccal films for pediatric use solution to the glass plate at a height of 1500 µm have already been proposed by several research using the applicator. The film solution was groups [1,2]. The incidence and prevalence of prepared by careful y mixing Na-alginate with hypertension in pediatrics have increased over water and plasticizer. During mixing with a the past 30 years, largely due to lifestyle propeller stirrer at 800 rpm, an ethanol solution changes in children and adolescents [3]. The of valsartan was introduced into the polymer. recommended starting dose of valsartan in A reduced combinatorial design was used to children is 1.3 mg/kg once daily (maximum study the effects of six factors. The amounts of dose 40 mg), which should be adjusted valsartan, Na-alginate, isomalt, xylitol, EtOH according to blood pressure response [4]. Some and water were varied and their effects on visual unique chal enges arise in the development of appearance, dissolution properties and buccal films, such as breaking of the film, crystal inity were studied. entrapment of bubbles, and inhomogeneous distribution of the drug. Al of these effects are a. Image analytics undesirable and difficult to evaluate. Images of final y produced films were Observation of the film provides information on embedded using a pre-trained CNN "Inception the homogeneity, presence of crystals, bubbles, v3". The distances between the image data were and mechanical properties of the films. With the evaluated by comparing the cosine distance and introduction of image analysis, unbiased the results were displayed in the dendrogram. comparison is possible without the risk of The clusters were connected using Ward's subjective evaluation and with repeatable linkage method. results. The use of multilayer convolutional neural networks (CNN) has been introduced in b. Other methods the field of image analysis. The trained neural Al films were also tested with Raman network behaves like the human visual cortex microscopy mapping (Xplora Plus Horiba, and can adapt its discrimination capabilities to a Japan), moisture content KF titration (Titroline® new chal enge. Using this approach, images of 7500, Germany), TGA and DSC (Met ler buccal films were compared and classified by Toledo TGA/DSC1), particle size distribution quality using a pre-trained CNN. The aim was (Mastersizer 3000) and dynamic contact angle to compare results with the drug release from evaluation (KRÜSS DSA 100). The release rate different films and evaluate their capability of of valsartan from the films was evaluated using films performance prediction. four different methods (USP I, USP II, modified USP IV and innovative cel for film release 2. MATERIALS AND METHODS (ICRF). 2.1. Materials Na-alginate (Protanal LF 10/60, Dupont, USA), xylitol (Xylisorb 100DC, Roqet e, France), isomalt (Beneo-Palatinit,Germany), valsartan 188 P22 3. RESULTS AND DISCUSSION potential for future optimization. Image analysis provided a complementary 3.1. Image analytics approach to visual inspection. Hierarchical The distances between the obtained data sets were compared and the results were presented clustering al owed characterization and grouping of the films by an independent in the form of a dendrogram (Figure 1). The data observer. This approach helps identify films or were hierarchical y clustered and four main groups of films with visual y similar clusters were identified at a height ratio of 45%. characteristics, assisting the researcher in decision making. 3.2. Dissolution testing The evaluation of the dissolution of the films revealed differences between the dissolution methods. In addition, some of the methods showed greater differences between formulations than others. The ICRF method was found to be superior than the modified USP IV method in terms of discriminability and repeatability. 4. CONCLUSION Figure 6: Dendrogram of image data distances. Image analysis proved to be a supportive technique in visual evaluation of film quality. Visual observation with the naked eye is usual y sufficient, but at the same time it is difficult to rank a large number of images by quality without a proper ranking tool. Even more important than ranking is finding the regions of the design space where the quality is within the desired parameters. Identifying these clusters of images that contain films of similar quality can help researchers choose the right path for further formulation development. 5. REFERENCES Figure 2: A representative from each cluster. 1. Khan S., et al., Formulation, Characterisation and Stabilisation of Buccal Films for Paediatric Figure 2 shows a single representative film from Drug Delivery of Omeprazole. AAPS each cluster. Films with trapped bubbles and PharmSciTech. 2015 Aug 1;16(4):800–10. broken edges were found in the C4 (orange) 2. Friedrichsdorf S, et al., Management of breakthrough pain in children with cancer. cluster. Broken edges formed during cut ing JPR. 2014 Mar;117. indicate that the film has poor mechanical 3. Ashraf M, et al., Pediatric hypertension: an properties and therefore tears easily during updated review. Clin Hypertens. 2020 handling. Trapped bubbles affect the Dec;26(1):22. homogeneity of the drug distribution in the film 4. Kapur G., Clinical utility of valsartan in the and have a poor esthetic appearance. Films with treatment of hypertension in children and a smal er and less dense distribution of bubbles adolescents. PPA. 2011 Mar;149. and sharp cut edges were located in cluster C2 (red). Cluster C3 (green) contained a film with ACKNOWLEDGMENT a white sheen, which could be due to light This research was funded by Slovenian scat ering by crystals on the film surface. Films Research Agency (ARRS), grant number [P1 with a smooth surface, homogeneity, sharp 0189]. edges, no bubbles, and no crystals were in cluster C1 (blue). The result shows that the design space of cluster C1 offers the greatest 189 P23 NEW HIGHLIGHTS OF THE DRUG CARRIER PEG-Β-CYCLODEXTRIN POLYMER Adam Haimhoffer1,2, Licia Dossi3, Ildikó Bácskai1,2, Ferenc Fenyvesi1 1Department of Pharmaceutical Technology, University of Debrecen, Hungary 2Institute of Healthcare Industry, University of Debrecen, Hungary 3Cranfield Defence and Security, Cranfield University, United Kingdom 1. INTRODUCTION 2.1. Materials Cyclodextrin and polymer binary systems are The β-cyclodextrin polymer cross-linked with used to improve the solubilization of poorly polyethylene glycol diglycidyl ether (βCPCD) soluble drugs when high amount of CD is was synthetized at Cranfield University in the needed for complexation. To achieve higher UK as previously described. Estradiol, cel degrees of drug solubilization, the synergistic culture and other reagents were from Sigma- solubilizing effect of a cyclodextrin and a Aldrich Ltd. (Budapest, Hungary). water-soluble polymer was used. 2.2. Cytotoxicity test To take advantage of ternary complexes, high The cytotoxic effects of the cyclodextrin molecular weight crosslinking agents are now polymers were evaluated using the MTT test. used for many cyclodextrin polymerizations The Caco-2 cel line and NIH/3T3 fibroblast [1]. The resulting polymers have more cel line were obtained from the European advantageous properties than the native CDs or Col ection of Authenticated Cel Cultures binary systems. (ECACC, UK). Cel s were maintained in Dulbecco’s modified Eagle’s medium at 37 °C Our aim was to test the water-soluble in an incubator containing 5 % CO2. 10,000 polyethylene glycol cross-linked β-CD polymer cel s/wel were seeded on 96-wel plates. After (fig 1.), which offers a combination of β-CD and 3 days, the medium was removed, and the cel s PEG properties [2]. Estradiol was chosen as a were incubated for 30 min with the solutions of model drug, which can be solubilized by cyclodextrin polymers at 37 °C. Then the cyclodextrin, cyclodextrin polymers and PEG samples were removed, and a 0.5 mg/ml MTT 400 too. solution was added to each wel . The plates were incubated for 3 h, then the MTT solution was removed and 0.1 ml of isopropanol – 1 M hydrochloride acid (25:1) was added to each wel to dissolve the formed formazan crystals. The absorbance of formazan was measured at 570 nm and the background was measured at 690 nm by Thermo-Fisher Multiskan Go (Thermo-Fisher, USA) microplate reader. 2.3. Phase-Solubility Test The phase-solubility test was performed by adding a fixed excess amount of β-estradiol powder (20 mg) to 2.0 ml aqueous solutions containing βCD, PEG, βCD and PEG mixture, and PEG-β-cyclodextrin polymer at increasing Figure 1. Schematic structure of PEG-β- concentrations (0.05 – 1.0 m/m %). The vials cyclodextrin polymer and API complex. The were vortexed for 30 seconds to achieve wel - yel ow colored API molecules are entrapped in mixed dispersions. They were rotated at room the βCD cavity and in the 3D cross-linked area. temperature at 50 rpm and protected from light. After 72 h, each vial was centrifuged at 11000 2. MATERIALS AND METHODS rpm for 20 min. The samples were taken from 190 P23 the clear supernatant, and the estradiol content Figure 2. Phase-solubility curve presents the of the samples was analysed by UV solubility of estradiol (mg/ml) in water versus spectrophotometer (Shimadzu UV-1900) at 280 the m/m % concentration of the βCD (red), PEG nm. (blue), βCD and PEG mixture (green), and PEG-β-cyclodextrin polymer (purple) 2.4. Dynamic light scattering (DLS) measurements of complexes 3.3. Dynamic light scattering (DLS) The effect of complexation on the average measurements of complexes particle size and particle size distribution of 0.5 The size of the complexes formed (270.70 ± m/m % solution of complexes was measured by 10.10 nm) after complexation is several times DSL. The cyclodextrin polymer solutions and bigger than the polymer size (49.76 ± 0.43 nm). complex solutions were measured using a During complex formation, the estradiol is able Malvern Nano-ZS Zetasizer (Malvern to connect to polymer chains and form a Instruments, UK) in purified water. coherent col oidal solution. 3. RESULTS AND DISCUSSION 4. CONCLUSION 3.1. Cytotoxicity test In terms of results, it can be said that the The cyclodextrin polymers were not cytotoxic polymer did not cause cel death in either the on Caco-2 cel s after 30 min of incubation up to Caco-2 or fibroblast cel line. Estradiol has 10.0 m/m % concentration. The cel viability bet er complexing properties with PEG-β- did not decrease below 60% compared to the cyclodextrin polymer, than the βCD or binary control. In the case of NIH/3T3 fibroblast cel , system. Furthermore, the formed matrix system the cell viability changed as expected. provides an opportunity to develop drug delivery systems. Our other goals include the 3.2. Phase-Solubility Test development and tests of a subcutaneous Based on the phase solubility curves, PEG formulation. cannot be used to increase the solubility of estradiol. When using βCD, a AN-type curve is 5. REFERENCES observed, the curve flat ens, and the precipitation of the formed complex is observed 1. Haimhoffer, A. et al.; Cyclodextrins in drug delivery systems and their effects on biological to form an over-saturated solution. There is no barriers. Sci. Pharm. 2019, 87, significant improvement with the concomitant 2. Haimhoffer, Á. et al.; Preformulation Studies use of PEG and βCD. In case of PEG-β- and Bioavailability Enhancement of Curcumin cyclodextrin polymer the line is AL-type, the with a ‘Two in One’ PEG-β-Cyclodextrin solubility of estradiol increased with increasing Polymer. Pharmaceutics 2021, 13, polymer concentration. Significantly bet er solubility was obtained, although the CD ACKNOWLEDGMENT content of the polymer was only around 50%. Project no. TKP2021-EGA-18 has been Thus, to focus to βCD component, it can be said implemented with the support provided from to have increased solubility of estradiol by more the National Research, Development and than 2-fold, as if ternary complexation had been Innovation Fund of Hungary, financed under used. the TKP2021-EGA funding scheme. 191 P24 PREPARATION AND EVALUATION OF SPRAY-DRIED MICROPARTICLES CONTAINING N-ACETYLCYSTEINE FOR LUNG APPLICATION Hana Hořavová1, Jan Gajdziok1 1Department of Pharmaceutical Technology, Faculty of Pharmacy, Masaryk University, Czech Republic 1. INTRODUCTION 180 °C, feed rate 10 – 20 device units, airflow Inhalation is the preferred administration route rate 35 device units, atomization pressure 2 of active substances for lung diseases (e.g., bars. Dry powders were evaluated by SEM cystic fibrosis). The basic therapeutic approach Tescan MIRA 3. Size distribution and median to this disease is the administration of size were determined using laser diffraction mucolytics. One of the commonly used is N- (HORIBA Partica LA-960). Aerodynamic acetylcysteine. Its administration in dry properties (MMAD = mass median microparticles can bring advantages like higher aerodynamic diameter, FPF = fine particle physical and chemical stability, bet er control of fraction) were determined using Smal -Scale particles’ size and deposition, and a reduced risk Powder disperser TSI 3433 and Aerodynamic of bronchoconstriction after inhalation Particle Sizer TSI 3321. compared to a liquid aerosol [1]. Spray drying 3. RESULTS AND DISCUSSION of N-acetylcysteine (NAC) was proved to be problematic due to its high hygroscopicity [2] In neutralized batches, particle median size and low glass transition temperature (Tg≈10°C) increased with increasing drying temperature, [3]. This work optimized the process by while MMAD decreased after the initial applying different drying conditions and increase (Fig. 2). That corresponds with the additives to obtain solid microparticles suitable increasing porosity of the particles observed in for inhalation. SEM images. Decreasing the feed rate led to more spherical and smal er particles. 2. MATERIALS AND METHODS A higher dextran concentration caused 2.1. Materials decreasing in both median size and MMAD Active agent N-acetylcysteine (NAC; with increased FPF. These particles were bet er Myprotein) is a mucolytic disrupting disulfide separated with a smoother surface and fewer bonds in the mucus structure. Mannitol (MAN; aggregates. However, the dextran concentration Penta) was used as a carrier material to dilute seemed not sufficient to increase the Tg enough. mucus by its high osmotic activity. Leucine Imamura et al. described a more significant (LEU; Myprotein) was used as an anti-adherent increase in Tg from 40% dextran content [4]. improving aerodynamic properties and Dextrose equivalent of maltodextrin has a reducing hygroscopicity. Sodium hydroxide nonlinear effect on the particles´ median size, (Dr. Kulich Pharma) solution was added to form but increased MMAD and FFP were observed a salt of NAC and thus reduce its with higher DE. That means smal er particles hygroscopicity. Dextran 10 (DEX; Carl Roth), creating larger aggregates or particles with maltodextrins (MD) with different dextrose different densities were formed. The more equivalents (DE = 2, 6, or 9; Roquet e), and wrinkled surface also influenced the results. low-molecular chitosan (CHIT; Sigma Aldrich) were used to increase the Tg. The composition In chitosan batches, the same dependencies as of prepared batches is listed in Table 1. in dextran batches were observed. On the other 2.3. Method hand, chitosan did not show a good ability to A 10% (w/w) aqueous solution was prepared for encapsulate NAC as its crystals were seen on al the materials in each batch ahead of drying. the SEM images. Subsequently, the solution underwent a spray- Al the described excipients showed a positive drying process using a LabPlant SD06. The effect on the formation of spherical parameters used were: inlet temperature 100 – 192 P24 microparticles compared to previous excipient- 4. CONCLUSION free products (not published). Nevertheless, the particles were stuck to aggregates. The only Although the addition of the excipients resulted approach leading to separated individual in spherical primary particles, the particles were particles was increasing the leucine content up present in aggregates and exceeded the to 30%. With increasing leucine content, both recommended size. Best results were found median size and MMAD decrease, and FPF with an increase in the leucine content. Batches increase with best results with 20 or 25% of with a concentration of 20 and 25% met the leucine (Fig. 1). The further increase did not requirements for inhalation particles best. These bring bet er aerodynamic properties, and a batches showed the largest fine particle higher number of col apsed particles was fractions and the smal est particle sizes, obtained. including narrow size distribution and appropriate morphology and shape. However, Table 1. Composition of prepared batches. further research is needed to achieve even smal er particle sizes. Sample MAN NAC LEU DEX MD CHIT name [g] [g] [g] [g] [g] [g] 5. REFERENCES NAC 1–5 30 15 neutr. 5 - - - ND 1 22,5 15 5 7,5 - - 1. Henke, M. O., et al., Mucolytics in cystic ND 2 15 15 5 15 - - NM 1 15 15 5 - 15 (2) - fibrosis. Paediatric Respiratory Reviews, 2007. NM 2 15 15 5 - 15 (6) - 8(1): 24-29. NM 3 15 15 5 - 15 (9) - 2. Lababidi, N., et al., Spray-drying of inhalable, NCH 1 27,5 15 5 - - 2,5 NCH 2 25 15 5 - - 5 multifunctional formulations for the treatment NL 10% 30 15 5 - - - of biofilms formed in cystic fibrosis. Journal of NL 15% 27,5 15 7,5 - - - Control ed Release, 2019. 314: 62-71. NL 20% 25 15 10 - - - NL 25% 22,5 15 12,5 - - - 3. Odziomek, M., et al., Conception, preparation NL 30% 20 15 15 - - - and properties of functional carrier particles for pulmonary drug delivery. International Journal of Pharmaceutics, 2012. 433(1-2): 51-59. 4. Imamura, K., et al., Water sorption and glass transition behaviors of freeze-dried sucrose– dextran mixtures. Journal of Pharmaceutical Sciences, 2002. 91(10): 2175-2181. ACKNOWLEDGMENT This work was supported by the project MUNI/A/1213/2020. Aerodynamic measurements were performed at the Faculty of Mechanical Engineering, Figure 1. SEM images (magnification 5000x) of University of Technology, Brno. batches a) NL 20%, b) NL 25%. Figure 2. Results of median size, MMAD, and FPF. 193 P25 THE EFFECT OF THE HIGH DRUG LOAD – THE EVALUATION OF THE PROPERTIES OF 3D PRINTED ORODISPERSIBLE TABLETS (ODTS) WITH 70% OF DRUG CONTENT Witold Jamróz1, Jolanta Pyteraf1, Mateusz Kurek1, Thao Tranová2, Jan Loskot3, Jitka Mužíková2, and Renata Jachowicz1 1Department of Pharmaceutical Technology and Biopharmaceutics, Jagiellonian University Medical College, Krakow, Poland; witold.jamroz@uj.edu.pl (W.J.) 2Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic; 3Department of Physics, Faculty of Science, University of Hradec Králové, Czech Republic 1. INTRODUCTION 2.4. 3D printing of ODTs Fused deposition modeling (FDM) is a 3D The ODTs projects were designed using printing (3DP) method that is considered as a Blender® 2.90 software and prepared for the potential tool for the preparation of printing in Voxelizer® slicing software. The personalized dosage forms [1]. In this method layer height was set at 0.1 mm with 10% of infil filaments containing API (active density and one outline. Tablets containing 50 pharmaceutical ingredient) are utilized in mg of FLU were manufactured by ZMorph® 2.0 dosage form preparation. The high drug content S (Poland), equipped with a 0.2 mm nozzle at in the filaments al ows for the preparation of 182°C and 10 mm/s of printing speed. Glass tablets with a variety of drug doses [2]. build plate temperature was 60°C. However, the stability and printability of the 2.5. Tablets disintegration high drug-loaded filaments after storage are Disintegration time of the tablets were questionable. evaluated by means of Pharmacopoeial (PhEur The aim of the studies was to verify the 10) method (2.9.1) in SAPO ED-2 apparatus possibility of ODF tablets preparation by means (Electrolab, India) in 700 mL of purified water. of the 3DP FDM method with the extremely Additionally, changes in the tablet’s height high (70%) API content filaments, which were during disintegration were recorded using in- freshly prepared (F) or after one-year storage house developed apparatus (BJKSN-13) [3]. (O) in the room conditions. 2.6. Dissolution study 2. MATERIALS AND METHODS Surface dissolution imaging apparatus SDi2 (Pion-Inc, UK) was applied to determine the 2.1. Materials Fluconazole (FLU, China) served as a model dissolution processes of API from printed tablets. Tablets were analysed at λ = 255 or 280 drug, while poly(vinyl alcohol) (PVA, Parteck® nm. Additional y, a real-time disintegration MXP, Merck KGaA, Germany) was used as a filament-forming polymer. process was observed by the UV camera. The amount of released FLU was assessed in-line 2.2. Hot-Melt Extrusion with UV-VIS Shimadzu 1800 Filament composed of 70% of FLU and 30% of spectrophotometer (Japan) at λ = 261 nm. PVA was prepared using a 12-mm corotating twin-screw, hot-melt extruder (RES-2P/12A 3. RESULTS AND DISCUSSION Explorer, Zamak Mercator®, Poland) equipped 3.1. 3D printed tablets with a 1.75 mm nozzle at 137°C. Filaments were characterized by good 2.3. Evaluation of filaments’ properties printability which resulted in smal weight Mechanical properties were evaluated with an variations (Tab. 1). During printing process the EZ-SX tensile tester (Shimadzu) while the tension between filament driving rol ers were microstructure was examined using a scanning reduced to avoid filaments fracture. electron microscope FlexSEM 1000 (Hitachi). Table 1. Tablets’ at ributes. F O 194 P25 Average mass (mg ± SD) 64.92 ± 3.79 71.07 ± 3.99 2,5 ) F 100 Dose (% 2,0 g) (mg ± SD) 48.35 ± 2.82 50.03 ± 2.81 80 Disintegration 1,5 time (s ± SD) 128 ± 7.81 139 ± 36.94 60 1,0 3.2. Disintegration and dissolution 40 rug release (mD characteristics. 20 0,5 ulative drug release In both cases: freshly prepared filament or after Cum 0 0,0 one-year storage, the disintegration time of 0 5 10 15 20 25 30 tablets was under 3 min (Tab. 1) which meets Time (min) the Pharmacopoeial requirements for ODTs. Changes in the tablet’ height during the 2,5 ) O 100 (% disintegration process were also similar Fig. 1. 2,0 g) 80 1,5 60 1,0 40 rug release (mD ulative drug release 20 0,5 Cum 0 0,0 0 5 10 15 20 25 30 Time (min) Figure 1. The disintegration profiles of 3D Figure 3. Drug dissolution profiles from 3DP printed tablets obtained by BJKSN-13 apparatus. tablets. Based on the surface dissolution imaging 4. CONCLUSION analysis (Fig. 2), the infil of O tablets was almost completely dissolved after 3 min. After The FDM technology allows for the fabrication 10 min, in both cases, only outline is visible. of high drug-loaded ODTs. The printability of the filament containing 70% of FLU after one year of storage is sufficient, and the tablets’ properties are comparable with tablets prepared with fresh filament. 5. REFERENCES 1. Zema, L., et al., Three-Dimensional Printing of Medicinal Products and the Challenge of Personalized Therapy. J. Pharm. Sci. 2017, 106: 1697–1705. 2. Pyteraf, J. et al., How to Obtain the Maximum Properties Flexibility of 3D Printed Ketoprofen Tablets Using Only One Drug-Loaded Filament? Molecules 2021, 26, 3106 3. Brniak, W., et al., The Practical Approach to the Evaluation of Methods Used to Determine the Disintegration Time of Orally Disintegrating Tablets (ODTs). Saudi Pharm. J. 2015, 23: 437– 443. Figure 2. UV views of the tablets in the initial phase, after 3, and 10 min of the study in SDi2, ACKNOWLEDGMENT λ2 = 280 nm. The authors acknowledge the National Science This differences in disintegration behaviour Centre, Poland for financial support (grant were observed in dissolution profiles. From O OPUS 16 no 2018/31/B/ST8/01327). tablets over 80% of API was released after 15 min whereas from F tablets 68%. However, over 95 % of FLU was released within 30 minutes of testing (Fig. 3). 195 P26 HCE-T CELL-BASED CORNEAL EPITHELIAL MODEL: SCALE-DOWN TO 96-WELL INSERT PLATES Bisera Jurišić Dukovski1, Josip Ljubica1, Petra Kocbek2, Luka Bočkor3, Jasmina Lovrić1 1Department of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia 2Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Slovenia 3Institute for Anthropological Research, Croatia 1. INTRODUCTION plate (PSHT004S5, Merck, Germany) pre- HCE-T cel line is the most extensively coated with rat tail type I col agen (Sigma- characterized human-derived cel line of Aldrich) and human fibronectin (Sigma- corneal epithelium. 3D HCE-T models are Aldrich). The cel s were cultivated submerged commonly cultured on 12-wel insert plates and in the medium (75 µL apical and 250 µL are widely used for permeability and basolateral volume) during 7 days and were biocompatibility testing of eye preparations [1, subsequently exposed to the air-liquid interface 2]. The aim of this research was transfer of a 3D (ALI) for 3 days. HCE-T model from 12 to 96-wel insert plate, 2.4. Histomorphological characterization in order to develop a high-throughput screening (HTS) model of human corneal epithelium. The cel s were fixed with 4% paraformaldehyde (Sigma-Aldrich) at different time points after 2. MATERIALS AND METHODS seeding and the cel nuclei were stained with 2.1. Materials DAPI. The membranes with cel s were mounted onto glass slides with mounting medium and the The cel s were stained using 4′,6-diamidino-2- cover slips were sealed with a nail polish. The phenylindole (DAPI, Invitrogen, USA). cel nuclei were imaged using confocal Fluoroshield™ mounting medium with DAPI fluorescence microscope (ImageXpress® Micro was purchased from Sigma-Aldrich (Germany) Confocal, Molecular Devices, USA) at 60× and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl magnification. tetrazolium bromide (MTT) was purchased from Carbosynth (UK). For nanoemulsion (NE) 2.5. Biocompatibility assay preparation the fol owing substances were used: Ophthalmic NEs were produced using ibuprofen (Hubei Biocause Pharmaceutical, microfluidizer (Microfluidics LM20, USA) at China), Miglyol® 812 (Kemig, Croatia), lecithin 1000 bar and 5 cycles and their composition is S45 (Lipoid, Germany), LMW chitosan shown in table 1. The NEs were diluted 10 times (Sigma-Aldrich), Kol iphor® EL (BASF, in HBSS buffer (pH 6) and the HTS model was Germany), glycerol (T.T.T., Croatia) and exposed to the diluted formulations for 30 min purified water (SG, Germany). at 37°C. The viability of the cel s was 2.2. Cell culture conditions determined with MTT assay and the results HCE-T cel s (RIKEN Cel Bank, Japan) were were compared with the results obtained cultivated in DMEM/F12 medium (Sigma- previously on a 12-well plate HCE-T model. Aldrich) supplemented with FBS (5%, Capricorn, Germany), insulin (5 μg/mL, Sigma- 3. RESULTS AND DISCUSSION Aldrich), dimethyl sulfoxide (0.5%, 3.1. Histomorphological features of HTS Applichem, Germany), epidermal growth factor HCE-T model (10 ng/mL, Sigma-Aldrich) and antibiotic- The HCE-T model was successful y cultivated antimycotic solution (Sigma-Aldrich) at 37°C on a 96-wel insert plate. After a 7-day in a humidified atmosphere with 5% CO2. cultivation period in submerged conditions a 2.3. Cultivation of a 3D HTS HCE-T model confluent monolayer was formed and 3 days The cel s were seeded (1×104 cel s per well) on after ALI exposure multiple layers were polycarbonate membranes of a 96-wel insert observed (Figure 1a). However, HTS HCE-T 196 P26 model was characterized with fewer cel layers 4. CONCLUSION (3-5) and uneven thickness compared to the 12- well plate model [1]. A HTS 3D HCE-T model of human corneal epithelium can be cultivated on 96-wel insert 3.2. HTS HCE-T model viability plates. However, slower cel proliferation and The viability of cel s in the HTS HCE-T model greater sensitivity to the ophthalmic NEs was after treatment with ophthalmic NEs was high observed in comparison to the standard 12-well (about 80%). However, it was lower than the plate HCE-T model. Thus, further optimisation viability of cel s in a 12-wel plate model treated of cultivation conditions is necessary to achieve with the same formulations, under the same higher similarity with the standard 12-wel plate conditions (Figure 1b) [2]. HCE-T model. 5. REFERENCES 1. M. Juretić et al. , HCE-T cel -based permeability model: A wel -maintained or a highly variable barrier phenotype?, Eur. J. Pharm. Sci. , 2017. vol. 104, pp. 23–30 2. B. Jurišić Dukovski et al. , Functional ibuprofen-loaded cationic nanoemulsion: Development and optimization for dry eye disease treatment, Int. J. Pharm. , 2020. vol. 576, 118979. ACKNOWLEDGMENT This work was supported by the project BeatDED (IP-2019-04-2174) funded by the Croatian Science Foundation and 2 projects funded by the European Regional Development Fund, namely FarmInova (KK.01.1.1.02.0021) and BIOANT (KK.01.1.1.02.0002). Josip Ljubica is recipient of a PhD fel owship Figure 1. Z-stack images of HTS HCE-T model captured with confocal fluorescence microscope from the Croatian Science Foundation prior ALI exposure (monolayer) and 3 days after (programme Young researchers' career ALI exposure (multilayer) (a); the viability of development project – training of new doctoral cel s treated with ophthalmic NEs on 12-wel and students). 96-wel insert plates (b). Table 1. The ophthalmic NE composition. The abbreviations in the brackets refer to the controls, i.e. drug-free formulations. Formulation Water phase Oil phase (%, w/w) (%, w/w) IN (N) Kol iphor® EL 0.25 Ibuprofen 0.2 Glycerol 2.5 Miglyol 812 2.5 Water up to 100 Lecithin 0.05 INC1 (NC1) Chitosan 0.05 Ibuprofen 0.2 Kol iphor® EL 0.25 Miglyol 812 2.5 Glycerol 2.5 Lecithin 0.05 Water up to 100 197 P27 FORMULATION AND CHARACTERIZATION OF MUCO-ADHESIVE ORAL FILMS CONTAINING LIDOCAINE HYDROCHLORIDE USING QUALITY BY DESIGN APPROACHES Mohammed Kalayi1, Berna Uzun1, Arkan Barbar1, Buket Aksu1 1Department of Pharmaceutical Technology, Altınbaş University, Turkiye 1. INTRODUCTION 2.2. Preparation of muco-adhesive film Mucoadhesive systems are the systems which formulations exist in a close connection with mucous Lidocaine loaded oral film were prepared by membrane, absorption tissue and site of action. solvent casting method, using HPMC and Mucoadhesive films are the oral dosage forms NaCMC sodium carboxymethyl cel ulose as which adheres to the mucosa and delivers the muco-adhesive polymer. After dissolving drug though it with a purpose of improvement lidocaine HCl in distil ed water, the polymers in local effects as wel as systematic effects [1]. were al owed to swel in 20 mL of distil ed Mucoadhesive drug delivery systems prolong water and stirred until completely dissolved. the residence time of the dosage form at the site Glycerol was used as plasticizer. After al of the of application or absorption. They provide rapid solutions were ful y dissolved, beakers were left absorption considering the surface area they in a refrigerator for a ful day to remove the air reach and high blood flow, so that they provide bubbles. The bubble-free solutions were effective treatment. QbD approach which careful y casted onto petri dishes. Films were provides a strategic process in product dried at 45 °C for 48 hours to evaporate the development used for this study. QbD solvent in a dry heat sterilizer. The dried films guarantees that, quality is offered by were packed in aluminum foil and stored in manufacturing instead of product testing. So desiccator at room temperature to keep their that, QbD ensures manufacturing of higher integrity. quality drugs within a shorter period and low costs [2]. Lidocaine, which is the API of this study, is a local anesthetic, cardiac depressant, 2.3. Optimization of muco-adhesive oralfilm and a pre-operation material for dental use. The of lidocaine hcl aim of the study was to ensure that the muco- adhesive films containing lidocaine HCl for After pre-formulation studies, optimization of dental usage has demanded quality at ributes the films was performed by Box Behnken during the product development with design using MODDE Pro version 12.1 implementation of QbD approach. software. Concentration of HPMC (X1), Na CMC (X2) both as muco-adhesive polymers 2. MATERIALS AND METHODS and concentration of glycerol (X3) as plasticizer were chosen as independent variable. 2.1. Materials Dependent variable pH (Y1), disintegration Lidocaine HCl (Doğa Ilaç, Istanbul, Turkey), time (Y2), tensile strength (Y3), and percentage HPMC (Sigma Aldrich, Switzerland), CMC elongation (Y4). Total of 12 film formulations (Aklar Kimya Ankara, Türkiye), Glycerol (F1- F12) shown in the Table 1were taken and (Tekkim Kimya, Bursa, Türkiye), Texture each film formulation was characterized in Analyzer (Stablemicrosystems, United triplicate. Kingdom), Dry Heat Sterilizer (Nüve, Turkey), Moisture Analyzer (Shimadzu, Japan), Orbital Shaker (Ika, Germany). 198 P27 3. RESULTS AND DISCUSSION F6 0 0.1 0.2 17.74 F7 0.4 0.1 0.08 17.46 3.1. Results and discussion of muco-adhesive F8 0.4 0.1 0.2 17.34 F9 0 0.2 0.14 17.7 lidocaine hcl film formulations F10 0.4 0 0.14 17.5 F11 0.4 0.2 0.14 17.3 F12 0.2 0.1 0.14 17.6 3.1.1. Mechanical Properties Tensile strength and elongation at break 4. CONCLUSION (mechanical properties) are vital for packing, handling procedures, dissolution behavior as By using QbD approaches different types of wel as stability of the film [3]. Tensile strength polymer and concentrations for polymers and of the oral films which prepared with high plasticizer are used as independent variable and amount of polymer are showed high results pH, disintegration time and mechanical compared with low concentrations. Using properties including tensile strength and NaCMC and HPMC in combinations showed percentage elongation are used as dependent higher tensile strength compared to using them variable to develop optimized formulation. alone. Using 1% of NaCMC and 2% HPMC MODDE Pro version 12.1 software was used showed higher tensile strength and using 0.5% for the optimization process and according to NaCMC alone showed the lowest tensile the results, design space was obtained. By using strength. Almost al of the results showed that, MODDE Pro version 12.1 software and linking increasing the amount of plasticizer leaded to it with QbD approaches, we understood the decrease in tensile strength. The percentage effect of polymer type and concentrations as elongation of oral films was found between wel as plasticizer concentration on the 10.554-99.402% (F1-F12). Increasing the disintegration time, pH, tensile strength, and amount of plasticizer are leading to increased percentage elongation. The results showed that amount of percentage elongation as showed in our optimum films exhibited good mechanical most of the formulations. The reason of increase properties including tensile strength and in elongation occurs since plasticizers decrease percentage elongation the inter-molecular bonds of polymer matrix 5. REFERENCES and change them with hydrogen bonds formed between polymer and plasticizer molecules. 1. Silva, B. M., et al., Mucoadhesive oral films: Formulation F3, F4, F10 and F11 showed good The potential for unmet needs. International elongation and tensile strength compared to journal of pharmaceutics, 2015. 494(1), 537- other formulations. 551. 2. Aksu, B., et al., Quality by design (QbD) for Table 1. Composition of film formulations pharmaceutical area. İstanbul Ecz. Fak. Derg. / J. Fac. Pharm. Istanbul, 2015.45(2), 233-251 Content (g) HPMC CMC Glycerol Lidocaine 3. Özcan Bülbül, E., et al., Product transfer from HCl F1 0.2 0 0.08 17.76 lab-scale to pilot-scale of quetiapine fumarate F2 0.2 0 0.2 17.64 orodispersible films using quality by design F3 0.2 0.2 0.08 17.56 approach. Journal of Drug Delivery Science and F4 0.2 0.2 0.2 17.44 F5 0 0.1 0.08 17.86 Technology, 2019. 54. 199 P28 STABILITY EVALUATION OF FLUCONAZOLE-LOADED FILAMENTS AND 3D PRINTED ORODISPERSIBLE TABLETS Mateusz Kurek1, Jolanta Pyteraf1, Witold Jamroz1, Justyna Knapik-Kowalczuk2, Marian Paluch2, and Renata Jachowicz1 1Department of Pharmaceutical Technology and Biopharmaceutics, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland; mateusz.kurek@uj.edu.pl 2A. Chelkowski Institute of Physics, University of Silesia in Katowice, ul. 75 Pulku Piechoty 1, 41-500 Chorzow, Poland 1. INTRODUCTION containing 50 mg of FLU were manufactured by Fused deposition modeling (FDM) is FDM ZMorph® 2.0 S (Poland) equipped with a considered as 3D printing method which has the 0.2 mm nozzle at 190 °C (20%, 40%) and 170 potential to be introduced as a preparation °C (70%) with 10 mm/s printing speed. method for personalized medicines. This 2.4. Evaluation of mechanical properties. technology utilizes filaments as a semi-product The uniformity of the filament was evaluated which should maintain its properties, i.e. using a two-dimensional laser diameter gauge mechanical and physicochemical properties, for (LDM25XY, Mercury-Tech Co., Ltd., China). a long time. 3D printed dosage forms should Mechanical properties were measured in a also be stable, but as the on-demand prepared stretching test for six randomly taken filament drugs, their stability can by significantly shorter pieces (n=6) carried out with an EZ-SX tensile e.g. two weeks. tester (Shimadzu, Japan). The purpose of the presented study was to analyze the impact of long-term storage on 2.5. Differential scanning calorimetry fluconazole-loaded filaments and short-term The thermal properties of the raw materials and storage of tablets printed with the fused the extrudates and tablets were examined using deposition modeling method. a DSC 1 STARe system (Met ler-Toledo, Switzerland). 2. MATERIALS AND METHODS 2.6. Disintegration time 2.1. Materials Pharmacopeial disintegration test was Fluconazole (FLU, 99.5%, Henan Tianfu performed in SAPO ED-2 apparatus Chemical Co. Ltd., China) was used as the (Electrolab, India) in 700 mL of purified water model active pharmaceutical ingredient (API) maintained at 37 °C. and poly(vinyl alcohol) (Parteck® MXP, Merck KGaA, Germany) as a filament-forming 2.6. Stability tests polymer. Al filaments were stored in string bags for 8 months, while tablets were kept in tablet 2.2. Hot-Melt Extrusion dispenser for 14 days under ambient conditions Filaments with 20%, 40% and 70% of (temp. 20-25 °C). After that, samples were fluconazole were successful y extruded using a reevaluated and the results were compared to 12-mm corotating twin-screw hot-melt extruder the freshly prepared filaments and tablets. (RES-2P/12A Explorer, Zamak Mercator®, Poland) equipped with a 1.75 mm nozzle at 3. RESULTS AND DISCUSSION 160°C, 147°C, and 137°C respectively. The Freshly prepared filaments were characterized filament was col ected on an air-cooled by good mechanical properties, which resulted conveyor belt and cut into approx. 2 m pieces. in their good printability. After eight months of 2.3. 3D printing storage, the breaking force decreased The tablets were designed with Blender® 2.90 significantly, while the elasticity specified by software (Blender Foundation, The the Young modulus was a lit le higher than for Netherlands) and sliced using Voxelizer® freshly prepared filaments. The loss of strength (version 1.4.18, Zmorph S.A., Poland). Printlets and increase in elasticity may be caused by water absorption during storage. The 200 P28 mechanical characteristic is presented in Table 40% aged 126.2 5.6 113 24 1. 70% fresh 64.9 3.8 128 8 Table 2. Mechanical properties of the filaments. 70% aged 71.1 4.0 136 37 Young The results of the disintegration time Filament Force measurement showed that tablets with slightly (N) SD modulus SD (MPa) longer disintegration times are produced with 20% aged filaments. It was possible to produce fresh 212.4 41.3 2070.9 117.9 tablets within mass variation pharmacopeial 20% aged 91.2 23.0 2219.9 141.4 limit using either fresh or aged filament. 40% fresh 152.1 22.5 1877.9 91.0 40% aged 68.6 20.7 2281.5 168.8 70% fresh 52.6 15.8 1341.3 185.2 70% aged 12.9 4.3 1634.3 247.7 Figure 8. Thermograms of fresh and 14-day-old tablets prepared from freshly extruded filaments. There were no significant differences in the thermal properties prepared from the freshly extruded and aged filaments. The 3D printed Figure 7. Comparison of thermograms of freshly prepared tablets after 14 days of storage under ambient and aged filaments. conditions remained almost unchanged in terms The DSC analysis revealed that fluconazole of crystal inity as the DSC curves were crystal ization occurs even in freshly prepared comparable. filaments with 70% fluconazole. After the 5. CONCLUSION storage time, an endothermic peak can be observed in the thermogram of the 40% The drug loading in the filaments and 3D fluconazole loaded filament while no sign of printed tablets has influenced its properties. crystal inity was found for aged filaments with However, the storage did not affect the 20% API. Despite the changes in filaments printability of the filaments and had only a mechanical properties and molecular order their slight impact on the 3D printed tablet at ributes, printability was maintained. In Table 2 the such as disintegration time. comparison of the tablets properties produced with fresh and aged filaments is presented. The ACKNOWLEDGMENT appearance of tablets produced with freshly extruded and aged filaments was comparable as The authors acknowledge the National Science al the physicochemical changes in the polymer Centre, Poland for financial support (grant matrix were reversed in the melting stage during OPUS 16 no 2018/31/B/ST8/01327). the 3D printing process. Table 2. Comparison of properties of selected tablets. Used Avg. mass filament (mg) SD Disintegr. time (s) SD 20% fresh 243.6 6.1 126 12 20% aged 239.5 1.8 134 1 40% fresh 121.1 2.2 88 3 201 P29 THE EFFECT OF NANO-MILLING ON DISSOLUTION/PERMEATION OF POORLY SOLUBLE DRUG COMPOUNDS Jakob T. Lynnerup 1,2, Jonas B Eriksen1, Ann Mari Holsæter2, Martin Brandl1 1Department of Physics, Chemistry & Pharmacy, University of Southern Denmark (SDU), Denmark 2Drug Transport and Delivery Research Group, Department of Pharmacy, UiT The Arctic University of Norway 1. INTRODUCTION AND AIM (v/v) trifluoracetic acid in purified water at a Many new drug entities arising are categorised flowrate of 1.0 mL min-1 and a column as class II compounds by the Biopharmaceutical temperature of 40 °C. Classification System, indicating low solubility 3. RESULTS AND DISCUSSION and fair permeability. These poorly soluble drug compounds show poor oral bioavailability due 3.1. Nano-milling to the low solubility. Different “candidate- nanoparticles were produced fast obtaining enabling” formulation principles are under nanoparticles after only 5 minutes (figure 1). investigation to either increase the solubility or Increasing the mil ing time led to smal er the dissolution rate of these compounds or both. particle sizes and a bigger partition of particles One of these is particle size reduction, such as in the nanosized range (figure 1 & 2). micronization and nano-mil ing [1]. The aim of this study was to produce nanoparticles of the poorly soluble model drug compounds Fenofibrate (FNB) and Cinnarizine (CNZ), further examining the combined dissolution and permeation behaviour of these compounds upon nano-mil ing. 2. MATERIALS AND METHODS 2.1 Nano-milling Figure 1. PSD as z-average diameter of FNB Wet bead mil ing using dual asymmetric (open bars) and CNZ (crossed bars) as wel as centrifugation was explored, using PDI (closed diamonds) of nanoparticle SpeedMixer™ DAC 150.1 FVZ-K (Synergy suspensions mil ed at 1500 rpm, measured at Devices Ltd, High Wycombe, U.K.). different timepoints, reported as mean ± SD (n 2.4 Combined dissolution/permeation- study = 3). A mil ing time of 90 minutes was dissolution/permeation setup was utilised: identified as optimum and used for further Firstly, 10 mg of raw API was added to the bot om chamber of jacketed Franz cel s and experiments. stirred by a magnetic stirrer V6A (PermeGear 3.2. Physical characterisation Inc). Cel ophane was used as the permeation The produced nanoparticles were crystal ine barrier. The bot om chamber was fil ed with with regards to DSC and XRPD (data not PBS and the top chamber was fil ed with 1 mL shown here). of acceptor medium. No obvious phase changes can be determined 2.5 API quantification based on these results, but further investigation by reverse phase high HPLC: FNB was is needed. quantified with a XSelect CSH C18 column (Waters Corp, Milford, MA, U.S.) with a mobile phase consisting of 80 % (v/v) of acetonitrile (ACN) and 20 % (v/v) of 0.1 % 202 P29 3.3. Combined dissolution and permeation Fig. 2. Dissolution in PBS and permeation profiles shown as the concentration of dissolved API in the donor compartment (closed circles), cumulative mass of permeated drug (open squares), and flux values (grey bars) of raw CNZ (a), CNZ nanoparticles (b), raw FNB (c), and FNB nanoparticles (d) at 37 °C, reported as mean ± SD (n = 3). __________________________________________________________________________________ Before conducting the combined dissolution 150.1 FVZ-K. The produced nanoparticles were and permeation experiments, the solubility of crystal ine and no phase changes could be the produced nanoparticles was investigated. observed, however further investigation of the No apparent difference in solubility could be physical characteristics are needed. observed after equilibration over 72 hours. The nanoparticles of Fenofibrate induced a The nanoparticles obtained showed an increase transient supersaturated state, thereby in dissolution and permeation when compared increasing the permeation. Cinnarizine to the pure compounds. FNB nanoparticles nanoparticles increased the dissolution rate and induced a transient supersaturated state. The thereby increased the permeation. Further amount of dissolved drug rapidly fel to the drug experiments are needed to determine the solubility over 120 minutes. This behaviour of partition of solubilizers on the dissolution and nanoparticles is similar to that found for the permeation behaviour. commercial product Lipidil 145 ONE® found 5. REFERENCES by Sironi et al. (2017) [2]. CNZ nanoparticles instead showed a faster dissolution rate. This 1. Buckley, S. T. et al., Biopharmaceutical increase in dissolved drug, when compared to classification of poorly soluble drugs with the pure compound, resulted in a higher respect to “enabling formulations” . European Journal of Pharmaceutical Sciences, 2013, 50. permeation and flux for both FNB and CNZ. 2. Sironi, D. et al., Dynamic dissolution/- The faster dissolution rate could also be permeation-testing of nano- and microparticle at ributed from the addition of solubilizers formulations of fenofibrate. European Journal of during the production of the nanoparticles. As Pharmaceutical Sciences, 2017, 96. the contribution of release of drug from ACKNOWLEDGMENT particles in suspension and solubilised drug is not differentiated in these experiments. The authors are grateful for a mobility grant to 4. CONCLUSION JTL and col aborative work between the two Nanoparticles of the poorly soluble drug institutions enabled by funding from Nordforsk compounds Fenofibrate and Cinnarizine could program Nordic University Hub (project be produced using the SpeedMixer™ DAC #85352) “NordicPOP”. Excellent lab-support by Kiril Jefimov is gratefully acknowledged. 203 P30 FREEZE-DRIED NANOCRYSTAL DISPERSION OF NOVEL DEUTERATED PYRAZOLOQUINOLINONE LIGAND (DK-I-56-1): PROCESS PARAMETERS AND CRYOPROTECTANT SELECTION THROUGH STABILITY STUDY Jelena Mitrović1, Maja Bjelošević2, Daniel E. Knutson3, Aleksandar Kremenović4, Dominique Lunter5, Pegi Ahlin Grabnar2, James M. Cook3, Miroslav M. Savić6, Snežana D. Savić1 1Department of Pharmaceutical Technology and Cosmetology, University of Belgrade–Faculty of Pharmacy, Serbia 2Department of Pharmaceutical Technology, University of Ljubljana, Faculty of Pharmacy, Slovenia 3Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, United States 4Laboratory of Crystallography, University of Belgrade-Faculty of Mining and Geology, Serbia 5Institut für Pharmazeutische Technologie, Eberhard-Karls Universität Tübingen, Germany 6Department of Pharmacology, University of Belgrade – Faculty of Pharmacy, Serbia 1. INTRODUCTION 2.1. Materials Nanocrystal dispersions are considered as the DK-I-56-1 was synthesized at the Department universal formulation strategy for brick dust of Chemistry and Biochemistry, University of substances. However, the stability of these Wisconsin—Milwaukee, USA. The fol owing systems to aggregation represents a big issue. other materials were used: polysorbate 80, To overcome this, nanocrystal dispersions are poloxamer 407, sucrose, mannitol (Sigma- usual y solidified by freeze-drying Aldrich Laborchemikalien GmbH, Germany) (lyophilization). During this process the risk of and trehalose (Carl Roth GmbH, Germany). aggregation is considered to be high, due to ice formation and/or water loss. To prevent the 2.2. Lyophilization aggregation, For the particle size preservation, Nanocrystal dispersions stabilized by therefore, it is necessary to add polysorbate 80 and poloxamer 407 were cryoprotectants/lyoprotectants, among which prepared by wet bal mil ing [3]. After addition sugars are most commonly used. To ensure of mannitol (M), sucrose (S), or trehalose (T) good structure of the cake, bulking agents are alone or in combination samples were freeze- often included in formulations, as wel [1,2], dried. Two processes were applied: (1) freezing although in nanocrystal ine dispersions the at -80 °C (3 h), primary drying at -10 °C, 0.340 combination of cryoprotectants and bulking mbar, secondary drying at 25 °C (24 h) or (2) agents is not frequent nor much investigated. freezing at -50 °C (3 h), primary drying at -45 °C, 0.2 mbar (21 h), secondary drying at 20 °C Nanocrystals of DK-I-56-1 (7‑methoxy‑2- (30 h). Samples were stored in crimped vials at (4‑methoxy‑d3-phenyl)-2,5-dihydro-3H- 25 °C (lyophilization 1) or 2-8 ºC pyrazolo[4,3-c]quinolin-3-one), patent (lyophilization 2) for three months. protected pyrazoloquinolinone ligand, have been developed recently, and characterized in 2.3. Physicochemical characterization terms of physicochemical properties and Particle size (z-ave) was measured by Zetasizer pharmacokinetics after intraperitoneal Nano ZS (Malvern Instruments, UK) and administration in mice. These formulations Mastersizer (Malvern Mastersizer 2000 were stable for three weeks [3]. Our aim in this Malvern, UK). Redispersibility index (RDI) study was to improve the stability by freeze- was calculated as z-ave (before)/z-ave (after) drying, and investigate the influence of different and expressed in percentages. Physical state of concentrations and physical form of samples was determined by differential cryoprotectants (sucrose, trehalose) and bulking scanning calorimetry (DSC1; Met ler Toledo, agent (mannitol) as wel as different primary Switzerland),powder X-ray diffraction (Rigaku drying conditions on the aggregation Smartlab X-ray Diffractometer) and polarized prevention. light microscopy (PLM) (Carl Zeiss ApoTome Imager Z1 microscope Zeiss, Germany). 2. MATERIALS AND METHODS 3. RESULTS AND DISCUSSION 204 P30 Right after preparation, nanocrystal dispersions containing it. Amorphous state of were with submicron particle size around 160 lyoprotectants al ows maximal hydrogen nm, and PDI below 0.2, suggesting narrow size bonding due to higher molecule flexibility and distribution. In the cryoprotectant screening availability of hydroxyl groups [3]. phase, sucrose and/or mannitol were added in Surprisingly, mannitol as a substance with high different concentrations. It was shown that 10% crystal ization tendency was with low of the total stabilizer concentration was needed crystal inity in lyophilizates. These for the particle size preservation: the achieved observations were confirmed by PLM. It is RDI was above 95%, while cakes with sucrose possible that it formed interactions with sucrose alone or in combination with mannitol in ratio or nanocrystal stabilizers [4]. 1:1 or 3:2 were also with satisfied appearance (Figure 1). 4. CONCLUSION Results from this study demonstrated freeze- drying as an important technique for the improvement of nanocrystals stability. However, the selection of cryoprotectant and bulking agent ratio beside process parameters (primary drying at -45 ºC) was crucial to get freeze-dried samples with good stability. Sucrose or trehalose in combination with mannitol (ratio 3+2) in total concentration 10% successful y hindered aggregation, thus prolonging the stability to 3 months at 2-8 ºC. 5. REFERENCES Figure 1. Particle size (z-ave), polydispersity index (PDI), redispersibility index (RDI) and 1. Van Eerdenbrugh, B., et al. Top-down cake appearance after lyophilization. production of drug nanocrystals: nanosuspension stabilization, miniaturization Lyophilization was conducted above or below and transformation into solid products. the glass transition temperature of the International journal of pharmaceutics, 2008. maximal y freeze-concentrated solution (Tg’) 364(1): 64-75. (around -39 ºC). When primary drying was 2. Trenkenschuh, E., and Friess, W. Freeze-drying performed at -10 °C, no aggregation was of nanoparticles: How to overcome colloidal noticed right after lyophilization, but particle instability by formulation and process size increased significantly, lowering down the optimization. European Journal of RDI to < 50%, after one month storage at 25 °C. Pharmaceutics and Biopharmaceutics, 2021.165: 345-360. This was confirmed by laser diffraction. In 3. Mitrović, J.R., et al. Overcoming the low oral lyophilization 2, with primary drying at bioavailability of deuterated temperature below Tg’, trehalose was also used pyrazoloquinolinone ligand DK-I-60-3 by in the same concentration as sucrose and in nanonization: A knowledge-based approach. combination with mannitol. Interestingly, in Pharmaceutics, 2021. 13(8): 1188. this process parameters setup, sucrose or 4. Kumar, S., et al. Sugars as bulking agents to trehalose alone did not prevent aggregation prevent nano-crystal aggregation during spray during freeze-drying. Particle size remained or freeze-drying. International journal of almost unchanged in formulation S+M 3+2 pharmaceutics, 2014. 471(1-2): 303-311. (RDI 95%) or slightly higher in T+M 3+2 (RDI ACKNOWLEDGMENT 90%), after three months storage, suggesting it was most probably the optimal combination for This research was supported by the Science the stabilization. Fund of the Republic of Serbia, grant No. 7749108, project Neuroimmune aspects of Physical state analysis revealed that sucrose and mood, anxiety and cognitive effects of mannitol in samples lyophilized by process 1 leads/drug candidates acting at GABAA and/or were in crystal ine state, as wel as sucrose sigma-2 receptors: In vitro/in vivo delineation when used alone in lyophilization 2. Trehalose, by nano- and hiPSC-based platforms- on the other hand was amorphous in al samples NanoCellEmoCog. 205 P31 PREPARATION OF ANISOTROPIC HOLLOW SILICA NANOSTRUCTURES Sebastjan Nemec1,2, Tanja Potrč2, Petra Kocbek2, Slavko Kralj1,2,3 1Department for Materials Synthesis, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia, 2Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia, 3Nanos SCI, Nanos Scientificae d.o.o., Teslova 30, 1000 Ljubljana, Slovenia 1. INTRODUCTION Nanos SCI (Slovenia) [4]. Additional coating of Nanostructures with hol ow compartments are a porous silica was deposited on the surface of particular class of nanomaterials [1]. The internal nanochains either by using the standard Stöber void offers a space for the incorporation of various silica synthesis procedure [3] or our own soft- functional cargo(s), such as drugs and catalysts. templating procedure [5]. However, the available synthetic procedures to 2.2. Methods prepare such complex nanostructures are scarce The acid etching procedures were carried out using and not wel elucidated. So far, various approaches 3 M or 12 M hydrochloric acid (HCl). Suspensions have been studied to prepare hol ow of nanochains with different silica coatings were nanostructures. The most widely used etching mixed with either 3 M or 12 M HCl to prepare methods rely on selective removal of sacrificial partial y hol ow or completely hol ow silica core from pre-synthesized nanoparticles while nanostructures, respectively. To terminate the core retaining the outer shel intact. If the sacrificial core removal process, the formed (partial y) hol ow is not removed completely, partial y hol ow nanostructures were centrifuged and quickly nanostructures are obtained which may offer washed several times with water, freezed in liquid combined functionalities of the shel and the nitrogen and dried using lyophilization. remaining core [2]. However, the procedures for the preparation of partial y hol ow nanostructures 2.3. Characterization are general y even more chal enging than obtaining Al types of nanochains, either treated with HCl or completely hol ow nanostructures, as the core not, were characterised using transmission electron removal process needs to be precisely control ed microscopy (TEM) and nitrogen sorption analysis. and should offer the possibility for rapid termination of the core removal procedure. Core- 3. RESULTS AND DISCUSSION shel nanoparticles provide an elegant way to control the core removal process. The shel that We chose thin-silica-coated iron oxide-based nanochains as starting nanomaterial for the surrounds the core provides a degree of protection to the core and may regulate the rate of core preparation of (partial y) hol ow nanostructures. removal process. By changing the thickness and To study the effect of different silica shel s on porosity of the shel , the core removal rate can be the removal rate of the iron oxide cores, we effectively control ed. coated the commercial nanochains with an additional ~30 nm thick low-porous silica shel Therefore, in our work we focused on the or a ~60 nm thick mesoporous silica shel with preparation of (partial y) hol ow nanostructures, i.e. pore diameter of ~15 nm. nanochains, based on core-shel superparamagnetic nanoparticles and investigation, how the thickness The combination of iron oxide core and silica and porosity of the silica shel affect the removal of shel is suitable for the preparation of (partial y) magnetic core. hol ow nanostructures using HCl etching method due to the differences in solubility of 2. MATERIALS AND METHODS both materials in HCl. In contrast to silica, iron oxide is wel soluble in HCl. This enabled us to 2.1. Materials use HCl etching to selectively (partial y) Three different types of silica coated nanochains remove the iron oxide cores and retain the outer were used for the preparation of the partial y hol ow silica shel s intact. Complete iron oxide core nanostructures. Commercial y available 1 μm long removal was achieved using concentrated 12 M nanochains coated with a thin 3 – 5 nm layer of HCl in al three different nanochain samples. low-porous silica (Fig. 1A) were provided by The etching was conducted until we achieved a 206 P31 complete removal of the core, which was 4. CONCLUSION confirmed by TEM (Fig. 1B-D). On the other hand, partial y hol ow nanostructures were We successful y prepared (partial y) hol ow prepared using 3 M HCl and precisely silica nanostructures using HCl etching from control ed etching times, that were determined silica-coated iron oxide-based nanochains. By experimental y. synthesizing an additional coating, we prepared thicker and/or mesoporous silica shel s on the Nitrogen sorption analyses showed increased commercial nanochains. Furthermore, we specific surface areas and pore volumes of the examined how different thicknesses and hol ow nanostructures, compared to their morphologies of the silica shel affect the corresponding starting non-etched nanochains. removal rate of the iron oxide cores. By The highest specific surface area and pore applying silica shel s which provide larger volume increase were determined for the degree of protection of the iron oxide against complete core removal of commercial acid dissolution, we are able to control the core nanochains, with an increase from 223 m2/g to removal process in order to obtain partial y 537 m2/g and 0.6 mL/g to 2.9 mL/g, hol ow nanostructures. The prepared partial y respectively. The time needed to achieve hol ow nanostructures wil be investigated in complete core removal was the longest (2 h) for future as magneto responsive delivery systems the thick low-porous silica-coated nanochains, for different types of drug molecules. whereas the etching times for the thin low- porous and mesoporous silica coated 5. REFERENCES nanochains were in both cases shorter, i.e., less than 1 min. Such time difference indicates the 1. Sharma, J., et al., Hollow silica particles: recent increased dissolution-protecting ability of the progress and future perspectives, Nanomaterials, 2020, 10(8). thick low-porous silica coating of nanochains 2. Priebe, M., et al., Nanorattles or yolk-shell towards HCl etching. Even though the nanoparticles – What are they, how are they mesoporous silica coated nanochains had the made, and what are they good for? , Chemistry thickest silica shel , the large number of centro- – A European Journal, 2015, 21(10): 3854- radial pores diminished the shell’s protecting 3874. ability. 3. Stober, W., et al., Controlled growth of monodisperse silica spheres in the micron size range, 1968, 26(01): 1599. 4. Kralj, S., et al., Magnetic Assembly of Superparamagnetic Iron Oxide Nanoparticle Clusters into Nanochains and Nanobundles, ACS Nano, 2015, 9(10): 9700-9707. 5. Nemec, S., et al., A Versatile Interfacial Coassembly Method for Fabrication of Tunable Silica Shells with Radially Aligned Dual Mesopores on Diverse Magnetic Core Nanoparticles, ACS Applied Materials & Interfaces, 2021, 13(1): 1883-1894. ACKNOWLEDGMENT The authors acknowledge the financial support from the Slovenian Research Agency (ARRS; Young Researcher Scheme 1000-18-0106, ARRS projects: J2-3043, J2-3040, J2-3046, J3- 3079 and Core Funding program P2-0089) and Figure 1. TEM micrographs of non-etched thin- the CENN Nanocenter for use of electron silica (A), thick low-porous silica (C), and microscopes. mesoporous silica (E) coated nanochains and their (partial y) hol ow counterparts: thin-silica (B), thick low-porous silica (D), and mesoporous silica (F) nanostructures. Scale bars: 500 nm. 207 P32 PREPARATION AND IN VITRO CHARACTERIZATION OF ONDANSETRON HYDROCHLORIDE LOADED LIPOSOME FORMULATIONS Zeliha Duygu Özdal1,2, Sevgi Takka1 1Gazi University, Department of Pharmaceutical Technology, Faculty of Pharmacy, Ankara, Turkey, 2Erzincan Binali Yıldırım University, Department of Pharmaceutical Technology, Faculty of Pharmacy, Erzincan, Turkey 1. INTRODUCTION type sonicator at 20% amplitude for 15 minutes Ondansetron hydrochloride (OND) is a at 2 s on and 3 s off intervals, then extruded. LPs hydrophilic drug used for management of were recovered by centrifugation at 18000 rpm chemotherapy, radiotherapy, and surgery for 45 min, and lyophilized. induced nausea and vomiting. OND has low Table 1. Composition of OND loaded LPs oral bioavailability and short half-life therefore it needs to administrate several times in a day Formulation PL:CHOL(molar PL [1]. OND has a pH-dependent solubility. The ratio) solubility decreases as the pH of the aqueous LP1 2:1 DPPC phase increases [2]. OND-loaded liposome LP2 4:1 DPPC (LPs) formulations have been prepared to LP3 6:1 DPPC reduce dosing frequency by prolonging the LP4 2:1 HSPC drug's residence time in circulation. This study LP5 6:1 DPPC was aimed to improve the encapsulation 2.3. Characterization of Liposomes efficiency by utilizing the pH-dependent solubility of OND. The mean particle size (PS), size distribution (PDI) and zeta potential (ZP) of LPs were 2. MATERIALS AND METHODS analysed by Zetasizer Nano ZS (Malvern Instruments Malvern, UK). 2.1. Materials Encapsulation efficiency (EE%) was 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, determined by lyophilized LPs formulations 16:0 PC (DPPC), hydrogenated soy dissolving in methanol. OND amount in this phosphatidylcholine (HSPC), and cholesterol solution was determined by HPLC analysis at were purchased from Avanti Polar Inc USA. 310 nm. Chloroform was purchased from Sigma. OND was a gift from Adeka Pharmaceutical The possible interactions between drug and the (Istanbul, Turkey). components of LPs were examined by Differential scanning calorimetry (DSC) and 2.2. Preparation of Liposomes Fourier-Transform Infrared Spectroscopy OND loaded LPs were prepared by film (FTIR). hydration method. Briefly, different molar ratio The in vitro release profile of OND loaded LPs of phospholipid (PL) and cholesterol were formulations and free OND were compared dissolved in chloroform (2 mL) as shown in using Franz cel with a dialysis membrane with Table 1. Chloroform was evaporated to obtain a molecular weight cut-off of 14 kDa at pH 7.4. the thin film. The film was hydrated using 5 mL The samples were withdrawn from receptor ultrapure water containing 4 mg OND. For LP5 phase at the determined time points of 0.5, 1, 2, formulation, drug was mixed with lipids and 3, 4, 6, 8, 12, 24 h and replaced with the fresh dissolved in chloroform. After evaporation the buffer. The samples were analysed by HPLC. film was hydrated using 5 mL phosphate buffer solution (pH 7.4) to increase the encapsulation 3. RESULTS AND DISCUSSION efficiency by reducing the drug leakage. In order to reduce particle size, the liposomal OND loaded sustained release LPs formulations suspension was first sonicated with a probe- were prepared successful y by film hydration 208 P32 method. The effect of type and molar ratio of 120,0 ) phospholipid and pH of hydration media on 100,0 formulation characteristic of the LPs were evaluated and presented in Table 2. The particle 80,0 size of the formulations from LP1 to LP4 was 60,0 measured between 178-217 nm and PDI values 40,0 below 0.15. However, the particle size of LP5 ulative drug release (% 20,0 formulation was 368 nm, PDI value was around cum 0.5. 0,0 0 5 10 15 20 25 The EE% of the LP1 and LP4 formulations time (hour) free OND LP1 LP2 prepared using different PL types were LP3 LP4 LP5 determined to be similar. Although the encapsulation efficiency increased as the amount of PL increased, it was stil low. The pH Figure 1. Release profile of OND loaded LPs and of the aqueous phase was altered to reduce drug free OND leakage and enhance the EE%. The EE% 4. CONCLUSION increased from 16 % to 38 % at pH7.4, where the solubility of OND is low. Results indicate that the molar ratio, type, and Tg of phospholipids affect the physicochemical Table 2. Physicochemical properties of LPs properties of LPs. Moreover, higher EE% was formulations obtained by altering the pH of the aqueous PS PDI ZP %EE phase. Al formulations were exhibited a LP1 178,2± 0,072± -15,2± 3,89± sustained-release characteristic profile. 2,6 0,004 6,2 0,25 5. REFERENCES LP2 217,0± 0,148± -11,4± 8,43± 6,3 0,047 3,0 0,13 1. Ye J.H., et al., Ondansetron: A Selective 5-HT3 LP3 212,3± 0,138± -7,38± 16,98± Receptor Antagonist and Its Applications in 5,5 0,022 1,9 0,36 CNS-Related Disorders. CNS Drug Reviews, 2001. 7:199-213. LP4 187,3± 0,082± -28,8± 3,50± 2. Duong, V. A., et al., Preparation of ondansetron 4,8 0,015 0,3 0,14 hydrochloride-loaded nanostructured lipid LP5 368,6± 0495±0, -17,1± 38,16± carriers using solvent injection method for 26,7 059 4,0 5,63 enhancement of pharmacokinetic properties. Pharmaceutical research, 36(10), 1-As a result of DSC and FTIR analysis, no 12 interaction was observed between the drug and 3. Chen J., et al., Influence of lipid composition on the phase transition temperature of liposomes excipients. composed of both DPPC and HSPC. Drug Free OND was almost completely released in 8 Development and Industrial Pharmacy, 2013; h while al LPs formulations were exhibited 39(2): 197–204. sustained release profiles (Fig 1). It was observed that increasing the molar ratio of phospholipid decreased the cumulative release ACKNOWLEDGMENT amount of drug in the LPs prepared with DPPC. This work has been supported by Gazi On the other hand, HSPC LPs had a slower University Scientific Research Projects release profile. The phase transition Coordination Unit under grant number TCD- temperature (Tg) of DPPC and HSPC are 41 and 2021-6957. 55°C, respectively. The lipid membranes with higher Tg demonstrate lower fluidity compared to membranes with lower Tg [3]. Therefore, lower drug released was obtained from HSPC LPs. There was no significant difference between the release profiles of the LP3 and LP5 formulations (p>0.05). 209 P33 DEVELOPMENT OF COMBINED INHALABLE FORMULATION OF IBUPROFEN AND MANNITOL FOR THE TREATMENT OF CYSTIC FIBROSIS Petra Party1, Rita Ambrus1 1Institute of Pharmaceutical Technology and Regulatory Affairs, University of Szeged, Hungary 1. INTRODUCTION aspirator capacity: 85%, airflow rate: 500 L/h, Ibuprofen (IBU) is a nonsteroidal anti- and feed pump rate: 10%. [3] inflammatory drug (NSAID), and inflammation 2.3. Investigation methods is a hal mark of cystic fibrosis (CF), which The investigation protocol of the dry powder contributes to lung destruction. IBU has a inhalers was fol owed during the significant effect on slowing the disease characterization of our products. [4] Laser progression and it is wel tolerated. [1] Mannitol diffraction was used to determine the particle (MAN) helps to dilute the thick, viscous mucus, size and the particle size distribution of the which is the main problem of CF.[2] Both of the microsuspension and the spray-dried samples mentioned drugs should be applied in high dose. (Malvern Mastersizer Scirocco 2000, Malvern Therefore, the preparation of inhalable IBU and Instruments Ltd., Worcestershire, UK). The MAN particles could be efficient for targeted active ingredient content and the solubility of lung delivery, therefore the applied dose could the spray-dried samples was determined be reduced. We aimed to combine IBU and spectrophotometrically (ATI-UNICAM UV/VIS MAN in a dry powder formulation to help Spectrophotometer, Cambridge, UK). To patients, who suffer from CF. establish the crystal ine character of the samples 2. MATERIALS AND METHODS X-ray powder diffraction (XRPD) spectra were recorded (BRUKER D8 Advance X-ray 2.1. Materials diffractometer, Bruker AXS GmbH, Karlsruhe, The active ingredient (API) was ibuprofen (Egis Germany). The bulk and tapped densities of the Pharmaceuticals PLC., Budapest, Hungary). formulations were measured using a tap density Poloxamer-188 (POL) (Sigma-Aldrich, tester (ETD-1020x, Electrolab, Mumbai, India). Darmstadt, Germany) D-mannitol (MAN), The in vitro aerodynamic properties were (Molar Chemicals Kft, Halásztelek, Hungary) investigated with Andersen Cascade Impactor and L-leucine (LEU), (AppliChem GmbH, (Apparatus D, Copley Scientific LTD., 250 Darmstadt, Germany) were the excipients. Nottingham, UK). 2.2. Preparation of the samples 3. RESULTS AND DISCUSSION Firstly, a presuspension was made, which As a result of wet mil ing IBU was micronized contained 2.00 g of pure IBU and 18.0 g of 1 % (Table 1.). The particle size of the spray-dried POL solution. The mil ing medium was 20.00 g formulations was in the required pulmonary of ZrO2 beads in a high performance mil size range in the case of LEU containing (Planetary Micro Mill Pulverisette 7, Fritsch, samples (Table 1.). LEU also had an increasing Idar-Oberstein, Germany Retsch Planetary Ball effect on the yield of spray-drying. The content Mill PM 100 MA, Retsch GmbH, Haan, of API was correlated with the theoretical Germany). Rotation speed (800 rpm), mil ing numbers. The solubility of the IBU was duration (4 cycles) was applied, each mil ing improved thanks to the micronization. The cycle comprised 15 min rotation fol owed by 10 compressibility index increased by adding more min pause. Different compositions were LEU (Table 2.): According to the XRPD prepared by adding MAN and LEU. Secondly, spectra, IBU became amorphous after the inhalable microparticles were produced by preparation process (Figure 1.). The best spray-drying (Büchi Mini Spray Dryer B-191, formulation (IBU1_POL_MAN2_LEU1) was Büchi, Flawil, Switzerland). The spray-drying chosen for the in vitro aerodynamic properties were the fol owing: inlet measurements, which were implemented using temperature: 70 °C, outlet temperature: 40 °C, Ezeeflo™ hydroxypropyl methylcellulose 210 P33 capsules (ACG-Associated Capsules Pvt. Ltd., Figure 1. XRPD results of the raw materials and the Mumbai, India) and gelatine capsules spray-dried samples. (Capsugel, Bornem, Belgium). The MMAD (mass median aerodynamic diameter and FPF Table 3. In vitro aerodynamic properties of the (fine particle fraction) were convenient for spray-dried sample pulmonary application (Table 3.). (IBU1_POL_MAN2_LEU1). Data are means ± SD (n = 3 independent measurements). Table 1. Particle size of the raw API, the suspension and the spray-dried samples. Data are Samples FPF (%) MMAD means ± SD (n = 3 independent measurements). (µm) Cellulose capsule 60.72±0.39 2.58±0.29 Samples D [0.5] (μm) Span SSA (m2/g) Gelatine IBU_POL_ 4.234± 3.922± 1.630± capsule 55.92±0.80 3.30±0.71 suspension 0.51 0.33 0.09 IBU1_POL 7.174± 4.212± 1.022± 4. CONCLUSION 1.06 0.28 0.13 IBU1_POL_ 8.071± 2.04± 0.994± We successful y formulated IBU and MAN MAN2 1.99 0.71 0.05 containing inhalable powders. We managed to IBU1_POL_ micronize the API and improve its solubility. MAN2_LEU0. 3.380± 3.513± 2.105± We observed the effect of LEU on the yield of 5 0.13 1.44 0.13 spray-drying, the particle size and the density. IBU1_POL_ 3.275± 1.657± 2.175± Based on the proper aerodynamic diameter and MAN2_LEU1 0.10 0.30 0.05 fine particle fraction, the formulations are promising for further characterization. In the future, we can provide a combined, innovative Table 2. Density measurements of the spray- treatment for CF. dried compositions. Data are means ± SD (n = 3 independent measurements). 5. REFERENCES 1. Sheikh, Z., et al., Is there a role for inhaled Bulk Tappe anti-inflammatory drugs in cystic fibrosis densit d treatment? Expert Opin. Orphan Drugs Samples y densit Carr 2018, 6, 69–84. (g/cm3 y index 2. Bilton, D., et al. Inhaled dry powder mannitol in cystic fibrosis: An efficacy and ) (g/cm3 ) safety study. Eur. Respir. J. 2011, 38, 1071– 1080. IBU1_POL 0.13 0.16 20.00 3. Party, P., et al. Formulation and In Vitro and IBU1_POL_MA In Silico Characterization of “ Nano-in- N2 0.17 0.25 33.33 Micro ” Dry Powder Inhalers Containing IBU1_POL_MA Meloxicam. Pharmaceutics 2021, 13, 1–18. N2_LEU0.5 0.22 0.35 37.14 4. Pal agi, E., et al. New aspects of developing a dry powder inhalation formulation IBU1_POL_MA applying the quality-by-design approach. N2_LEU1 0.17± 0.28± 38.00 ± Int. J. Pharm. 2016, 511, 151–160. ACKNOWLEDGMENT The work was supported by Gedeon Richter’s Talentum Foundation, and Gedeon Richter Plc. Centennial Foundation (Gyömrői 19-21, Budapest, H-1103, HU), and Project no. TKP2021-EGA-32 implemented with support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development, and Innovation Fund, financed under the TKP2021-EGA funding scheme, and Hungarian-Slovenian Bilateral Project 2019-2.1.11-TÉT-2020-00147. 211 P34 ORAL DOSAGE FORMS WITH CARVEDILOL FABRICATED BY SELECTIVE LASER SINTERING (SLS) 3D PRINTING TECHNIQUE Nikola Pešić1, Mirjana Krkobabić2, Ivana Adamov1, Svetlana Ibrić1, Branka Ivković3, Đorđe Medarević1 1Department of Pharmaceutical Technology and Cosmetology, Faculty of Pharmacy, Belgrade, Serbia 2Pharmaceutical and Chemical Industry, Zdravlje AD, Leskovac, Serbia 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Belgrade, Serbia 1. INTRODUCTION When it comes to pharmacy, 3D printing has Powder for 3D printing was obtained by mixing gained immense popularity in recent years due al the components of the formulation and to its revolutionary use in printing drugs tailored sifting through a sieve with a diameter of 180 to individual patient needs [1,2]. Selective laser µm. sintering (SLS) is an industrial 3D printing 2.3. 3D printing of oral dosage forms technique which uses a powder bed to build up the 3D object thanks to a laser which binds the A cylindrical 3D models of the printed tablets powder particles together. Advantages of SLS (8.00 mm diameter and 2.00 mm thickness) technique include the fact that it is a solvent-free were designed with Autodesk Fusion 360 process and offers relatively fast production. software version 2.0.8809 (Autodesk Inc, San Until today, a limited number of studies Rafael, CA, USA), exported as a investigating the production of drug dosage stereolithography file (.stl) and printed with forms using SLS have been reported [2,3]. Sintratec Kit 3D printer (Sintratec AG, Switzerland). The printing parameters were 2. MATERIALS AND METHODS control ed using Sintratec 3D printer software. 2.1. Materials After a series of variations in temperature and laser speed, the optimal values of these Carvedilol (CRV) was used as a model parameters used in the 3D printing process were substance in this study and it was donated by established and shown in Table 2. Hemofarm (Vršac, Serbia). The following excipients used to obtain 3D printing tablets: Table 2. SLS 3D printing process parameters polyvinyl alcohol (PVA, Merck), mannitol Surface Chamber Laser (Parteck® M, Merck), Ludipress® Temperature Temperature speed Hatch space (coprocessed excipient consisting of 93% ( ◦C) ( ◦C) (mm/s) lactose monohydrate, 3.5% crospovidone 80 ºC 70 ºC 60 250 µm (Kol idon® CL) and 3.5% povidone K30 2.4. Mechanical properties of 3D tablets (Kol idon® 30), BASF), talc (Merck) and candurin (Candurin® Gold Sheen, Merck). Tablets (n = 10) were weighed on a Sartorius BP 210 D analytical balance (Sartorius, Goet ingen, 2.2. Preparation of formulations Germany) and measured (diameter and The compositions of the formulations are thickness) using a digital caliper (Vogel, shown in Table 1. Kevelaer, Germany). Table 1. Composition of the formulations 2.5. Powder X-ray diffraction analysis (PXRD) Material Formulation 1 Formulation 2 CRV 10% 10% PXRD analysis was performed to assess PVA 55% 55% whether the laser induced amorphization of any Parteck® M 30% / of the compounds, especial y amorphization of Ludipress® / 30% poorly soluble CRV. Samples were col ected Talc 2% 2% Candurin® using a Philips PW-1050 (Philips, The Gold Sheen 3% 3% Netherlands) diffractometer, operated at 40 kV and 30 mA, using Ni-filtered Cu Kα radiation. 212 P34 2.6. Dissolution and Drug Release Analysis 3.4. Dissolution and Drug Release Analysis Dissolution testing was performed under non- sink conditions using mini paddle apparatus (Erweka DT 600, Germany) with a paddle rotation speed of 50 rpm for 8 h, in 100 ml of phosphate buffer (pH 6.8). The amount of dissolved CRV was determined by HPLC method using Dionex Ultimate 3000 (Thermo Scientific, USA) HPLC system. 3. RESULTS AND DISCUSSION Figure 2. Dissolution profiles of 3D printing 3.1. 3D printing process tablets 4. CONCLUSION It was shown that SLS printer was able to fabricate 3D tablets with CRV, as wel as that SLA represents a new chapter in 3D printing of success of the printing process depended on the solid oral dosage forms and in individualized used printing parameters. therapy in particular. By adjusting the formulation and process parameters, it was 3.2. Mechanical properties of 3D tablets possible to produce SLS tablets with co- amorphous CRV and PVA as a main polymer. The dimensions of the obtained 3D tablets were Complete drug release was achieved under non in accordance with the defined values of the sink conditions after 8 hours in phosphate created 3D models (F1: 8.10 ± 0.08 mm buffer. The tailoring of drug release might be diameter and 2.10 ± 0.13 mm thickness, F2: achieved by varying formulation factors as wel 8.13 ± 0.09 mm diameter and 2.10 ± 0.12 mm as process parameters, although it could be thickness). Significant variations in tablet governed by the composition of the whole weight between formulations were not observed formulation. (m1=0.146 ± 0.04; m2=0.136 ± 0.03). 5. REFERENCES 1. Goynez, A., et al., 3D Printing of Medicines: 3.3. Powder X-ray diffraction analysis Engineering Novel Oral Devices with Unique (PXRD) Design and Drug Release Characteristics. Molecular pharmaceutics, 2015. 12(11): 4077- 4084. 2. Fina, F., et al., 3. Goyanez, A., et al., Effect of geometry on drug release from 3D printed tablets. International Journal of Pharmaceutics, 2015(494):657-663. Figure 1. The X-ray powder diffraction of F1 and F2. 213 P35 DISSOLUTION KINETICS OF GLIBENCLAMIDE AMORPHOUS SOLID DISPERSIONS IN BIORELEVANT MEDIA Vladimir Petkov, Zahari Vinarov, Slavka Tcholakova Department of chemical and pharmaceutical engineering, Sofia university „Saint Kliment Ohridski “Bulgaria” 1. INTRODUCTION 3. RESULTS AND DISCUSSION A substantial part of modern drugs exhibit poor 3.1. Solid state analysis aqueous solubility, which leads to low or highly variable oral bioavailability. One of the The spray-dried formulations were approaches to overcome this issue is the characterized by polarized light microscopy, preparation of amorphous solid dispersions, WAXS and differential scanning calorimetry. which can form supersaturated solutions that The results showed that the drug was in an drive liquid-liquid phase separation. amorphous state in al studied systems. The The aim of the current study was to investigate used polymers ensured stability for > 90 days. the supersaturation propensity and liquid-liquid phase separation of glibenclamide ASDs prepared by spray-drying with hydroxypropylcel ulose with low (HPC-SSL) or high molecular weight (HPC-L) and hydroxypropylmethylcel ulose acetate succinate (HPMCAS-HG). 2. MATERIALS AND METHODS 2.1. Materials Glibenclamide (99 %, Alfa Aesar), HPC-SSL and HPC-L (NISSO Chemical Europe GmbH), HPMCAS-HG (Shin-Etsu), NaCl (Sigma- 1000 Aldrich), KCl (Sigma-Aldrich), NaHCO3 /g 500 (Sigma-Aldrich), Hydrochloric acid 37% , W SPD-GLB:HPMCAS (Honeywel ), bovine bile 50% (Sigma-Aldrich) 0 SDP GLB 2.2. Methods -500 SPD-GLB:SSL SPD-GLB:L Glibenclamide ASDs were prepared by alized heat flow -1000 dissolving drug and polymer (at 1:3 drug to Norm -1500 polymer ratio) in a mixture of dichloromethane GLB and methanol at a total concentration of 5 wt %. -2000 40 60 80 100 120 140 160 180 Al solutions were spray dried by using Buchi Temperature, OC mini spray drier B-290. The samples were spay dried at 10ml/min flow rate, 70% aspiration and Figure 1. PLM images and DSC analysis of the 50 °C inlet temperature. Differential scanning spray-dried formulations. calorimetry, polarized light microscopy (PLM) 3.3. Amorphous solubility determination and wide-angle X-ray scat ering (WAXS) were The amorphous solubility of glibenclamide in used to characterize the solid state of the different media was evaluated using a solvent obtained formulations. High performance liquid shift method. The results showed that the chromatography (HPLC), was used to obtain amorphous solubility is highly affected by the the dissolution kinetics in biorelevant media. presence of bile salts and polymers in the UV-Vis spectroscopy was used to determine the solution. When present, bile salts increase the amorphous solubility of the drug and dynamic amorphous solubility to around 500 µg/ml light scat ering (DLS) was used to study the compared to a simple phosphate buffered with col olids in the supersaturated solutions. 214 P35 pH-7 in which phase separation occurs at around 250µg/ml. Polymers can also have an impact on the amorphous solubility of the drug. In presence of bile salts, HPC SSL raises the amorphous solubility to around 575 µg/ml and HPMCAS lowers the amorphous solubility to 440 µg/ml. 600 l 500 400 300 orphous solubility, ug/m Am 200 pH-7 Bile Bile+ SSL HPMCAS+BILE Figure 2. Effect of media on amorphous solubility. Figure 3 . Dissolution kinetics of glibenclamide 3.2. Drug release kinetics in biorelevant formulations in (A) single stage intestinal model media. and (B) gastrointestinal transfer model The spray dried formulations showed dramatic increase in the dissolved drug concentrations 4. CONCUSION compared to the crystal ine compound at the Spray-drying was successful y used to prepare single-stage intestinal conditions model (Figure amorphous glibenclamide formulations with 3A) and also in the gastro-intestinal transfer excel ent storage stability. The ASDs enhanced model (Figure 3B). Interestingly, the neat the drug release by several orders of magnitude, amorphous glibenclamide (obtained after spray compared to the crystal ine drug. The studied drying) showed very high drug concentrations, polymers (HPC-SSL, HPC-L and HPMCAS- similar to polymer-based formulations, in the HG) stabilized the amorphous state of the drug single-stage model. In contrast, very low drug during storage, but also during dissolution in the concentrations, similar to the crystal ine drug stomach compartment. Best performance was were measured in the gastrointestinal transfer observed for the low molecular weight HPC-L, model, indicating crystal ization of the where concentrations up to 300 µg/mL were amorphous glibenclamide in the gastric phase. measured. The measured amorphous In respect to the effect of the polymer type, the solubilities in biorelevant media (which exceed low molecular weight HPC-SSL showed the drug concentrations measured during ASD superior performance compared to HPMCAS- dissolution) and the DLS results suggest that HG in both models. The high-molecular weight liquid-liquid phase separation does not occur in HPC-L showed similar drug release kinetics to these systems and the drug is molecularly HPC-SSL in the single-stage model (intestinal dissolved. conditions only), whereas slightly slower drug ACKNOWLEDGMENT release was measured in the gastrointestinal transfer model. Al formulations were stable Part of the research is done using equipment during dissolution, showing no sign of bought for project bg05m2op001-1.002-0012 precipitation. financed by op neig sufinanced by the European union via european structural and investment funds. 215 P36 TOPICAL FORMULATION OF LYOPHILIZED P. CORONARIUS FLOWER AND LEAF EXTRACTS, ANTIMICROBIAL ASSESSMENT OF THE PLANT Ágota Pető1,2, Dóra Kósa1,2, Ádám Haimhoffer1, Dániel Nemes1,, Pálma Fehér1, Zoltán Ujhelyi1, Judit Váradi1, Ferenc Fenyvesi1, Miklós Vecsernyés1, Zoltán Tóth3, Annamária Pallag4, Tünde Jurca4, Ildikó Bácskay1,2 1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Debrecen, Hungary 2Institute of Healthcare Industry, University of Debrecen, Hungary 3Department of Medical Microbiology, Faculty of Medicine, University of Debrecen, Hungary 4Department of Pharmay, Faculty of Medicine and Pharmacy, University of Oradea, Romania 1. INTRODUCTION 3. RESULTS AND DISCUSSION The medicinal use of herbs is very popular, and 3.1. Texture analysis there is a high demand for herbal preparations Texture analysis revealed that the compositions among patients because of their beneficial have adequate consistency, those formulations, effects [1]. P. coronarius is widely used in folk which contain Tefose 63 have slightly harder medicine for the treatment of various diseases, consistency. its antimicrobial effects are wel known, but scientifical y less investigated and no external 3.2. In vitro release preparation is available of the herb yet [2]. The The results of in vitro release studies by Franz objective of our work was to formulate O/W diffusion chamber apparatus revealed, that the emulsion ointments using lyophilized P. best release profile was achieved by that coronarius flower or leaf extracts with the preparation, which contained SP70 sucrose addition of different penetration enhancers ester, closely fol owed by those ones, which [3,4]. were prepared with Tefose 63. 2. MATERIALS AND METHODS 3.3. Antimicrobial testing P. coronarius flower was not able to to inhibit 2.1.Materials or delay the growth of bacteria or fungi, but the SP70 sucrose ester was kindly gifted by Sisterna leaf was able to delay the growth of C. albicans (Roosendaalc, The Netherlands). Cetostearyl and S. aureus compared to the control. alcohol, propylene glycol, stearic acid, 3.4. MTT test isopropyl myristate, conservant solution were obtained from Hungaropharma Ltd. (Budapest, The results of MTT experiments demonstrated Hungary). HaCaT cel s were supplied from Cel that the selected excipients and the preparations Lines Service (CLS, Heidelberg, Germany). are safe under in vitro conditions. Transcutol, Tefose 63, Sedefos 75 was a kind gift from Gattefossé (Lyon, France). 3.5. Bioactive compound content 2.2. Method The leaf of P. coronarius contains a high After the preparations were formulated in vitro amount of delphinidin 3-rutinoside chloride release studies and texture analysis of the (0.3354 mg/100 mg), as wel as luteolin 7- ointments were formulated. In addition, glucoside (0.2528 mg/ 100 mg) and 7- antimicrobial testing of the lyophilized extracts methoxycoumarin (0.2061 mg/100 mg) and biocompatibility investigation of the compared to the other components. The flower selected excipients were carried out. Bioactive contains bergapten in a high amount (2.8370 compound content had been determined by mg/100 mg), as wel as caffeic acid (1.8407 HPLC method. mg/100 mg), delphinidin 3-rutinoside chloride (1.7928 mg/100 mg), 7-methoxycoumarin (1.6725 mg/100 mg). The flower contains delphinidin 3-rutinoside chloride and 7- methoxycoumarin in a much higher amount, than the leaf. 216 P36 4. CONCLUSION According to the results the composition and the selected excipients of the ointments have a great impact on the drug release, texture and bioavailability of the preparation. During microbiological testing, P. coronarius leaf was effective against E. coli and S. aureus. P. coronarius is a promising herb, and its topical application in antimicrobial therapy can be a useful addition to modern medical therapy. 5. REFERENCES 1. Klecáková, J.; Chobot, V.; Jahodár, L.; Laakso, I.; Víchová, P. Antiradical Activity of Petals of Philadelphus Coronarius L. Cent. Eur. J. Public Health 2004, 12 Suppl, S39-40. 2. Valko, V.; Fickova, M.; Pravdova, E.; Nagy, M.; Grancai, D.; Czigle, S. Cytotoxicity of Water Extracts from Leaves and Branches of Philadelphus Coronarius L. Biomed. Pap. Med. Fac. Univ. Palacky. Olomouc. Czech. Repub. 2006, 150, 71–73, doi:10.5507/bp.2006.007. 3. Nagy, M.; Grancai, D.; Jantova, S.; Ruzekova, L. Antibacterial Activity of Plant Extracts from the Families Fabaceae, Oleaceae, Philadelphaceae, Rosaceae and Staphyleaceae. Phyther. Res. 2000, 14, 601–603. 4. Hano, C.; Tungmunnithum, D. Plant Polyphenols, More than Just Simple Natural Antioxidants: Oxidative Stress, Aging and Age- Related Diseases. Medicines 2020, 7, 26, doi:10.3390/medicines7050026. ACKNOWLEDGMENT Project no. TKP2021-EGA-18 has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the TKP2021-EGA funding scheme. The research was carried out under the project Debrecen Venture Table S Catapult (EFOP- 3.6.1-16-2016-0002). The project was supported by the European Union and co- funded by the European Social Fund. The work is supported by the GINOP-2.3.4-15-2020- 00008 project. The project is co-financed by the European Union and the European Regional Development Fund. The present work was supported by grant from The Ministry of Hungary Domus Hungarica 2019. The research was co-financed by the Richter Gedeon Talent Foundation. 217 P37 FENOFIBRATE ORODISPERSIBLE TABLET MADE WITH GRANULATED MESOPOROUS SILICA Odon Planinšek, Ana Baumgartner, Blaž Grilc 1Department for Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION 2.4. Orodispersible tablet formulation The fabrication of amorphous solid dispersions 3% of AcDiSol was added to impregnates is an approach to improve the dissolution of granules and compressed into tablets containing sparingly soluble active substances in water. 140 mg of fenofibrate using 12 mm flat punches Among hydrophilic water innsoluble excipients at 25 kN compression force. for the production of solid dispersions, 2.5. Granules and solid dispersions mesoporous silicon dioxide is the most evaluation promising [1]. Many research works have a) Specific surface area of nonimpregrated shown the feasibility of incorporating sparingly Syloid and of fenofibrate impregnated granules soluble active ingredients into mesoporous was determined with BET Surface area analyzer materials, with the main disadvantage of Nova 2000, Quantachrome, Germany. obtained amorphous solid dispersions having poor flowability and compressibility. In our b) Particle size analysis of powder samples was study, we solved this problem by granulating of performed by laser diffraction method, mesoporous silicon dioxide with erythritol and Mastersizer 3000, Malvern, UK using Aero S subsequent impregnation with the poorly water- dry powder disperser. soluble active ingredient, fenofibrate. c) Thermal properties of produced solid 2. MATERIALS AND METHODS dispersion were determined with Differential scanning calorimeter DSC1, Met ler Toledo, 2.1. Materials Switzerland. a) Model active pharmaceutical ingredient: Fenofibrate-Biosynth, Carbosynth, Great d) Orodispersible tablet disintegration time Britain. determination was determined in water using b) Mesoporous silicon dioxide: Syloid 244FP, Erweka ZT320 disintegration tester (Germany) Grace, Germany. at 37ᴼC. c) Silicon dioxide binder: Erythrytol, Instantina, Austria. e) Dissolution of pure fenofibrate, of fenofibrate d) 2-propanol: Merck, Germany. physical mixtures with Syloid 244FP and e) Croscarmel ose Sodium, Ac-Di-Sol®, erythritol and from solid dispersions containing Dupont, USA. the same proportion of active ingredient as physical mixtures was determined in water 2.2. Syloid 244FP granulation phosphate buffer at pH 6,8. Concentrations at Syloid 244FP was granulated with Erythrytol six time points from 5 to 60 min were and water as a granulating liquid in high shear determined with Hewlet Packard 8453 UV-VIS granulator ProCept 4M8-TriX (Belgium). spectrophotometer (USA) (λ=250 nm) Syloid to Erythritol proportion in dry granules was 2:1. 3. RESULTS AND DISCUSSION 2.3. Granules and Syloid244FP impregnation 3.1. Specific surface area Granules were impregnated with 20% of 26,7% Syloid has large specific surface area (SSA) 377 of fenofibrate from 2-propanol solution in m2/g (Table 1) which can adsorb significant rotavapor (Büchi R-114, Switzerland). Pure amount of active pharmaceutical ingredient. Syloid 244FP particles were impregnated with Granulation of silica decreased this property to fenofibrate in the same way as eythritol 217 m2/g. Theoretical y SSA should be of 248 granules. m2/g. Difference between these two values can be at ributed to the portion of erythritol that 218 P37 impregnated pores. As expected, impregnation 2-melting of erythritol crystals at about 110 ºC. of silica pores with fenofibrate decreased SSA of granules. Higher the amount of fenofibrate, 3.3. Tablet disintegration time lower the SSA of the product (Table1). Disintegration time of tablets was less than a However, for al the solid dispersions minute. (granulated and non-granulated) specific 3.4. Dissolution of fenofibrate from solid surface area is much larger than for pure dispersions fenofibrate (0.5 m2/g). 3.2. Particle size Granulation of silica increased particle size from 4 µm to 65 µm. This property of granules enabled their tableting which could not be performed with pure silica particles or with fenofibrate impregnated silica particles. Table 1. BET specific surface area and average particle size. Sample BET area Average (m2/g) size (µm) Figure 2: Dissolution profiles of pure Syloid 244FP 377 3,8 fenofibrate, solid dispersions and tableted Fenofibrate 0.5 41.5 granulated solid dispersions Granulated Syloid 217 65 FP244+Erythritol 2:1 Formulation of solid dispersions significantly 20% solid dispersion 121 4,0 improved the dissolution of fenofibrate (Figure 30% solid dispersion 80 3,7 2). Although the orodispersible tablet 20% Tableted gran. 125 85,9 formulation shows inferior dissolution solid dispersion compared to the pure solid dispersions, it is 26,7% Tableted gran. 82 42,6 reasonable to develop a patient friendly dosage solid dispersion form which stil perform much bet er than pure fenofibrate. 3.2.Thermal pr p ope p rties 4. CONCLUSION Co-processing of Syloid 244 FP with erythritol enables formation of a solid dispersion of sparingly soluble fenofibrate with improved dissolution and can be easily compressed into anorodispersible tablet. 5. REFERENCES Figure 1. DSC curves of impregnated granules 1. Baumgartner A., Planinšek O. Application of composed of Syloid 244FP and erythritol with commercial y available mesoporous silica for drug fenofibrate. A -20% fenofibrate content, B-26,7 dissolution enhancement in oral drug delivery. % fenofibrate content. European Journal of Pharmaceutical Science 2021, 167, 1-13. DSC curve for impregnated granules with 20 % of fenofibrate shows three peaks: ACKNOWLEDGMENT 1-melting of fenofibrate nanoparticles located The authors acknowledge financial support in mesopores. from the Slovenian Research Agency (Research 2-melting of fenofibrate crystals at 80 ºC. Core Funding, No. P1-0189). 3-melting of erythritol crystals at about 110 ºC. DSC curve for impregnated granules with 26,7 % of fenofibrate shows two main peaks: 1-melting of fenofibrate nanoparticles located in mesopores. 219 P38 EX VIVO BIOFILM MODEL ON PIG SKIN TO TEST THE EFFICACY OF ELECTROSPUN ANTIMICROBIAL DRUG-LOADED FIBER MATERIALS AS WOUND DRESSING Kaisa Põhako1, Kairi Lorenz1, Marta Putrinš1,2, Külli Kingo3, Tanel Tenson2, Karin Kogermann1 1 Institute of Pharmacy, University of Tartu, Estonia, 2 Institute of Technology, University of Tartu, Estonia, 3 Dermatology Clinic of Tartu University Hospital 1. INTRODUCTION 2.2. Ex vivo pig skin wound infection model Chronic wounds are a growing problem to development healthcare systems al over the world. Often Ex vivo wound infection models were chronic wounds may become infected with developed on pig skin using pathogenic wound bacteria. The presence of bacterial biofilms in bacteria (Fig 1.). A wound was created using a combination with the lack of suitable topical biopsy punch needle and infected with bacteria antimicrobial treatments makes the wound care (E. coli, S. aureus, S. epidermidis). Electrospun even more chal enging.1 fiber matrices were place over the infected wound and the whole construct was incubated In the last decades, scientists are searching for up to 72 h at 37°C. novel wound care approaches. One possible solution could be antimicrobial electrospun 2.3. Colony forming unit (CFU) fiber dressings for the local treatment of determination wounds. Electrospinning is a fiber production To indicate the inhibitory effect of the method, which enables to incorporate different electrospun fiber materials on bacterial growth, drugs and antimicrobial agents into the fiber CFU detection was carried out. A pig skin and structure. This feature makes electrospun fibers electrospun fibers from wound model were a desirable material to be used as a wound washed with 1xPBS and formed biofilm was dressing.2 disrupted by sonification and vortexing. Samples containing disrupted biofilm were With new potential medical devices (e.g. wound plated on LB plates, incubated at 37°C for 24 h dressings), it is very important to test their and counted. safety, efficacy and biocompatibility. Variety of skin and wound infection models are developed 2.4. Microscopy and used to test novel wound dressing materials. The biofilm formed in the model and the Such novel materials require more biorelevant efficacy of antimicrobial fiber matrices were testing methods in order to understand their visualized using scanning electron microscopy quality and real potential for the treatment of (SEM). Firstly, samples were fixed with infected wounds.3 formaldehyde and dried with ethanol, then SEM imaging was carried out. The aim of this work was to develop ex vivo infected wound model on pig skin to test the 3. RESULTS AND DISCUSSION antimicrobial efficacy of electrospun fiber An antibiotic (Ab) loaded and antimicrobial dressings. peptide (AMP) loaded fiber matrices and their 2. MATERIALS AND METHODS respective pristine polymeric fiber matrices were successful y produced by monoaxial 2.1. Preparation of electrospun dressings electrospinning. The measured fiber diameters Monoaxial electrospinning was used to create (n=100) were al above 1 micrometer (Table 1). four different fiber matrices - two antimicrobial model drug loaded matrices and two of their respective controls. Exact compositions of the fibers together with fiber diameters are presented in Table 1. 220 P38 Table 1. Description of electrospun fiber materials used. Sample Composition Diameter name size (µm) Mat A mixture of 2.2 ± 5.7 hydrophilic and hydrophobic polymers Mat A+Ab mixture of 2.9 ±1.1 hydrophilic and hydrophobic polymers and antibiotic (Ab) Mat B hydrophobic 3.6 ± 0.5 polymer Mat B+AMP hydrophobic 1.3 ± 0.2 polymer and Figure 1. Experimental set up of infected ex vivo antimicrobial wound model on pig skin. peptide 4. CONCLUSION (AMP) In conclusion, developed ex vivo wound infection model on the pig skin is suitable for The ex vivo wound infection model enabled to testing the antimicrobial effect of electrospun mimic the biorelevant conditions of infected fiber matrices. Both tested antimicrobial fiber wound and the formation of bacterial biofilm matrices hold potential to be used as was detected (Figure 1). The model results antimicrobial wound dressings for the treatment revealed, that pristine fiber matrices were of wound infections. Further studies wil reveal suitable surfaces for bacteria to grow on and their in vitro safety and in vivo efficacy and form a biofilm. The lat er is important to keep biocompatibility. in mind while designing novel wound dressings. Fibrous matrices with antimicrobial agents ACKNOWLEDGMENT prevented the biofilm formation and significantly inhibited the bacterial growth in The study was funded by the Estonian Research the wound model due to the release of drugs Council project PRG 1507. from electrospun fibers and antibiofilm REFERENCES properties of the dressings. CFU counting and microscopy enabled to understand the biofilm 1. Percival, S. L., McCarty, S. M. & Lipsky, B. formation and effect of antimicrobial fiber Biofilms and Wounds: An Overview of the matrices. Evidence. Adv. Wound Care 4, 373–381 (2015). 2. Cui, W., Zhou, Y. & Chang, J. Electrospun nanofibrous materials for tissue engineering and drug delivery. Sci. Technol. Adv. Mater. 11, 014108 (2010). 3. Ud-Din, S. & Bayat, A. Non-animal models of wound healing in cutaneous repair: In silico, in vitro, ex vivo, and in vivo models of wounds and scars in human skin. Wound Repair Regen. 25, 164–176 (2017). 221 P39 RELATIVE BIOAVAILABILITY ENHANCEMENT OF SIMVASTATIN VIA DRY EMULSION SYSTEMS: COMPARISON OF SPRAY DRYING AND FLUID BED LAYERING TECHNOLOGY Mitja Pohlen1; Jurij Aguiar Zdovc1, Jurij Trontelj1, Janez Mravljak1, Mirjam Gosenca Matjaž1, Iztok Grabnar1, Tomaž Snoj2, Rok Dreu1, 1Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, SI-1000 Ljubljana, Slovenia 2Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1000 Ljubljana, Slovenia 1. INTRODUCTION For a comprehensive description of the process In recent decades more and more newly and formulation parameters, the reader is discovered drugs are active pharmaceutical directed to two studies [2, 3]. ingredients (APIs) exhibiting poor Briefly, coating experiments were performed biopharmaceutical characteristics, especial y using the GPCG-1 process equipment (Glat ® low water solubility [1]. GmbH, Germany) utilising a modified Wurster- The aim of this study was to compare two dry type process chamber equipped with swirl emulsion forming techniques, i.e. spray drying generator design. For the SPD process a Mini (SPD) and fluid bed layering (FBD) in terms of spray dryer B-290 (Büchi, Switzerland) was process, physicochemical product employed. characteristics and bioavailability enhancement Emulsion component limits, where the of the model drug, simvastatin (SV). The formulations were processable, were set for critical quality at ributes (CQAs) were both technologies and were used as the compared based on two previously conducted experimental space for the DoE study (Table 1). Design of Experiment studies (DoE) [2, 3]. A Table 1. Excipients weight proportion limits for final pharmacokinetic (PK) study was DoE study. conducted in rats to evaluate the in vivo performance of optimised products from each Weight proportion limits (%) technique and compared with a SMEDDS FBD SPD formulation, a spray dried formulation with SV Formulation Low High Low High glyceride mimetic and a physical powder Oil 27.86 33.17 27.00 40.00 mixture resembling a generic SV tablet. Mannitol 52.00 65.15 49.04 61.84 2. MATERIALS AND METHODS HPMC 5.42 14.21 8.46 10.66 Tween® 20 0.50 2.50 0.50 2.50 2.1. Materials 2.2.2. Physicochemical characterization and Simvastation (pharmaceutical grade, Krka d.d.), in vivo evaluation 1-oleoyl-rac-glycerol (technical grade ׽40% Dry emulsions were compared according to (TLC)), Tween® 20 (Merck, Germany), process yield (PY), drug content, stability, Pharmcoat 603 (ShinEtsu, Japan), Miglyol® encapsulation efficiency (EE), particle size, 812 (Sasol, Germany), mannitol (Roquet e, morphology, redispersibility and dissolution France), Cel ets 200 (Harke Pharma GmbH, performance. For the in vivo evaluation, Wistar Germany), Avicel® PH 101, lactose mesh 200 female rats were divided into 5 groups of 12 (Lek d.d., Slovenia), Magnesium stearate animals and administered oral y by gavage (1 (Merck KGaA, Darmstadt), SV-D6 standard mg of SV per 100 g of body weight). and Simvastatin hydroxy acid (SVA)-D6 (Toronto Research Chemicals, Canada). Al 3. RESULTS AND DISCUSSION solvents for UPLC analysis were of HPLC grade. Al other reagents used were of analytical 3.1. Process and product comparison grade. The comparison of EE, PY, drug content and 1 month stability is shown in Table 2. In SPD, a 2.2.1. Process and formulation development much higher inlet/product temperature was 222 P39 needed to successful y perform the process, compared to FBD, which can degrade API, thus lowering the EE. Additional y, FBD, which produced bigger particles provided significantly (α = 0.05) higher one month relative drug content stability. Table 2. Process yield, drug content and one month stability of dry emulsion products (average values). SPD FBD Encapsulation efficiency (%) 68.4 80.0 Process yield (%) 71.5 83.3 Drug content (mg/g) 22.2 9.34 Figure. 2: The schematic representation of the One month stability (%) 85.5 93.8 final population PK model for SV and SVA. The individual particles of the spray dried dry 4. CONCLUSION emulsion powder were round in shape (Fig. 1. Two dry emulsion production techniques, i.e. a), but they were physical y bound and FBD technology and SPD technology were combined into fractal agglomerates (median compared. FBD showed significantly bet er particle size d50 = 56 µm), which makes them process yield, encapsulation efficiency, less suitable for further processing, compared to improved stability and bet er morphology for round shaped (Fig. 1. b) and bigger (median size further processing into a final dosage form. SPD of d50 = 336 µm) dry emulsion layered pel ets. al owed us to broaden the limits of formulation component variables, resulting in a higher drug content. Fluid bed layered dry emulsion pel ets provided the highest increase in relative bioavailability within the group of five formulations, confirming the superiority of FBD over SPD for potent/low dose APIs formulated as dry emulsion systems. Figure 1: Dry emulsion products: a) spray dried dry emulsion powder; b) dry emulsion layered 5. REFERENCES pellets. 1. Buckley S.T., et al., Biopharmaceutical Regardless of the reconstitution parameter used, classification of poorly soluble drugs with differences between optimised products of both respect to ‘enabling formulations. EJPS, 2013. technologies are marginal. Dissolution tests 50: 8–16. showed a more than 18- and 20- fold increase 2. Pohlen M., et al., Preparation, Physicochemical (compared to crystal ine SV) in drug dissolution Characterisation and DoE Optimisation of a Spray-Dried Dry Emulsion Platform for for fluid bed layered dry emulsion pel ets and Delivery of a Poorly Soluble Drug, Simvastatin. spray dried dry emulsion powders, respectively. AAPS PharmSciTech. 2020. 21: 119. 3.2. In vivo study 3. Pohlen M., et al., A redispersible dry emulsion system with simvastatin prepared via fluid bed A one-compartment model with two paral el layering as a means of dissolution enhancement first order absorption processes for SV, and of a lipophilic drug. International Journal of additional absorption process for SVA, was Pharmaceutics, 2018. 549: 325-334. chosen to describe the distribution of both SV and SVA. Fluid bed dry emulsion layered ACKNOWLEDGMENT pel ets provided the highest increase in relative The authors thank Krka, d.d. Novo Mesto, bioavailability (215%) within the group of five Slovenia and the Faculty of Pharmacy, formulations. University of Ljubljana, Slovenia (Slovenian Research Agency under contract number P1- 0189), for supporting this study. 223 P40 COMPARISON OF THE RELEASE PROFILES OF MELATONIN FROM MATRIX TABLETS CONTAINING POLY(ε-CAPROLACTONE) AND COPOLYMERS Chrystalla Protopapa1, Marilena Vlachou1, Angeliki Siamidi1, Evi Christodoulou2, Nikolaos D. Bikiaris2 1Division of Pharmaceutical Technology, Department of Pharmacy, National and Kapodistrian University of Athens, Greece 2Department of Chemistry, Laboratory of Polymer Chemistry and Technology, Aristotle University of Thessaloniki, Greece 1. INTRODUCTION 2.1. Materials Τhe pineal hormone that regulates the circadian The polymeric materials (neat PCL, mPEG- rhythm is melatonin (MLT, Fig. 1). In the PCL [1:3] and mPEG-PCL [1:1]) were newly elderly, though, its excretion is significantly synthesized (by ring-opening polymerization of lowered, and exogenous administration of the ε-caprolactone) and kindly donated by hormone is needed to compensate for its Professor Dimitrios Bikiaris’s group, diminished concentration. However, due to Laboratory of Chemistry and Technology of MLT’s poor bioavailability and short biological Polymers and Dyes, in the Department of half-life, modified release formulations are Chemistry of the Aristotle University of needed [1]. Our research group has been studied Thessaloniki. Other excipients used for the a plethora of biopolymers with diverse chemical tablets’ production included lactose structures, as excipients in per os administered monohydrate, Avicel PH102, and magnesium solid dosage forms [2-5]. In the context of this stearate. The commercial y available drug study, MLT matrix tablets, containing Circadin® was purchased from a local polycaprolactone (PCL) and its copolymer, pharmacy. methoxy poly(ethylene glycol)-co-PCL 2.2. Methods (mPEG-PCL) (Fig. 1), have been developed and evaluated with respect to their dissolution The tablets were prepared using the excipients, profile, aiming at dealing with sleep onset shown in Table 1, by direct compression. A and/or maintenance dysfunctions, caused by USP apparatus II was used for the dissolution experiments, which were carried out in aqueous low MLT concentrations in the body media, A: pH 1.2, for 2 h, and B: pH 6.8, for 6 h, in order to simulate the conditions in the gastrointestinal track. The samples were analysed using a UV spectrophotometer (λmax= 278), and dissolution curves were constructed. Table 1. Formulations (F1-F3) of MLT Ingredients F1 (mg) F2 (mg) F3 (mg) Melatonin 2 2 2 Neat PCL 150 mPEG-PCL [1:3] 150 mPEG-PCL [1:1] 150 Lactose Monohydrate 10 10 10 Figure 1. Chemical structures of MLT, PCL Avicel PH 102 36 36 36 and mPEG-PCL. 2. MATERIALS AND METHODS 224 P40 3. RESULTS AND DISCUSSION 4. CONCLUSION The aqueous dissolution test results obtained for We have shown that the formulations developed MLT from the developed formulations and for the MLT’s effective release lead to its 100% MLT from the commercial y available drug release during the first 2 hours (F1 and 4 hours Circadin® are presented in Figure 1. F2 and F3). These formulations are considered appropriate for treating sleep onset dysfunctions, in contrast to the commercial y available drug Circadin®, which is suitable for 100 treating combined sleep onset/sleep maintenance problems. 80 5. REFERENCES 60 1. Arendt J, Melatonin and human rhythms, 40 F1 Chronobiol. Int., 2006, 23, (1-2), 21-37 F2 2. Vlachou M, Stavrou G, Siamidi A, et al. N- F3 Acetylserotonin vs Melatonin: In vitro control ed Circadin release from hydrophilic matrix tablets LDDD, 2019, 16(3): 347-352. 0 3. Vlachou M, Siamidi A, Anagnostopoulou D, et al. 0 120 180 240 300 360 420 480 Modified release of the pineal hormone melatonin t (min) from matrix tablets containing poly(L-lactic acid) and its copolymers. Polymers, 2022, 14(8), 1504. Figure 1. In vitro % release of MLT from Circadin® 4. Vlachou M, Kikionis S, Siamidi A, et al. Modified vs. time, at pH 1.2 (0-120 min) and at pH 6.8 (120- in vitro release of melatonin loaded in nanofibrous 480 min) (SD<2) (n= 3). electrospun mats incorporated into mono-layered and three-layered tablets. J Pharm Sci, 2019, 108(2):970- From the release curves seen in Fig. 1, it can be 976. assumed that the MLT release from formulation 5. Vlachou M, Tragou K, Siamidi A, et al. Modified F1, containing neat PCL, resembles the MLT in vitro release of the chronobiotic hormone release from the commercial y available drug melatonin from matrix tablets based on the marine sulfated polysaccharide ulvan. J DDST, 2018, 44:41- Circadin®, during the first 2 hours at pH 1.2. 48. Also, it was observed that the release of MLT from formulation F2, containing mPEG-PCL [1:3], resembles the release profile of formulation F1 that contains neat PCL through the whole dissolution process (8 hours). On the other hand, the hormone’s release from the formulation F3, that contains mPEG-PCL [1:1], was particularly high (100% at 2 hours), indicating that the larger the amount of the hydrophilic mPEG-segment onto the PCL backbone, the more enhanced the MLT’s release. 225 P41 HARMIQUINS, NOVEL POTENT ANTIPLASMODIAL HITS Zrinka Rajić1, Goran Poje1, Lais Pessanha de Carvalho2, Jana Held2, Ivana Perković1, Tana Tandarić3, Robert Vianello3 1Department of Medicinal Chemistry, University of Zagreb Faculty of Pharmacy and Biochemistry, Croatia 2Institute of Tropical Medicine, University of Tübingen, Germany 3Ruđer Bošković Institute, Croatia 1. INTRODUCTION 2.2. In vitro drug sensitivity assay against Malaria is the deadliest protozoan infectious erythrocytic stages of P. falciparum disease widely spread in the tropical and Antiplasmodial activity was evaluated against subtropical areas [1]. As existing antimalarials four laboratory P. falciparum strains (3D7, CQ- are losing their efficacy due to the emergence of sensitive; 7G8, CQ-resistant; Dd2 and K1, resistant Plasmodium strains, there is an urgent multidrug-resistant,) using the histidine-rich need for novel agents. Molecular hybridization, protein 2 assay [3]. fusion of at least two pharmacophores with individual activity, represents a popular 2.3. In vitro cytotoxicity assay approach to obtain dual-acting antimalarial Cytotoxicity against a human cel line (HepG2) agents [2]. We decided to employ that strategy was evaluated using the neutral red assay [3]. and to combine two scaffolds with known 2.4. Inhibition of heme polymerisation antimalarial activity, harmine and chloroquine, The inhibition assay of heme polymerisation to produce hybrid agents, harmiquins. was performed as previously described [4]. 2. MATERIALS AND METHODS 2.5. Computational details The starting point of our molecular dynamics 2.1. General synthetic procedures simulations was a P. falciparum heat shock Triazole-type (TT) harmiquins 18-25: A protein 90 (P f Hsp90) N-terminal domain suspension of harmine-/7-chloroquinoline-structure obtained from the Protein Data Bank based alkyne (0.230 mmol), the corresponding (accession code 3K60). The analysis was 7-chloroquinoline-/harmine-based azide (0.253 performed as previously described [3]. mmol), catalytic amount of Cu(OAc)2 in MeOH (5 mL) was stirred at rt for 24 h, and solvent 3. RESULTS AND DISCUSSION evaporated. Amide-type (AT) harmiquins 26, 27: A solution of a harmine-based carboxylic 3.1. Chemistry acid (0.176 mmol), DIEA (0.352 mmol), HATU A library consisting of TT and AT harmiquins (0.176 mmol) and 7-chloroquinoline-based was prepared. Structural diversity was achieved amine (0.160 mmol) in DCM (4 mL) was stirred by derivatizing harmine's -carboline core at 5 at rt for 18 h, and solvent evaporated. AT different positions, namely C-1, C-3, O-6, O-7 harmiquins 28-32: A suspension of 7- and N-9. TT harmiquins were prepared by chloroquinoline-based carboxylic acid (0.211 Cu(I)-catalysed azide-alkyne cycloaddition, mmol), harmine-based amine (0.192 mmol), using the Cu(II) acetate precatalyst in methanol. TEA (0.422 mmol) and T3P (0.211 mmol) in On the other hand, AT harmiquins were DMF (2 mL) was stirred at rt for 18 h, followed synthesized by a simple and straightforward by dropwise addition of 5% NaOH. The formed coupling reaction, using eighter HATU/DIEA precipitate was filtered off. or T3P/TEA. Crude products were purified by column 3.2. In vitro evaluation of antiplasmodial chromatography, fol owed by trituration with activities diethyl ether. Harmiquins exhibited remarkable activity against the erythrocytic stage of P. falciparum life cycle, in nanomolar range. The results have 226 P41 shown that AT harmiquins and most TT nM), at least 15.9-fold higher activity than CQ harmiquins were significantly more active than against P. falciparum CQ-resistant strains and the parent compound harmine and that the had a very high selectivity index (4450). Two optimal position for the substitution of the - possible mechanisms of action, inhibition of carboline ring is N-9. In general, AT harmiquins heme polymerisation and binding to Pf Hsp90, were more active than TT harmiquins against al might play a role in the inhibition of strains of P. falciparum tested. Although Pf 3D7 Plasmodium intraerythrocytic development. strain was the most sensitive (9/15 compounds showed activity less than 100 nM), 10/15 5. REFERENCES harmiquins exhibited higher activity than CQ 1. World Malaria Report 2021, World Health against Pf Dd2 and 7/15 against Pf K1 and Organisation 2021. Pf 7G8. 2. Agrawal, S., et al., Association of a novel 3.3. In vitro evaluation of cytotoxicity mutation in the Plasmodium falciparum Screening of the cytotoxic activity of chloroquine resistance transporter with harmiquins against HepG2 was performed with decreased piperaquine sensitivity, Journal of Infectious Diseases 2017. 216: 468–476. the aim of evaluating their selectivity against 3. Perković, I., et al., Harmicines - harmine and Plasmodium over mammalian cel s. The cinnamic acid hybrids as novel antiplasmodial cytotoxicity of al compounds tested was hits, European Journal of Medicinal Chemistry similar, and the majority of harmiquins 2020. 187:111927. exhibited IC50 values in the 5-20 µM range. 4. Olafson, K. N., et al., Mechanisms of hematin Since most compounds showed antimalarial crystallization and inhibition by the antimalarial activity in the nanomolar range, we concluded drug chloroquine, Proceedings of the National that harmiquins have significant selectivity Academy of Sciences 2015. 112: 4946-4951. against Plasmodium. 3.4. Possible mechanism of action ACKNOWLEDGMENT The ability of harmiquins to inhibit heme The authors acknowledge the financial support polymerization was examined in vitro. The by the Croatian Science Foundation (research results have shown that TT harmiquins project UIP-2017-05-5160), University of significantly inhibited heme polymerisation Zagreb (support for 2020), and Fundação para a (6/8), whereas AT harmiquins were not as Ciência e Tecnologia, Portugal (FCT) (grant effective (2/7). On the other hand, 02/SAICT/2017/29550). computational analysis was utilized to provide an insight into the binding of a representative set of derivatives towards P f Hsp90. Calculated ∆ G BIND values were al exergonic, thereby indicating a favourable binding of al derivatives towards P f Hsp90, which agrees with their demonstrated biological activities. Our analysis confirmed the highest activity of compound 32, with the calculated affinity of – 38.2 kcal mol–1, thereby demonstrating its optimal positioning within the P f Hsp90 interior. 4. CONCLUSION Here we present design and synthesis of AT and TT harmiquins, evaluation of their biological activity and possible mechanism of action. Harmiquins displayed remarkable activity against the erythrocytic life stage of CQ- sensitive, as wel as CQ-resistant Plasmodium strains. The most active compound, AT harmiquine 32, displayed a 5.5-fold higher activity against Pf 3D7 than CQ (IC50 = 2±0.3 227 P42 FORMULATION AND PERMEABILITY STUDIES OF FENUGREEK (TRIGONELLA FOENUM-GRAECUM) CONTAINING SEDDS Dávid Sinka1, Enikő Doma1, Mercédesz Varga1, Pálma Fehér1, Liza Józsa1, Zoltán Ujhelyi1, Ildikó Bácskay1 1Deptartment of Pharmaceutical Technology, University of Debrecen, Hungary 1. INTRODUCTION 4. CONCLUSION Fenugreek is used as a spice and a traditional Based on our results, a modern, non-toxic, herbal medicine for a variety of purposes, given cytocompatible fenugreek SEDDS formulation its antidiabetic and antioxidant effects. Self- with high antioxidant capacity was developed in emulsifying drug delivery systems (SEDDS) of order to improve the permeability and herbal drugs are targets of extensive research bioavailability of al components. aiming to increase bioavailability and stability. [1,2] The study’s objective was to formulate SEDDS 5. REFERENCES containing Trigonel a foenum-graecum extract 1. Snehlata, H.S.; Payal, D.R. Fenugreek (Trigonel a to improve the stability of herbal extract and to foenum-graecum L.): An overview. Int. J. Curr. increase their permeability through a Caco-2 Pharm. Rev. Res. 2011, 2, 169–187. monolayer. 2. Pavoni, L.; Perinel i, D.R.; Bonacucina, G.; Cespi, 2. MATERIALS AND METHODS M.; Palmieri, G.F. An Overview of Micro- and Nanoemulsions as Vehicles for Essential Oils: A characterized fenugreek dry extract was used Formulation, Preparation and Stability. for the formulations, while the SEDDS Nanomaterials 2020, 10, 135. properties were examined by particle size analysis and zeta potential measurements. Permeability assays were carried out on Caco-2 cel monolayers, the integrity of which was ACKNOWLEDGMENT monitored by fol ow-up trans-epithelial electric Project no. TKP2021-EGA-18 has been resistance measurements (TEER). Cytocompatibility was tested by the MTT implemented with the support provided from method, and an indirect dissolution test was the National Research, Development and performed, using DPPH antioxidant reagent. Innovation Fund of Hungary, financed under the TKP2021-EGA funding scheme. 3. RESULTS AND DISCUSSION Two different SEDDS compositions were formulated from a standardized fenugreek dry extract at either the micro- or the nanoemulsion scale with sufficient stability, enhanced bioavailability of the compounds, and sustained release from HPMC capsules. 228 P43 SOLUBILITY ENHANCEMENT OF MEGESTROL-ACETATE VIA MICELLE AND POLYMERIC MICELLE FORMULATION Bence Sipos1, Ildikó Csóka1, Piroska Szabó-Révész1, Gábor Katona1 1University of Szeged, Faculty of Pharmacy, Institute of Pharmaceutical Technology and Regulatory Affairs, Hungary 1. INTRODUCTION °C. The solubilizers were dispersed individual y Oral drug administration faces a number of at this temperature. Each sample was cooled chal enges, especial y for drugs with poor water down at 80 °C and MGA was dispersed in them. solubility and/or permeability. Megestrol- Before the total solidification of the melt at 33 acetate (MGA) is characterized by these factors °C, the mass was dispersed through a sieve with leading to high required dosages with lower a mesh size of 1.2 mm. patient adherence. By utilizing surfactant type 2.3. Solid state characterization excipients, these parameters can be optimized The thermal behaviour was investigated via for the bet er, however choosing the proper differential scanning calorimetry and solubilizing agent is of paramount importance. thermogravimetry. Crystal inity was A comparison study was conducted in order to determined by X-ray powder diffraction and (i) investigate the processability of the micel e- granule size and distribution was measured with forming Cremophor® RH 40 and the polymeric laser diffraction. micel e former Soluplus® via melt technology; (i ) differentiate the structure characteristics of 2.4. Micelle characterization these two surfactant-based granules and (i i) to Micel e size and distribution was measured by determine the col oidal and in vitro gastric dynamic light scat ering. The thermodynamic behaviour of the micel e carrier systems [1]. solubility was determined by the oversaturation method and the solubility related parameters 2. MATERIALS AND METHODS were calculated. Surface free energy and 2.1. Materials polarity was measured by contact angle determination. The carrier forming materials D-xylitol (XYL), β-D-mannitol (MAN) and polyethylene glycol 2.5. In vitro gastric drug release study 6000 (PEG 6000) were purchased from Sigma- The modified paddle method was used to Aldrich (Budapest, Hungary). The solubilizing examine the rate of drug release from the agents Cremophor® RH 40 (CR 40) and surfactant-free and the surfactant-containing Soluplus® (SP) were obtained from BASF MGA granules. Two separate dissolution media GmbH (Ludwigshafen, Germany). Megestrol- were used: fasted and fed state simulated gastric acetate (Farmabios S. p. A., Pavia, Italy) was fluid. Six mathematical drug release models chosen as the model material for this were fit ed with the obtained cumulative drug experimental work. Powders for the biorelevant release vs. time curves to describe the kinetics. gastric fluids were purchased from 3. RESULTS AND DISCUSSION Biorelevant.com Ltd. (London, UK). 3.1. Characterization of MGA in the carrier On the DSC thermogram of crystal ine MGA, 2.2. Development of the carrier the characteristic melting point at 217.95 °C can For the preparation of the carrier, melt be observed, which cannot be found on the other granulation was applied. XYL, MAN and PEG thermograms of the granule formulations. This 6000 were melted together on a sand bath at 112 229 P43 supports the successful melting and dispersing 3.3. In vitro gastric release study of MGA in the molten carrier system. The peak The proper nanoparticle characteristics around 60 °C is assigned to the melting point of contributed to higher dissolution rate both in PEG 6000 and the peak around 91 °C is the fasted and fed state drug release media (Figure eutectic melt of XYL-MAN (Figure 1.) 3.). The formulation fol owed Higuchi kinetics which is typical for a rapid drug release system and for polymeric micel es. Figure 1. DSC thermograms of MGA and the granules. XMP, the carrier system; MGA – SP and MGA – CR 40, the surfactant-loaded systems; MGA – XMP, surfactant-free granule. Figure 3. Example drug release curve of MGA and the granule systems in fasted state gastric The diffractograms of the granules support this drug release media. Data are presented as means result, as the characteristic crystal ine peak of ± SD. MGA cannot be found in the granule systems 4. CONCLUSION (Figure 2.). Al in al , it can be concluded that the polymeric micel e forming Soluplus® has bet er potential in the enhanced drug release and delivery of MGA over classic micel e forming surfactants. 5. REFERENCES 1. G, K., et al., Characterizing the Drug-Release Enhancement Effect of Surfactants on Megestrol-Acetate-Loaded Granules. Pharmaceuticals, 2022. 15(2): 113 ACKNOWLEDGMENT The work was supported the Gedeon Richter Figure 2. XRPD diffractograms of MGA and the Plc. Centennial Foundation (Gyömrői 19-21, granule systems Budapest, H-1103, HU) and Project no TKP 3.2. Micelle characterization 2021 EGA 32 implemented with support The MGA-SP formulation prevailed in case of provided by the Ministry of Innovation and successful micel e preparation with optimal Technology of Hungary from the National nanocharacteristics. The average hydrodynamic Research, Development, and Innovation Fund, diameter was 102.27 nm, with a distribution financed under the TKP 2021 EGA funding expressed as polydispersity index of 0.259. The scheme. monodisperse polymeric micel es had a zeta potential of -12.99 mV. 230 P44 VISUALISATION OF SIMVASTATIN CORE-SHELL PARTICLES PREPARED BY ELECTROSPRAYING METHOD USING STIMULATED EMISSION DEPLETION MICROSCOPY Barbara Sterle Zorec1, Hana Kokot2, Stane Pajk3, Janez Štrancar2, Rok Dreu1 1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Slovenia 2 Department of Condensed Matter Physics, Jožef Stefan Institute, Ljubljana, Slovenia 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION 2. MATERIALS AND METHODS In recent decades, biodegradable drug-loaded 2.1. Materials polymeric nano/microparticles have gained Polyvinylpyrrolidone (PVP, pharmaceutical considerable at ention in the field of drug grade, donation by Lek d.d.) of 50,000 daltons delivery and are constantly being investigated (PVP K30), polycaprolactone of 14000 daltons for their efficacy in modifying drug release, (PCL, Sigma Aldrich, USA), simvastatin (SIM, improving drug solubility and/or stability [1]. pharmaceutical grade, donation by Krka d.d.), The preparation of core-shel particles can 2-propanol (Merck KGaA, further improve the above properties, making Germany),chloroform (Merck KGaA, them an at ractive carrier for drug loading. Germany), Prolong Gold Antifade mountant Various methods have been presented for the (ThermoFisher Scientific), and #1.5H cover preparation of core-shel particles, but glasses (Paul Marienfeld) were used in this electrospraying seems to be the most promising work. Al solvents and other reagents used for with its numerous advantages. Electrospraying UPLC analysis were of HPLC grade. can be used to generate size-control ed, nearly monodisperse core-shel particles by optimising 2.2. Particle preparation the process parameters and solution properties Nanoparticles were fabricated by [2]. However, visualisation of such core-shel electrospraying using vertical y positioned particles remains a major chal enge. Scientists core-shel nozzle setup (Fluidnatek LE-100 mainly use scanning electron microscopy apparatus; Bioinicia, Spain) at temperature of (SEM) and transmission electron microscopy 25 ± 2 °C and relative humidity of 35 ± 2%. (TEM) to visualise core-shel particles. In Positive high voltage (8.8–13.4 kV) was applied contrast, stimulated emission depletion to the nozzle while the col ector was charged microscopy (STED) has not been used so far. negatively with -3.0 kV. Drug and polymer STED produces images with resolution down to formulation included core (PVP of 4.5% (w/V) 20 nm by selectively deactivating fluorophores, and SIM of 7.5% (w/V) dissolved in 2- which minimises the il umination area at the propanol) and shel (PCL of 2% (w/v) and focal point and thus increases the achievable fluorescent probe SAG38 of 0.02 mM) solution. resolution for a given system, enabling the The process parameters involved different core observation of structures smal er than the (0.3 – 0.6 mL/h) and shel solution flow rates diffraction light limit. However, only (1.0 – 1.5 mL/h), while the distance of the photostable fluorescent molecules with nozzle to col ector remained the same appropriate spectral characteristics can be used throughout al the experiments (20 cm). The for this purpose. The present study was devoted particles were sprayed directly onto #1.5H to STED visualisation of core-shel particles cover glasses and mounted with Prolong Gold containing simvastatin as a poorly soluble drug Antifade after drying. prepared by the coaxial electrospraying method. For this purpose, an efficient fluorescent probe 2.5. STED microscopy was synthesised and introduced into the shel The high-resolution STED micrographs of the polymer solution for subsequent STED fluorescent core-shel particles were measured visualisation. on a custom-built STED microscope (Abberior Instruments) [3] with 80 MHz pulsed lasers and a 60x water immersion objective (NA 1.2). The 231 P44 fluorophores were excited at 561 nm and their shel flow rate is too low (Fig. 2a). However, if emission was depleted by a STED laser at 775 the shel flow rate is increased sufficiently, a nm, shaped to obtain the best possible 3D completed shel can be observed that resolution. After passing through a pinhole (1.1 completely encases the core components (Fig. A.U), the emit ed photons with wavelengths in 2b). Further increasing the flow rate of the shel the range of 580–625 nm were detected on an solution resulted in shrinkage of the core (Fig. APD (filters by Semrock). The pixel size was 2c) and numerous particles containing only the 30 nm, and the dwel time and laser powers shel polymer (Fig. 2d). The flow rate that were optimised for each sample. Image proved to be optimal was 0.3 ml/h for the core Acquisition and basic analysis were performed and 1.2 ml/h for the shel . with Imspector Software (v16.3, Abberior Instruments). 3. RESULTS AND DISCUSSION 3.1. Fluorescent probe Figure 2. Typical super-resolution STED Fluorescent probe SAG38 (Fig. 1) is a micrographs of core-shel particles containing derivative of coumarin 6. Compared to the fluorescent probe in shel solution. The flow ratio lat er, SAG38 is more lipophilic and has between the core and shel solution decreases significantly red-shifted excitation and from left to right. emission spectra with their respective maxima at 547 nm and 625 nm. Importantly the emission 4. CONCLUSION of SAG38 at 775 nm is large enough to be successful y depleted by the 775 nm STED The results presented show that STED laser, and the probe is very photostable, making microscopy is an important alternative for it ideal for STED microscopy. visualisation of core-shel particles, where the addition of a suitable fluorescent probe in the shel at the right concentration is crucial. To achieve that the shel completely envelops the core of the particle, the flow rate between the two is of great importance. 5. REFERENCES 2. Kumar, B., et al., Recent advance in nanoparticles-mediated drug delivery. Journal of drug delivery science and technology, 2017. Volume 41: p. 260-268. 3. Sterle Zorec, B. et. al., Particle properties and Figure 1. Left: excitation (yel ow) end emission drug metastable solubility of simvastatin (green) spectra of SAG38. Right: structure of containing PVP matrix particles prepared by SAG38. electrospraying technique. European Journal of Pharmaceutical Sciences,2021. Vol.158:p.1-10. 3.2. Particle visualisation and morphology 4. Kokot, H. et. al.,. Prediction of Chronic The concentration of the fluorescent probe in Inflammation for Inhaled Particles: the Impact the shel solution was very important and of Material Cycling and Quarantining in the influenced the intensity of the final microscopic Lung Epithelium. Adv. Mater. 2020, 32. images. The probe concentration varied, with 0.02 mM being the most optimal. ACKNOWLEDGMENT Core-shel particles were prepared, varying the flow ratio between core and shel solution to The authors grateful y acknowledge the obtain particles whose core components are financial support provided by the Slovenian completely encased in shel polymer. At the Research Agency (Program P1-0189). same time, the flow rates were also varied according to the process stability to get a nicely stable Taylor cone. A look at Figure 2 shows that the particle shel is not complete when the 232 P45 ELECTROSPUN NANOFIBERS AS A DELIVERY SYSTEM FOR VAGINAL PROBIOTICS Spase Stojanov1,2, Tina Vida Plavec1,2, Julijana Kristl2, Špela Zupančič2, Aleš Berlec1,2 1Department of Biotechnology, Jožef Stefan Institute, Slovenia 2Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION were performed immediately after The female vagina is dominated by species of electrospinning and after 56 days of storage. the genus Lactobacillus. A decrease in their 2. MATERIALS AND METHODS numbers al ows overgrowth of opportunistic pathogens, leading to vaginal infections. The 2.1. Electrospinning of lactobacilli use of vaginal lactobacil i as probiotics can Lactobacil i (1010-1014 CFU/ml) were mixed restore the disturbed vaginal microbiota and with different polymer solution (PEO, stop the development of the infections. The lack PEO/alginate and PEO/alginate/sucrose) and of a proper delivery system and techniques to fil ed into 5 mL syringe at ached to an study the functionality of lactobacil i hinders electrospinning machine. The flow rate of the their use as probiotics. Nanofibers, produced by polymer dispersion was 150-250 µl/h and the electrospinning, are a promising delivery applied voltage was 10-13 kV. The nanofibers system for biological drugs [1] and vaginal were col ected on aluminium foil. lactobacil i [2, 3]. Electrospinning is a 2.2. Genetic engineering of lactobacilli technique in which a high voltage is applied to Four genes encoding fluorescent proteins with a polymer solution, resulting in elongation of different spectral properties, e.g. IRFP, GFP, the polymer and evaporation of the solvent [4]. mCherry and mTagBFP2, were cloned under The aim of our study was to address two major the control of the ldh promoter and inserted into limitations of vaginal probiotics, namely, the pNZ8418 plasmid. The four plasmids were development of a solid nanofiber delivery then loaded into lactobacil i using cel wal system for local administration, and techniques weakening agents and different electroporation for the studies of probiotic release and protocols [2]. distribution. We studied three vaginal lactobacil i ( L. crispatus, L. gasseri and L. 2.3. Viability assays jensenii) and L. plantarum as a control. For the The viability of lactobacil i was analysed in first part of our research we genetical y polymer solutions, in nanofibers immediately engineered the four species to express after electrospinning and after storage (56 fluorescent proteins with different spectral days). Series of ten-fold dilutions of bacteria- properties (IRFP, GFP, mCherry and polymer dispersions and dissolved nanofibers mTagBFP2) and incorporated them into loaded with bacteria were prepared. Bacterial polyethylene oxide (PEO) nanofibers [2]. In the viability was tested using the drop plate method second part, we tested the stability of non- [5], whereby 10 µL drops of each dilution were transformed vaginal lactobacil i in three pipet ed onto MRS agar plates and incubated different polymer solutions: PEO, PEO/alginate anaerobically at 37 °C for 2-3 days. and PEO/alginate/sucrose. The three formulations were used to prepare nanofibers with incorporated lactobacil i. Viability tests 233 P45 3. RESULTS AND DISCUSSION some viability after 56 days. L. gasseri showed 3.1. Nanofiber morphology the highest viability, fol owed by L. jensenii and L. crispatus. Incorporation of lactobacil i into nanofibers was confirmed by scanning electron (Fig. 1) and 4. CONCLUSION confocal microscope. Incorporation was In this research, we provide two solutions for observed with specific thickenings in the higher applicability of vaginal probiotics. We nanofibers. Incorporated lactobacil i and have shown that electrospun nanofibers are a addition of excipients changed the mean suitable delivery system for vaginal probiotics diameter of the nanofibers. that can preserve bacterial viability. Fluorescent proteins enable easy and real-time tracking of bacteria after their release. With this study we contribute to the development of novel medicine against vaginal infections. 5. REFERENCES 1. Stojanov, S., et al., Electrospun nanofibers as carriers of microorganisms, stem cells, proteins, and nucleic acids in therapeutic and other applications. Frontiers in bioengineering and biotechnology, 2020. 8: 130. Figure 1. Scanning electron microscope of a 2. Stojanov, S., et al., Engineering of vaginal mixture of four lactobacil i incorporated into lactobacilli to express fluorescent proteins PEO nanofibers. enables the analysis of their mixture in nanofibers. International journal of molecular 3.2. Fluorescent detection of lactobacilli after their release from nanofibers sciences, 2021. 22(24): 13631. 3. Silva, J.A., et al., Immobilization of vaginal Lactobacil i retained their fluorescence after Lactobacillus in polymeric nanofibers for its release from the nanofibers. Fluorescence incorporation in vaginal probiotic products. European journal of pharmaceutical sciences, signals depended on species, with L. plantarum 2021. 105563. demonstrating the highest fluorescence and L. 4. Pelipenko, J., et al., Critical attributes of crispatus the lowest. Fluorescent proteins were nanofibers: preparation, drug loading, and used to distinguish lactobacil i in a mixture and tissue regeneration. International journal of for studying individual lactobacil i distribution. pharmaceutics, 2015. 484 (1): 57-74. 5. Herigstad, B., et al., How to optimize the drop 3.3. Viability of lactobacilli in different plate method for enumerating bacteria. Journal polymer and nanofiber formulations of microbiological methods, 2001.44(2):121- 129. Lower temperatures (4 °C) and the addition of sucrose to the polymers improved the viability ACKNOWLEDGMENT of the lactobacil i. Bacterial viability in polymer solution reflected the stability of bacteria during Slovenian Research Agency (grants N3-0184, electrospinning. Sucrose also protected L. P1-0189 and P4-0127) and Public Scholarship, crispatus and L. jensenii during electrospinning Development, Disability and Maintenance Fund and L. gasseri and L. jensenii during storage. of the Republic of Slovenia – Ad Futura for S.S. The protective effect of sucrose might be related to its interaction with the bacterial proteins, as we showed using DSC and FTIR methods. Al lactobacil i species in al formulations showed 234 P46 EVALUATION OF DEXAMETHASONE CONTAINING IN SITU GELLING MUCOADHESIVE EYE DROPS Boglárka Szalai1, Mária Budai-Szűcs1, Orsolya Jójárt-Laczkovich1 1Institute of Pharmaceutical Technology and Regulatory Affairs, University of Szeged, Hungary 1. INTRODUCTION Adhesive force and work of adhesion were Eye drops are commonly used for the treatment determined based on the force-distance curves. of ocular diseases. The complex elimination 2.4. In vitro drug release study mechanisms of the eye cause poor The dialysis sac method [1] was used to bioavailability of this route of administration. investigate the in vitro drug release of the Dexamethasone (DXM) is frequently used to formulations. The studies were carried out at treat non-infectious inflammatory ocular 35℃, the release medium was simulated tear diseases. The low water-solubility and fluid. The released amount of DXM was penetration ability of dexamethasone decrease measured by HPLC method. DXM suspension its biological effectiveness. This work aims to and DXM-HPBCD solution were used as formulate in situ gel-forming mucoadhesive eye reference preparations. drops containing dexamethasone-cyclodextrin inclusion complex to improve the residence 3. RESULTS AND DISCUSSION time and the solubility of the active 3.1. Rheological studies pharmaceutical ingredient (API). The gel ing temperature and the gel ing time of the samples ranged from 23 to 37℃ and from 0 2. MATERIALS AND METHODS to 7 min., respectively. Both parameters were 2.1. Materials determined by the concentration of P407: For the complexation of DXM increasing the P407 concentration decreased the (2 - hydroxypropyl)-ߚ-cyclodextrin (DS׽4.5; gel ing temperature and the gel ing time. HPBCD) was chosen. The lowest necessary Formulations with a gel ing temperature above amount of HPBCD was applied to solubilize the room temperature but under body temperature therapeutic concentration of DXM. and with rapid gelation are desired. Poloxamer 407 (P407) was used to form 3.2. Tensile test thermosensitive in situ gel ing eye drops, and 2 Depending on the composition of the mucoadhesive polymers were combined with it. formulations the adhesive force and work of The concentration of the 3 polymers was adhesion values were between 800 and investigated on 3 levels. 2100 mN and 40 and 130 mN.mm, respectively. 2.2. Rheological studies Most of the formulations exhibited adequate Rheological studies were carried out to mucoadhesive properties. investigate the gel ing properties of the gels: 3.3. In vitro drug release study gel ing temperature and gel ing time were During the investigation, DXM was completely measured. released from the DXM-HPBCD solution while 2.3. Tensile test only about 51% of the API was released from Mucoadhesivity of the eye drops were the suspension. Regarding the in situ gels, the examined with a texture analyzer applying a released amount of DXM was higher compared mucoadhesion test rig. A mucin covered surface to the suspension; moreover, the dissolution was used to imitate the surface of the eye. wasn’t complete after 6 h. The dissolution 235 P46 curves suggested that more API might have dissolved over a longer period. 4. CONCLUSION We managed to formulate in situ gel ing mucoadhesive eye drops containing DXM- HPBCD inclusion complex. The complexation ensured the dissolution of the therapeutic concentration of DXM in the formulations. The eye drops had proper gel ing properties: the gelling temperature was neither too low nor too high, also the sol-gel transition was rapid enough not to be washed away easily. Satisfying mucoadhesivity was achieved by adding mucoadhesive polymers to the formulations. The gels displayed bet er in vitro drug release properties than the DXM suspension while providing prolonged dissolution. These results may be promising in formulating ocular in situ gels with prolonged residence time due to their mucoadhesive properties, thus increasing the bioavailability of eye drops. 5. REFERENCES 1. Kiss, E. L., et al., Development and Characterization of Potential Ocular Mucoadhesive Nano Lipid Carriers Using Full Factorial Design. Pharmaceutics, 2020. 12(7): 682. ACKNOWLEDGMENT Project no. TKP2021-EGA-32 has been implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021-EGA funding scheme. 236 P47 SOLUBILITY ASSESSMENT OF CANNABIDIOL IN DIFFERENT SOLVENTS Eva Tavčar1 1The Department of Pharmaceutical Biology, University of Ljubljana, Faculty for Pharmacy, Slovenia 1. INTRODUCTION 2.3. HPLC analysis Cannabidiol (CBD) is a cannabinoid, natural y CBD concentration was determined by the occurring in cannabis ( Cannabis sativa, L.). It HPLC method, using the Phenomenex exhibits pharmacological effects on Kinetex C18 column (100 mm/4,6 mm/2.7 vertebrates by acting on cannabinoid µm) and HPLC system Shimadzu, Kyoto, receptors, without causing mental impairment Japan (DGU-20As, LC-20AD XR, SIL-20AC [1]. Over the last ten years, the use and XR, CTO-20AC, SPD-M20A). Mobile phase recognition of CBD have increased A was water with 2 % acetonitrile and 0.1 % significantly among the general public. Apart trifluoroacetic acid. Mobile phase B consisted from being an active pharmaceutical ingredient of acetonitrile with 2 % water and 0.1 % in medicines, such as Epidyolex®, Sativex®, and trifluoroacetic acid. The gradient started with magistral preparations, CBD is being 44 % B, rising to 100 % during the fol owing increasingly used in food supplements and 10 minutes with a flow rate of 2 mL/min at 40 cosmetic products [2]. Nevertheless, there is °C. The injection volume was 5 µL. The lit le data in the literature describing its analytes were detected at 220 nm. technological properties. Therefore, we researched the solubility of CBD in the solvents 3. RESULTS AND DISCUSSION that are most important for its pharmaceutical 3.1. Solubility of CBD in laboratory solvents use. Measured solubility was highest in acetone, 2. MATERIALS AND METHODS methanol, and diethyl ether, fol owed by other laboratory solvents. Solubility in water was 2.1. Materials not observed within the limits of detection Cannabidiol isolate of 98,62 % purity (Dr. (Fig. 1). Ehrenstorfer, Germany). Solvents of p.a. purity: Propylene glycol, triethyl citrate, PEG 400, triacetin, ethanol, methanol, isooctane, acetone, isopropanol, DMSO, 1-butanol, dimethylformamide, cyclohexane, dichloromethane, diethyl ether. Oils, purchased as food products: Medium- chain triglycerides, hemp seed oil, olive oil, and chia seed oil. Solvents for HPLC analysis of HPLC purity: acetonitrile, water, and trifluoroacetic acid. 2.2. Solubility Solubility was determined by using the shake- flask method. Firstly, CBD suspensions were prepared (at room temperature), then shaken for 48 h on a shaker, filtered, and diluted. 237 P47 Figure 1. Solubility of CBD in laboratory 4. CONCLUSION solvents. The best laboratory solvent for CBD is acetone, 3.2. Solubility of CBD in pharmaceutical fol owing methanol. Among edible solvents pharmaceutical solvents, CBD is wel soluble in CBD was also very soluble in the solvents that triacetin, PEG 400, triethyl citrate, and are often used for the production of CBD propylene glycol, following edible oils. CBD is products. Among them, solubility was highest not soluble in water and even smal addition of in triacetin, PEG 400, and triethyl citrate. It water has a great impact on its solubility in other was slightly worse in propylene glycol. By solvents. adding water to propylene glycol, we discovered that even a smal addition of water 5. REFERENCES greatly reduces the solubility of CBD (Fig. 2). 1. Bonini S. A. et al.: Cannabis sativa: A comprehensive ethnopharmacological review of a medicinal plant with a long history. Journal of mg CBD/g solvent Ethnopharmacology 2018; 227: 300-315. 2. Francke N. M. et al.: Formulation of Cannabidiol in Colloidal Lipid Carriers, Glycerin with 5 % water 0 Molecules 2021, 26. Propylene glycol with 86 10 % water Propylene glycol with 5 270 % water Propylene glycol 781 Triethyl citrate 1173 PEG 400 1317 Triacetin 1334 0 400 800 1200 Figure 2. Solubility of CBD in pharmaceutical solvents. 3.3. Solubility in vegetable oils Among vegetable oils, CBD was most soluble in MCT oil and least soluble in olive oil. This indicates that the longer the triglyceride chain of the oil, the poorer is CBD solubility (Fig. 3). mg CBD/mL oil Olive oil 380 Hemp seed oil 400 Chia seed oil 410 MCT oil 681 0 200 400 600 Figure 3. Solubility of CBD in vegetable oils. 238 P48 DEVELOPMENT OF CHITOSAN AND ALGINATE BASED NANOFIBERS FOR WOUND HEALING APPLICATION Biljana Temova1,2, Betka Krampelj2, Petra Kocbek2 1Agency for Medicinal Products and Medical Devices of the Republic of Slovenia (JAZMP), Ljubljana, Slovenia 2Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION CS/PEO 2000 kDa (C), ALG/PEO 2000 kDa The primary goal of innovative wound healing (D), and ALG/PEO 2000 kDa with CaCl2 (E) is to create conditions that enable a fast formation of healthy tissue by stimulation of the normal restoration processes. Novel approaches are thus evolving in the field of advanced therapeutical medicinal products. The preparation of such medicines involves isolation of allogeneic or autologous cel s, their proliferation in vitro and seeding on/in a biocompatible three-dimensional matrix, which provides physical support to cel s. Nanofibers * with 0.7% and 2.0% (w/w) CaCl2,; **with 0.5%, 1.2% and 1.5% (w/w) CaCl have at racted significant interest in the 2; ***with 0.5%, 0.9%, 1.2% and 1.5% (w/w) CaCl2 preparation of such scaffolds, since they can 2.3. Electrospinning serve as a drug delivery system for different Fluidnatek LE-100 (Bioinicia, Valencia, Spain) bioactive molecules [1], mimic the structural was used for nanofiber preparation. A 10 mL building blocks of the natural extracel ular syringe was fit ed with a metal needle 0.7 mm matrix and provide a suitable microenvironment in diameter. The distance to the col ector was for cel adhesion, growth, and proliferation [2]. fixed to 15 cm for CS/PEO and to 20 cm for To achieve this nanofiber morphology should ALG/PEO solutions. The pump rate was set to be stable in wet tissue environment. Thus, the 100-800 µL/h. A voltage was varied from 10 kV aim of our study was development of such to 25 kV. Al nanofibers were electrospun at nanofibers. Chitosan (CS) and alginate (ALG) 37±2 °C and RH of 16±2 %. were selected as polymer matrix formers, since 2.4. Characterization of nanofibers they are biocompatible, non-toxic, natural The morphology of the electrospun products polymers with adequate water absorption and was analyzed using scanning electron poly(ethylene oxide) (PEO) was added to microscopy (SEM) (Supra35 VP; Carl Zeiss, improve the spinnability of polymer solutions. Oberkochen, Germany). The mean diameters of To increase the water stability of nanofibers selected nanofibers were determined based on a calcium ions were added to the ALG/PEO, representative SEM images using the ImageJ which have cross-linked ALG polymer chains. 1.53k software (NIH, USA). The nanofibers were electrospun onto glass 2. MATERIALS AND METHODS coverslips for evaluation of their stability in 2.2. Preparation of polymer solutions water. The coverslips with nanofibers were then The polymer solutions (Table I) were prepared soaked in 20 mL of purified water. Changes in just prior electrospinning. CS and PEO were their morphology were examined under an dissolved in 3% (v/v) acetic acid and Triton X- inverted light microscope 1 h, 24 h and 48 h 100 (0.3%, w/v) was added. ALG and PEO since the beginning of the experiment. were dissolved in purified water and Triton X- 100 (0.3%, w/v) or CaCl 3. RESULTS AND DISCUSSION 2 (0.5-2.0%, w/wALG) was added before electrospinning. The focus of our work was to develop Table 1: The composition of polymer solutions: hydrophilic nanofibers, stable in aqueous CS/PEO 400 kDa (A), CS/PEO 900 kDa (B), environment, as a novel drug delivery system 239 P48 for bioactive molecules for wound healing. A morphology of nanofibers prepared from 4.0% variety of polymer solutions based on CS and (w/v) ALG/PEO (70/30) solution with Triton ALG was thus prepared and electrospun to X-100 is shown in Figure 1B. The crosslinked investigate their spinnability and formation of ALG/PEO nanofibers were more stable in water nanofibril ar product. than ALG/PEO nanofibers, however their morphology was not preserved during 24 h 3.1. Chitosan/PEO nanofibers incubation. This indicates that 2.0% (w/w) CS/PEO nanofibers were prepared from CaCl polymer solutions containing polymers in 2 in polymer solution did not enable weight ratios 60/40, 50/50, 40/60 and 30/70, formation of nanofibers sufficiently resistant to aqueous environment and further research with respectively. The total polymer concentration was 3.0%-4.0% (w/v) in the case of PEO 400 higher concentrations of CaCl2 is thus needed. kDa and PEO 900 kDa and 3.0%-3.5% (w/v) in Table 2: Average diameters of CS/PEO and the case of PEO 2000 kDa. The diameters of the ALG/PEO nanofibers. selected electrospun nanofibers are presented in Table 2. SEM image of nanofibers prepared from 3.0% (w/v) CS/PEO 2000 kDa (40/60) solution with Triton X-100 shows smooth nanofibril ar morphology, without any beads (Figure 1A). The persistence of nanofibers morphology in water correlated with the amount of CS in the polymer solution. The fibril ar morphology of CS/PEO 2000 kDa (40/60) nanofibers was preserved during 48 h 4. CONCLUSION incubation in water, indicating their potential for wound healing application. CS/PEO and ALG/PEO nanofibers were successful y electrospun. CS/PEO 2000 kDa (40/60) nanofibers showed preserved A morphology (48 h) in aqueous environment, but morphology of ALG/PEO nanofibers with CaCl2 was not preserved, thus their composition should be further optimized for potential application in wound healing. 5. REFERENCES 1. Bertoncelj V., et. al., Development and bioevaluation of nanofibers with blood-derived B growth factors for dermal wound healing. European Journal of Pharmaceutics and Biopharmaceutics, 2014. 88 (1): 64-74. 2. Lee V. K., et. al., Design and fabrication of human skin by three-dimensional bioprinting. Tissue Engineering. Part C, Methods, 2014. 20 (6): 473-84. ACKNOWLEDGMENT Figure 1. Representative SEM image of (A) The authors grateful y acknowledge the CS/PEO and (B) ALG/PEO nanofibers. financial support provided by the Slovenian Research Agency (Program Codes P1-0420 and 3.2. Alginate/PEO nanofibers P1-0189) and JAZMP. ALG/PEO 2000 kDa nanofibers were prepared from polymer solutions containing polymer in weight ratios 80/20, 70/30, 60/40 and 50/50, respectively. The total polymer concentration was 2.0-4.5% (w/v). The diameters of selected nanofibers are presented in Table 2. The 240 P49 APPLICATION OF SUPPORT VECTOR MACHINE LEARNING FOR ORODISPERSIBLE FILMS DISINTEGRATION TIME PREDICTION Erna Turković, Ivana Vasiljević, Dragana Vasiljević, Svetlana Ibrić, Jelena Parojčić Department of Pharmaceutical Technology and Cosmetology, University of Belgrade – Faculty of Pharmacy, Serbia 1. INTRODUCTION by dispersing HPC in ethanol:glicerol solution Orodispersible films (ODF) have emerged as fol owed by continuous stirring on the magnetic innovative dosage forms that provide wide stirrer. Prepared dispersions were: (i) casted on variety of advantages for patients and a unit-dose plexiglas plates, or (i ) printed using manufacturers over conventional dosage forms. Ultimaker 2+ (Ultimaker, , Netherlands). ODFs The prominent characteristic of ODFs is fast were characterized in terms of mechanical disintegration fol owed by good patients properties using Z-LX Table-Top Testing acceptability [1]. Therefore, relevant Machine (Shimadzu, Japan) and DT using disintegration time (DT) is usual y considered adapted compendial tester (Erweka ZT52, as ODF critical quality at ribute. Extensive Germany) with a weight. research on ODFs is generating a lot of data, but 3. RESULTS AND DISCUSSION lack of standardization is the main obstacle that limits their comparative evaluation. The 3.1. Data pre-processing fol owing work aims to explore literature data 274 papers (without reviews) were identified on ODFs characteristics using the predictive via search, of which 112 were included in the data-classification algorithm Support vector database. machine (SVM) and assess its applicability in pharmaceutical development based on the set of Table 1. Classification of disintegration testing experimentally obtained data. and manufacturing methods 2. MATERIALS AND METHODS Disintegration testing methods Manufacturing methods 2.1. Materials Compendial method for solid dosage Solvent casting – 1 forms – 1 Hydroxypropyl cel ulose (Klucel GF, Ashland, Adapted compendial method (with Inkjet printing – 2 USA), ethanol (≥99.8%, Honeywell, Charlotte, clamp/frame) – 2 NC, USA) and glycerol, 85% (w/w) (Ph. Eur.) Adapted compendial method (with 3D printing – 3 were used for preparation of printing and clamp/frame and weight) – 3 casting dispersion. Drop method / Slide frame method Electrospinning – 4 (without weight) – 4 2.2. Data pre-processing Drop method / Slide frame method Hot-melt extrusion Comprehensive data exploration has been (with weight) – 5 – 5 conducted in the PubMed database using most Petri dish / Beaker / Glass vial method common synonyms for ODFs with fifteen (with agitation) – 6 synonyms in singular and plural. Built database Petri dish / Beaker / Glass vial method (without agitation) – 7 had fol owing at ributes: manufacturing Other – 8 approach, polymer selection, polymer molecular weight (KDa), polymer load (%), Nominal data from literature was transformed mechanical properties (tensile strength (MPa), into numerical, using coding operator so that Young's modulus (MPa), elongation at break each nominal data had corresponding numerical (%)), disintegration method and disintegration value. Critical at ributes for films fast time (DT) (s). disintegration were derived. 18 polymers were included as categorical data and were further 2.3. ODF preparation and characterisation differentiated on the basis of molecular weight. Polymer dispersions for solvent casting and Values for most commonly evaluated semi-solid extrusion 3D printing were prepared mechanical properties were included as 241 P49 numerical data. Different DT methods were classified in seven classes (Table 1), while the manufacturing methods were classified in five classes. RapidMiner Studio 9.10 (RapidMiner, Dortmund, Germany) was used to transform data and employ SMV algorithm. 3.2. SVM model prediction At ributes with the highest weight were polymer load and DT method employed (Figure Figure 2. Model simulator used to predict DT of 1). The polymer type and characteristic did have 3D printed HPC-based films conclusive effects on DT as their weight varied during data mining. This can be at ributed to When solvent casting method was considered, inconclusive data provided in papers and lot of predicted DT value was remarkedly higher than missing values for those at ributes. Mechanical the experimental y obtained one, indicating bad properties had low weight, which can be predictability. It might be assumed that good explained with the broad value range for those predictability obtained in the case of 3D printed at ributes. Different research groups had films is associated with lower data variability due different approach to disintegration testing, to more simple sample composition and robust which lowered model precision as it was preparation method. In the case of casted films, reported that SVM does not have high accuracy data was much more complex due to a higher when data is imbalanced [3]. Relative error number of research papers and approaches to value was 20%, which can be considered as characterisation. high, but, having in mind great diversity in 4. CONCLUSION presented data and methodology, obtained value is stil acceptable for the pilot study. The obtained results indicate that SVM algorithm can be employed to predict ODF DT value based on the dataset created using literature data. However, in order to obtain meaningful predictions, larger dataset, with fewer inconsistences and less missing values would be advantageous. 5. REFERENCES 1. Scarpa, M., et al., Key acceptability attributes of orodispersible films. European Journal of Figure 1. At ribute weight in predicting model Pharmaceutics and Biopharmaceutics, 2018. 125: 131-140. 3.3. Experimental validation 2. Preis, M., et al., Comparative study on novel test HPC-based films prepared by 3D printing had systems to determine disintegration time of tensile strength, elongation at break and orodispersible films. Journal of Pharmacy and Young’s modulus of 3.5 MPa, 137% and 5 Pharmacology , 2014, 66: 1102–1111. MPa, respectively. Average DT was 69 s. For 3. Wu,G and Chang,E.Y. Adaptive feature-space casted films, relevant values were 3.4 MPa, conformal transformation for imbalanced-data 105% and 3 MPa, and DT was 27 s. learning. Proceedings of the 20th ICML, Experimental y obtained results were entered Washington DC, 2003. 816-823. into model simulator (Figure 2) to simulate situation reflecting the experimental set up in which HPC-based films were prepared by 3D ACKNOWLEDGMENT printing and solvent casting, and relevant This work was financial y supported by the at ribute values obtained by samples Ministry of Education, Science and characterization. In the case were manufacturing Technological Development of the Republic of method was set to be 3D printing (coded as 1) Serbia (project 451-03-68/2022-14/200161). predicted DT value was close to experimental y obtained value, i.e. 71.7 and 69 s, respectively. 242 P50 DEVELOPMENT OF A PREPARATION METHOD FOR NANOCAPSULES USING FACTORIAL DESIGN Luca Éva Uhljar1, Balázs Kürtösi1, Rita Ambrus1 1Institute of Pharmaceutical Technology and Regulatory Affairs, University of Szeged, Hungary 1. INTRODUCTION The aim of the present research was to prepare In the production of medicines, particular NCs incorporating diverse active at ention should be paid to the quality of the pharmaceutical ingredients (API) by solvent product. For this reason, FDA proposes the use displacement method while specifying the of Quality by design (QbD), a process that optimal ranges of preparation parameters, thus al ows quality to be built into medicines [1]. As facilitating the creation of a future Design part of the QbD, the Critical Quality At ributes Space. and Critical Process Parameters are determined 2. MATERIALS AND METHODS in order to define the Design Space of the production. Factorial design is a research 2.1.Materials methodology, also used in QbD, that al ows us The drugs tested were lamotrigine, to obtain reliable results and correlations from a ciprofloxacin, and propranolol. The adjuvants smal er number of experiments. forming the core of the NCs were PEG-stearate- 40, Transcutol, and oleic acid, while the shel Nowadays, nano-drug delivery systems have was made up of chitosan. The aqueous phase become prominent. Polymer nanocapsules was ultra-purified water. (NCs) are innovative drug delivery systems that are composed of an oil-fil ed core and a 2.3. Preparation of the nanocapsules biodegradable polymer shel . NCs have several One of the criteria for factorial experiment advantages, such as increased drug stability, design is to have measurable and control able solubility and permeability, and reduced production parameters. For this purpose, toxicity, as wel as tailored therapy through continuous mixing was ensured by a magnet control able drug release. NCs can be stirrer with digital display. The oil phase and formulated mainly for alternative after the polymer solution were dripped through administration routes, e.g., topical, parenteral, a needle into the aqueous phase via a peristaltic nasal and pulmonal. pump with adjustable flow rate. NCs can be produced in several ways, of which 2.2. Setting up a factorial design solvent displacement method is a simple three- step process. In the first step, a nanoemulsion of As part of the QbD, the Critical Quality the oil and aqueous phases is formed by At ributes and Critical Process Parameters that continuous mixing. In the second step, the determine the quality of NCs have been nanoemulsion droplets are coated with the col ected and systematized in an Ishikawa- shel -forming polymer, also by dripping and diagram during a previous work of our research continuous mixing. The third step is the group [2]. In this work, the process parameters purification of the NCs by ultracentrifuge. The affecting product quality were the mixing rate properties of the NCs (e.g., size, size (rpm), the flow rate (mL/min) of the oil phase, distribution, stability) can be control ed by and the diameter of the needle (G). For the varying the preparation parameters. By factorial design, we chose these parameters as choosing the appropriate parameters, NCs with independent variables and then varied them at optimal properties can be produced. three levels, set ing up an 33-1 factorial design (Table 1). 243 P50 The most important characteristics of NCs are 500 0,35 particle size and size distribution (PdI), which 0,3 400 can be measured by dynamic light scat ering 0,25 ) (DLS), and stability, which can be determined 300 0,2 from the surface charge (Zeta potential). 0,15 200 Size (nm Therefore, these properties were chosen as the 0,1 100 0,05 dependent variables in the factorial design. 0 0 1 2 3 4 5 6 7 8 9 Table 1. Independent variables of the 33-1 lamotrigine - size ciprofloxacin - size factorial design propranolol - size lamotrigin - PdI ciprofloxacin - PdI propranolol - PdI diameter of 14 mixing speed flow rate the needle 12 (rpm) (mL/min) (G) V) 10 200 0.35 18 8 6 200 1.06 23 Zeta potential (m 4 200 1.76 20 2 0 850 0.35 18 1 2 3 4 5 6 7 8 9 lamotrigine ciprofloxacin propranolol 850 1.06 20 Figure 1. Investigated properties of the NC 850 1.76 23 samples 1500 0.35 20 4. CONCLUSION 1500 1.06 18 NCs containing lamotrigine, ciprofloxacin and 1500 1.76 23 propranolol were prepared. Most of the samples meet the criteria, therefore the investigated method was considered robust. Among the 3. RESULTS AND DISCUSSION preparation parameters, the mixing speed was found to be the most relevant factor. It is NCs containing lamotrigine, ciprofloxacin and recommended to keep it high during the propranolol were prepared and examined production of NCs by solvent displacement according to the 33-1 factorial design. To ensure method. that the concentration of the API would not influence the results, NCs were prepared using 5. REFERENCES 2 mg/mL solutions of al three APIs. 1. Yu, L. X., et al., Understanding Pharmaceutical For critical properties, the acceptance criteria Quality by Design. The AAPS Journal, 2014. were set to a maximum of 400 nm for size, 0.2 16(4): 771-783. 2. Gieszinger, P., et al., Preparation and for PdI, and a minimum of 5 mV for Zeta characterization of lamotrigine containing potential (Fig.1). nanocapsules for nasal administration. European Journal of Pharmaceutics and The encapsulation of lamotrigine and Biopharmaceutics, 2020. 153(2020): 177-186. ciprofloxacin resulted in a product with satisfactory properties over almost the entire ACKNOWLEDGMENT range of the chosen adjustment parameters. This work was supported by the UNKP-21-3-II. Overal , a higher mixing rate is recommended. New National Excel ence Program of the This is also recommended for propranolol, but Ministry for Innovation and Technology. Also, even so, a homogeneous particle size 2019-2.1.11-TÉT-2020-00147 and Gedeon distribution cannot be ensured. Richter Plc. Centenial Foundation are acknowledged. 244 P51 DEVELOPMENT AND EVALUATION OF FDM PRINTED NASAL DEVICE FOR SOLID NANOPARTICLES Zoltán Ujhelyi1 2, Dóra Kósa1 2 3, Ágota Pető1 2 3 ,Thinh To Quoc2, Ildikó Bácskay1 2 3 1Department of Pharmaceutical Technology, University of Debrecen, Hungary 2Doctoral School of Pharmaceutical Sciences, University of Debrecen, Hungary 3Healthcare Industry Institute, University of Debrecen, Hungary 1. INTRODUCTION performed on RPMI 2650 immortalised nasal Nasal drug administration has been in the focus epithelial cell line by MTT viability assay. of scientific interest for decades. A number of 2.3. Formulation of solid nano carriers drug delivery systems and devices have been already on the market. Their advantageous Solid phase nanoparticles were formed by properties are not questionable. Most nasal BUCH B 90 Nano Spry dryer apparatus. The preparations are liquids that can cause physical properties of the formed systems were discomfort to patients during the application. examined by two methods. The particle size The aim of the research was to develop and distribution of the formed nanoparticles was characterise novel nanoparticles that provide investigated in dispersion state using Malven improved bioavailability of incorporated ZSPNano Zetasizer. SEM images were taken to pharmaceutical compounds. Finely divided examine the morphology of the formed nanoparticles were formed by Büchi B90 Nano nanoparticles. Spray dryer. A major step in the development of the drug carrier was the selection of the 2.3. Preparation of nasal filters and nasal appropriate inert carrier compounds. Besides drug delivery devices the selection of suitable carriers physical and The devices were manufactured using FDM 3D biocompatibility parameters had been evaluated printing technology after 3D computer design, as wel . Moreover, in connection with the upon the anatomical features. Performed project, an individual drug delivery device and devices had been refined upon the kind a protective nasal filter were also developed suggestions of our volunteers with FDM 3D technology. 3. RESULTS AND DISCUSSION 2. MATERIALS AND METHODS 3.1. Formulation of SNEDD systems 2.1.Materials In our experiments, five surfactant Chlorpromazine Lauroglycol FCC, combinations were created by titration with Kol iphorEL, Transcutol HP, Miglyol 840, self-assembling ability. Al compositions were DMSO, Labrafil 1944, Glycerolum 85%, stable after two week standing at RT. Labrasol, Propylene glycol, PVA, Mannitolum, Maltodextrin, BSA, RPMI 2650 nasal ephitelial Smix A Lauroglycol FCC cel line, MTT cel viability assay kit, PLA Cremophor EL filaments. Transcutol HP 2.2.Formulation of SNEDD systems SmixB Miglyol 840 The formation of self-assembling emulsion Cremophor EL systems was performed by titrometric dilution (DMSO:glicerin, 1:3) of the appropriate combination of surfactants (Fig. 1). Biocompatibility assays of selected compounds and performed systems had been 245 P51 SmixC Labrafil 1944 3.3. Preparation of nasal filters and nasal Labrasol drug delivery devices Cremophor EL Propylene glycol PLA base nasal filter devices and individual nasal delivery device had been developed by 3D SmixD Labrasol FDM technique. Transcutol Cremophor EL (DMSO glicern, 1:3) SmixE Lauroglycol 90 Cremophor EL Transcutol HP Table 1. Compositons of performed SNEDDS. Figure 3. Computer draft and FDM printed model of nasal device 4. CONCLUSION Our result might ensure useful data for further development strategies in nasal drug delivery system development 5. REFERENCES Figure 1. Pseudoternary diagrams of performed 1. Flavia Laffleur et al. Progress in nasal drug SNEDDS. delivery systems,International Journal of Pharmaceutics,Volume 607, 2021, 120994 3.2. 2. Patrícia Henriques et al. Spray Dried Powders Formulation of solid nano carriers For Nasal Delivery: Process And Formulation PVA based chlorpromazine solid nano carriers Considerations, European Journal Of Pharmaceutics And Biopharmaceutics, Volume had been formulated. SEM investigations 176, 2022, Pages 1-20, confirmed the apprpiate morphology of the 3. Niklas Sandler Salmela et al. Towards particels. Size distribution measurements fabrication of 3D printed medical devices to confirmed that the size of the performed prevent biofilm formation, International Journal particals is between 30 and 300 nm in case of of Pharmaceutics, Volume 459, Issues 1–2, 2014, Pages 62-64. different compositions. ACKNOWLEDGMENT Research was supported by GINOP-2.3.4-15- 2016-00002 and project no. TKP2021-EGA-18 Has been implemented with the support provided from the National Research, Development And Innovation Fund of Hungary, financed under the TKP2021-EGA Figure 2. Size distribution of composition B funding scheme. combined PVA particles 246 P52 INVESTIGATION OF THE EFFECTIVENESS OF CHITOSAN COATING ON PROBIOTIC MICROCAPSULES AND INTERACTION WITH LACTOBACILLUS PLANTARUM Judit Váradi1, Lóránd Erdélyi1, Ferenc Fenyvesi1, Gábor Vasvári1, Ádám Haimhoffer1 Ilona Bereczki2, György Vámosi3, Miklós Vecsernyés1, Ildikó Bácskay1, Renátó Kovács4 1Department of Pharmaceutical Technology, University of Debrecen, Hungary 2Department of Pharmaceutical Chemistry, University of Debrecen, Hungary 3Department of Biophysics and Cell Biology, University of Debrecen, Hungary 4Department of Medical Microbiology, University of Debrecen, Hungary 1. INTRODUCTION performed by adding 0.4 w/w% LMW chitosan Chitosan is a mucoadhesive polymer, a solution. Wet alginate and chitosan coated commonly used gel ing agent. However, it has microcapsules were lyophilized to increase their antibiotic activity, which is used as an adjuvant shelf life. A portion of the dried alginate in addition to antifungal and antibiotic agents in microcapsules was coated with Eudragit L100- topical formulations. In the present study 55 in Mini-Glat fluid bed system. chitosan-coated and Eudragit L100-55 coated 2.3. Dissolution Test in Simulated alginate microspheres were produced to Gastrointestinal Fluids encapsulate Lactobacillus plantarum probiotic The dissolution test was performed in simulated bacteria. gastric (pH 2.0) than intestinal fluids (pH 7.4). According to our previous results, the chitosan Samples were col ected every hour, and the used as a coating significantly reduced the microcapsules were dissolved in simulated number of germs, which may result in reduced gastric juice for 60 minutes and in simulated efficacy of the probiotic preparation. intestinal fluid for an additional 180 minutes. Concentration-dependent interactions with 2.4. Live/Dead Cell Determination Dissolved Lactobacillus plantarum and the resulting from Coated and Uncoated Microcapsules bacteriostatic/bactericidal effect were Al dissolution samples were centrifuged, and investigated. the bacterial cel s were stained with SYTOX 2. MATERIALS AND METHODS Green reagent. Samples were analyzed with Guava Easy Cyte 6HT-2L flow cytometer 2.1. Materials (Merck Ltd., Darmstadt, Germany). Released Lactobacillus plantarum subsp. plantarum cel numbers and live /dead ratios were (ATCC 14917) was bought from ATCC (USA). evaluated (1). Low (50-190 kDa, LMW), medium (MMW) 2.5. Determination of Dissolved Chitosan and high (310-375 kDa, HMW) molecular Coat weight chitosan were purchased from Sigma- FITC‐labeled LMW chitosan was used to Aldrich (Budapest, Hungary). quantify the chitosan coating of the 2.2. Preparation and Coating of Alginate microcapsule. The preparation of the Microcapsules microcapsules, coating and dissolution test Probiotic bacterium-loaded alginate were same as previously described. However, ¼ microcapsules were prepared by gelation partial y FITC-label ed chitosan was used for method. Planktonic bacterium suspension was coating. The fluorescence of the samples was added to sterile sodium alginate solution. The examined by microplate reader (1). vibration nozzle technology was used by Buchi 2.6. Determination of Minimum Inhibitory 390-Pro equipment. Microcapsules were Concentration (MIC) al owed to harden in calcium chloride solution. The chitosan coating of wet microcapsules was Chitosan MICs were determined using the CLSI standard broth microdilution method. MICs 247 P52 were read after 24 h using the partial inhibition criterion as outlined in the guidelines (at least 50 % growth reduction as compared to the growth control) (2). 2.7. Time-Kill Assay In time-kil assays the effect of LMW, MMW, HMW chitosan was investigated on Lactobacillus plantarum. The starting inoculum was 106 CFU/mL, the chitosan concentrations were 0,003-0,125 %. Al tubes were plated at 0, 4, 8, 12, and 24 h and CFU were determined (2). 3. RESULTS AND DISCUSSION Figure 2. Representative time-kil curve of 3.1. Live/Dead Cell Determination Dissolved LMW chitosan. from Coated and Uncoated Microcapsules The time-kil assay was performed with al three chitosan samples. Based on our results, with increasing molecular weight, we observed a decrease in CFU at earlier time points at the same concentrations. This assay samples were investigated paral el by flow cytometry (method described in 2.4. section). The results of the time-kil assay were confirmed by the flow cytometric results. 4. CONCLUSION The antimicrobial activity of chitosan has not Figure 1. Viability of released L. plantarum. been studied in the probiotic formulations in Uncoated alginate (A), chitosan-coated (C), and which it is a disadvantage. In our studies, we Eudragit-coated (E) microcapsules were revealed that chitosan used as a coating can investigated in triplet. Data are presented as form concentrations above the MIC means ± SDs, n = 3 (1) concentration in the dissolution medium. In our latest studies, we confirmed the interaction of 3.2. Determination of Dissolved Chitosan chitosan with L.plantarum, which resulted a Coat bacteriostatic and bactericidal effect depending on the concentration. In addition to the time- In view of the labeling efficiency and the kil ing assay, the interaction was also confirmed labeled chitosan ratio in the coating solution, the by confocal microscopy. concentration of chitosan in the dissolution medium was 0.043% (1). 5. REFERENCES 3.3. Determination of Minimum Inhibitory 1. Erdélyi, L., et al., Investigation of the Role and Concentration (MIC) Effectiveness of Chitosan Coating on Probiotic Microcapsules. Polymers, 2022. 14(9):1664. The low molecular weight chitosan (LMW) 2. Papp Z., et al., Unpredictable In Vitro Killing MIC value were determined: 0,015%. The Activity of Amphotericin B against inhibitory concentration of chitosan decreases Four Candida auris Clades. Pathogens, 2021 with increasing molecular weight. 10(8):990. 3.4. Time-Kill Assay ACKNOWLEDGMENT Project no. TKP2021-EGA-18 has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the TKP2021-EGA funding scheme. 248 P53 INVESTIGATION OF MELOXICAM POTASSIUM CONTAINING NANOPARTICLES FOR INTRANASAL ADMINISTRATION Patrícia Varga1, Csilla Bartos1,Rita Ambrus1 1Faculty of Pharmacy, Institute of Pharmaceutical Technology and Regulatory Affairs, University of Szeged, 6720 Szeged, Hungary 1. INTRODUCTION 2.2. Preparation of the formulations Nose is a potential route to deliver active The formulations (HPβCD_MEL-P; pharmaceutical ingredients (APIs) to the HPβCD_MEL-P_PVA; αCD_MEL-P; systemic circulation or the central nervous αCD_MEL-P_PVA) were prepared by nano system. The absorbed drugs avoid the first pass spray drying using a BÜCHI Nano Spray Dryer hepatic metabolism and rapid onset of action B-90 HP (BÜCHI Labortechnik AG, Flawil, may occur [1,2]. However, the excipients and Switzerland) applying 80 °C of inlet air the dosage form need to be careful y chosen temperature. because of the hindering effect of the 2.3. Characterization of the formulations mucociliary clearance and the limited The size, shape and surface characteristics of absorption of certain molecules on the the prepared particles were observed with bioavailability [3,4]. Mucoadhesive polymers scanning electron microscopy (SEM). can increase the contact time of the APIs with The crystal inity of the products and the the mucosa, and permeability enhancers can physical mixtures (PMs) as controls was improve the absorption of the drugs so that investigated with X-ray powder diffractometry higher bioavailability can be achieved [5,6]. (XRPD). Among nasal dosage forms, nasal powders The thermal properties of the prepared samples appear to be a bet er choice than drops, sprays and the physical mixtures (PMs) as controls or ointments, because their residence time is were determined by differential scanning higher on the mucosal surface, and their calorimetry (DSC). stability is higher because of their low moisture The determination of secondary interactions content. Also, there is no need to use between MEL-P and the excipients was carried preservatives during their preparation process out with Fourier-transformed infrared which are often irritating for the sensitive nasal spectroscopy (FTIR). mucosa [7]. The in vitro diffusion of MEL-P was In our work, we aimed to produce nasal y investigated using a modified horizontal applicable meloxicam potassium containing diffusion cel which model ed the nasal cyclodextrin nanospheres by spray drying to conditions. An artificial membrane soaked in induce rapid analgesic effect. isopropyl myristate was applied to il ustrate the 2. MATERIALS AND METHODS lipophilic mucosa. The ex vivo permeation studies were performed 2.1.Materials using human nasal mucosa as the membrane in the modified horizontal diffusion cel . The test Meloxicam potassium monohydrate (MEL-P) was carried out on the formulations that showed was used as the API. (2-Hydroxy)-propyl-β- the best in vivo results. cyclodextrin, (HPβCD) α-cyclodextrin (αCD), poly(vinylalcohol) (PVA) and hyaluronic acid 3. RESULTS AND DISCUSSION (HA) were used as permeability enhancing and mucoadhesive excipients. As a result of spray drying, smooth surfaced, round shaped, nanosized particles were successfully prepared (Figure 1). 249 P53 On the XRPD diffractograms of the spray dried 4. CONCLUSION samples, the disappearance of the diffraction peaks assigned to crystal ine MEL-P and αCD Spherical nanoparticles containing amorphous indicated their amorphization and the formation MEL-P were successful y prepared and the of the inclusion complexes. formation of API-cyclodextrin complexes were According to the DSC measurements, the confirmed. Based on the results of in vitro and endothermic peaks of crystal ine MEL-P on the ex vivo permeation studies, the highest amount curves disappeared in al of the spray dried of MEL-P diffused from the αCD_MEL- samples compared to the PMs. This suggests the P_PVA sample. The prepared formulations may amorphization of the drug and the formation of be suitable for delivering MEL-P to the inclusion complexes with the cyclodextrins. systemic circulation through the nasal route and Besides, on the FTIR spectra, the shifting of the relieve pain rapidly after further optimization. peaks assigned to the stretching and the bending 5. REFERENCES vibration of H-O in the samples could indicate the formation of hydrogen bonds between the 1. Ugwoke, Michael I., et al. Nasal mucoadhesive MEL-P and the cyclodextrins. drug delivery: background, applications, trends and future perspectives. Advanced drug Based on the results of in vitro and ex vivo delivery reviews, 2005. 57(11): 1640-1665. permeation studies, the presence of HA resulted 2. Mathias, NR.; Hussain, MA. Non-invasive in the decrease of MEL-P permeation, while systemic drug delivery: developability PVA seemed to affect beneficial y the diffusion considerations for alternate routes of of MEL-P under nasal conditions. The highest administration. Journal of pharmaceutical amount of MEL-P permeated from the sciences, 2010, 99(1): 1-20. 3. Il um, L. Nasal drug delivery—possibilities, αCD_MEL-P_PVA sample both in vitro and ex problems and solutions. Journal of control ed vivo (Figure 2). release, 2003. 87(1-3), 187-198. 4. Inoue, D., et al. The relationship between in vivo nasal drug clearance and in vitro nasal mucociliary clearance: Application to the prediction of nasal drug absorption. European Journal of Pharmaceutical Sciences, 2018. 117, 21-26. 5. Jiang, L., et al. The application of mucoadhesive polymers in nasal drug delivery. Drug development and industrial pharmacy, 2010. 36(3): 323-336. 6. Davis, S. S., & Il um, L. Absorption enhancers Figure 1. SEM images of the spray dried for nasal drug delivery. Clinical pharmacokinetics, 2003. 42(13), 1107-1128.. samples 7. Kublik, H., & Vidgren, M. T. Nasal delivery systems and their effect on deposition and absorption. Advanced drug delivery reviews, 1998. 29(1-2), 157-177. ACKNOWLEDGMENT This work was supported by the Ministry of Human Capacities, Hungary grant TKP2021- EGA-32 and GINOP 2.2.1-15-2016-00007 Projects. Figure 2. Ex vivo diffusion of MEL-P 250 P54 HOW FORMULATION PARAMETERS AFFECT COMPRESSION BEHAVIOUR OF MULTIPARTICULATE UNITS PREPARED BY SELECTIVE LASER SINTERING? Ivana Vasiljević, Erna Turković, Jelena Parojčić Department of Pharmaceutical Technology and Cosmetology, University of Belgrade – Faculty of Pharmacy, Serbia 1. INTRODUCTION Model Polymer Colorant Size Selective laser sintering (SLS) represents novel Sample drug (87%) (3%) (mm) 3D printing technology recently introduced in (10%) drug fabrication. It is applicable in different C1/I1 1 dosage forms preparation, including C2/I2 CAF / EC 2 multiparticulate units (MPUs) (1). The C3/I3 IBU CGS characteristics of the obtained MPUs remain to MA-EA 1 C4/I4 2 be described, particularly their compression behaviour and mechanical properties of the Table 1. Multiparticulate units composition obtained compacts. The aim of this work was to investigate 2.3. Multiparticulate units compression compaction suitability of MPUs prepared by MPU compacts (100 mg) were prepared on an SLS printing and investigate the effect of model instrumented tablet press GTP series D drug type, polymer type and MPU size as wel (Gamlen Tableting Ltd, UK) in the single as compression pressure on the compression- compression mode, under the compression related parameters (detachment and ejection loads of 250 and 500 kg, using 6 mm diameter stress, net work of compression) and the flat punch, at the compaction speed 30 mm/min. obtained compacts characteristics (out-of-die The supporting software enabled complete elastic recovery, solid fraction and tensile visualization of the upper punch position and strength). force in real time. The measured force- displacement curves were used to calculate nett 2. MATERIALS AND METHODS work of compression, friction force between lower punch and tablet during detachment 2.1. Materials phase (detachment stress) and friction force The MPUs were prepared using either ethyl between die and tablet in the ejection phase cel ulose (EC, Ethocel, Fluka, Switzerland) or (ejection stress). methacrylic acid-ethyl acrylate copolymer (1:1) Compact dimensions were determined 24 hours (MA-EA, Eudragit L 100-55, Evonik, after compression. Caliper was used to measure Germany) as polymer forming matrices. the out-of-die compact thickness (t), while Ibuprofen (IBU) and caffeine (CAF) were used compact diameter (R) and hardness (F) were as model drugs and Candurin® Gold Sheen measured using the hardness tester Erweka (CGS, Merck KGaA, Germany) was added as TBH 125D (Erweka GmbH, Germany). The pharmaceutical grade colorant. obtained values were used to calculate compact tensile strength, solid fraction and out-of-die 2.2. Multiparticulate units preparation elastic recovery. Spherical 3D models were designed and In order to statistical y investigate the input imported as print job file (.stec) to desktop SLS parameter effects (polymer type, model drug printer Sintratec Kit (Sintratec AG, type and MPU size), experimental design was Switzerland). Samples composition is presented applied, using software Design-Expert v.7.0 in Table 1 (C-samples contain CAF, while I- (Stat-Ease Inc, USA). samples contain IBU). 251 P54 3. RESULTS AND DISCUSSION drug type-polymer type and model drug type- compression pressure interaction (p<0.0001). 3.1. Multiparticulate units compression Higher compression pressure increased the The prepared compact tensile strength was elastic recovery values more notably in the case general y higher than 1 MPa and acceptable (2), of IBU or EC samples. Model drug type, as represented in Fig. 1, while solid fraction polymer type and compression pressure ranged from 67.67 (C4) to 89.46% (C1 and I1). affected net work (p<0.001), as wel as model MPUs containing CAF and MPUs with MA-EA drug-compression pressure and polymer type- exhibited higher increase in solid fraction and compression pressure interaction. Based on the tensile strength when compression load was investigated MPU samples, software-aided increased, in comparison to samples prepared prediction recognized IBU, MA-EA and 1 mm- with IBU or EC, respectively. This indicates MPUs size as desirable for obtaining compacts bet er tabletability and compressibility. MPU with high tensile strength, but also low elastic samples with MA-EA or 1 mm size exhibited recovery, low detachment and ejection stress higher net work of compression, but also higher and high net work values. values of elastic recovery. Higher energy input corresponds to higher compressibility and 4. CONCLUSION susceptibility to particle consolidation. Ejection The multiparticulate units were successful y stress values did not exceed 3 MPa, which is compressed into compacts with good tensile associated with compact defect propensity (3), strength values (higher than 1 MPa, general y), while detachment stress was lower than 4 MPa. low detachment and ejection stress (lower than This indicates that the prepared samples do not 3 and 4 MPa, respectively). MPUs containing stick to punch and die and may be easily CAF and MPUs with MA-EA exhibited higher detached. tabletability and compressibility in comparison to samples prepared with IBU or EC, respectively. Polymer type and compression pressure affected al the investigated compact characteristics (tensile strength, detachment and ejection stress, out-of-die elastic recovery and net work of compression), while MPU size impact on the observed parameters was the lowest. MPUs containing IBU and MA-EA, with 1 mm size were recognized as preferable for obtaining compacts with favourable characteristics. Figure 1. The obtained MPU tensile strength values 5. REFERENCES Al of the investigated factors (model drug type, 1. Awad, A., et al. 3D printed pellets polymer type, MPU size and compression (miniprintlets): A novel, multi-drug, controlled pressure), as wel as model drug-polymer type release platform technology. Pharmaceutics, and model drug-compression pressure 2019. 11(4): 148. interaction significantly affected compact 2. Hancock, B.C., Identifying Candidates for Direct Compression Using Material-Sparing tensile strength (p<0.0001). In the case of Formulation Tools, presented at AAPS, 2004. MPUs containing CAF as model drug and EC 3. Pit , K.G., et al., Compression prediction as polymer, higher compression pressure accuracy from small scale compaction studies increased tensile strength more notably. to production presses. Powder Technology, In the case of detachment stress, model drug 2015. 270: 490-493. type, polymer type and compression pressure ACKNOWLEDGMENT were found as relevant factors (p=0.0013), while ejection stress was affected by polymer This work was financial y supported by the type, compression pressure and their interaction Ministry of Education, Science and (p=0.0097). Elastic recovery was impacted by Technological Development of the Republic of al the investigated parameters, as wel as model Serbia (project 451-03-68/2022-14/200161). 252 P55 FLAXSEED-OIL BASED LYOTROPIC LIQUID CRYSTALS: INFLUENCE OF MICROSTRUCTURE ON BETAMETHASONE DIPROPIONATE RELEASE PROFILE Mercedes Vitek1, Alenka Zvonar Pobirk1, Žiga Medoš2, Mirjam Gosenca Matjaž1 1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Slovenia 2Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia 1. INTRODUCTION Organica, Slovenia), and bidistil ed water. BD In recent years lyotropic liquid crystals (LLCs) was from ACA Pharma NV, Belgium. have at racted great interest as an outstanding Beloderm® (reference medicinal product) was drug delivery platform. They can form a wel - from Belupo d.o.o., Slovenia. defined and complex microstructure. LLCs with 2.2. Sample preparation lamel ar structure are especial y suitable for LCCs (Table 1) were prepared from dermal drug delivery due to similarity with homogeneous mixtures of lipid-surfactant intercel ular lipids of stratum corneum. Thus, phase and BD (0,64 mg/g) that were adequately they can provide improved bioavailability, diluted with bidistil ed water. target the site of action, and hence limit the Table 1. Composition [w/w %] of the studied secondary effects of the incorporated drug. In LLCs. addition, when composed of ingredients with skin supportive properties, they can restore skin LCC Flax- Lecithin + Lecithin + Water barrier function [1, 2, 3]. seed Tween 80 Montanov Considering al the advantages, which improve oil 68 1 28 42 / 30 patient adherence, lamel ar LLCs are especial y 2 20 30 / 50 suitable for the treatment of chronic skin diseases, which represent a particularly heavy 3 16 / 24 60 burden for the patients. In that regards, atopic 4 8 / 12 80 dermatitis (AD) is one of the most common 2.3. SAXS analysis chronic skin diseases that greatly impairs a Analysis was performed at 25, 32, and 37 °C by patient’s life [4]. Kratky camera system (Anton Paar, Austria), The aim of this study was to evaluate and at ached to a conventional X-ray generator compare flaxseed oil-based LLCs loaded with (Bruker AXS, Karlsruhe, Germany), equipped betamethasone dipropionate (BD). Flaxseed oil with a sealed X-ray tube (Cu-anode target type). is known for its anti-inflammatory activity and BD represents the mainstay drug for mild to 2.4. In vitro release study moderate AD treatment. The studied LLCs BD release through Strat-M® membranes was varied in type of surfactants and water content. evaluated using Franz diffusion cel s (n=6) with Microstructure of the systems was investigated a diffusion area of 0.785 cm² for 24 hours at 32 by smal -angle X-ray scat ering (SAXS) °C by HPLC (Agilent 1100 Series, USA). method and linked with the interpretation of the 3. RESULTS AND DISCUSSION BD release profile using Franz diffusion cells. 3.1. SAXS analysis 2. MATERIALS AND METHODS Structural evaluation by SAXS (Fig. 1 and Fig. 2.1. Materials 2) showed that temperature did not have a significant effect on the microstructure of the LLCs consisted of flaxseed oil (A.C.E.F. s.p.a., studied LCCs therefore being preserved Italy), mixture of Lipoid S-100® (Lipoid GmbH, fol owing skin application (i.e., 32 °C). Further, Germany) and Tween 80® (Merck KGaA, the microstructure was not altered by Germany) or MontanovTM 68 (Factory incorporation of BD. As regards LCC1 and LLC2, values of the scat ering vector q showed 253 P55 the ratio q1:q2=1:2, indicative for lamel ar 25 mesophases. For the LCC3 and LCC4, the ratio LCC1 was q LCC2 1:q2:q3=1:2:3 and q1:q2:q3:q4=1:2:3:4, 2] 20 LCC3 respectively. The ratio is also representative for ass of LCC4 lamel ar LCCs, however, the terminal part of 15 [μg/cm Beloderm D the scat ering curves indicated presence of ulative m 10 micel e structures as well [3, 5]. m um 5 C released B 0 0 4 6 8 10 12 24 Time [h] Figure 3. Release profile of BD from studied LCCs. 4. CONCLUSION In the present work, release profile of BD was evaluated for LCCs with different type of surfactants and water content in relationship to their microstructure determined by SAXS. Figure 1. SAXS curves of LCC1 and LCC2. Obtained data show great potential for further in vitro evaluation on keratinocyte cel line and prospective therapeutic efficacy of these systems. 5. REFERENCES 1. Silvestrini, AVP., et al., Advances in lyotropic liquid crystal systems for skin drug delivery. Expert Opinion on Drug Delivery, 2020. 17(12): 1781-1805. 2. Vitek, M., et al., A comparative study of lipid-based drug delivery systems with different microstructure for combined dermal administration of antioxidant vitamins. Journal of Dispersion Science and Technology, 2022. Figure 2. SAXS curves of LCC2 and LCC4. Ahead-Of-Print: 1-14. 3. Gosenca, M., et al., Lecithin based lamellar 3.2. In vitro release study liquid crystals as a physiologically acceptable BD was released from al studied LCCs in dermal delivery system for ascorbyl palmitate. greater extent compared to Beloderm® (with the European Journal of Pharmaceutical Sciences, same BD content) (Fig. 3.). 2013. 50(1): 114-22. 4. Weidinger, S., et al., Atopic dermatitis. Nature The drug release was significantly influenced Reviews Disease Primers. 4(1): 1. by microstructure of the individual systems. 5. Lombardo, D., et al., Evidence of premicellar The release of BD was the fastest and the aggregates in aqueous solution of amphiphilic highest after 24 hours in case of LCC4 with the PDMS–PEO block copolymer. Physical most numerous and pronounced presence of Chemistry Chemical Physics. 21(22): 11983-91. micel es in coexistence with lamel ar ACKNOWLEDGMENT mesophases as confirmed by SAXS analysis. This work was supported by the Slovenian Research Agency under Grant P1-0189. 254 P56 BY INCLUSION COMPLEXATION WITH SULFOBUTYL ETHER β- CYCLODEXTRIN Lamija Hindija1, Jasmina Hadžiabdić1, Merima Sirbubalo1, Amina Tucak-Smajić1, Ognjenka Rahić1, Stanko Srčič2, Edina Vranić1 1Department of Pharmaceutical Technology, University of Sarajevo – Faculty of Pharmacy, Bosnia and Herzegovina 2Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION 2.3. Attenuated Total Reflectance - Fourier Dimenhydrinate (DMH) is an antiemetic drug Transform Infrared Spectroscopy made of diphenhydramine and 8- (ATR/FTIR) chlorotheophylline. It’s a slightly soluble drug ATR/FTIR spectra of pure DMH, SBE-β-CD, with water solubility of 3 mg/mL their physical mixture and inclusion complexes [1].Cyclodextrins (CDs), amphiphilic prepared by kneading and solvent evaporation oligosaccharides, make inclusion complexes methods were recorded (Cary 360 FTIR (ATR) with poorly water-soluble drugs. Host-guest spectrophotometer, Agilent). The spectra were interaction occurs by expel ing water molecules col ectedfrom 32 scans, in 4000-650 cm-1 from inner hydrophobic CD cavity and creating scanning range,at 4 cm-1 resolution. non-covalent bonds with guest molecules. 3. RESULTS AND DISCUSSION Complexationprovides solubility and stability improvement, physical separation of 3.1.Phase solubility study incompatible drugs and taste masking. Limited Phase solubility diagram shows linear solubility of natural β-cyclodextrin can be increment of DMH solubility in solutions of exceeded by substitution of reactive hydroxyl SBE-β-CD, indicating an AL-type isotherm.The groups and using sulfobutyl ether β- slope was less than unity, suggesting a cyclodextrin (SBE-β-CD) to increase formation of a 1:1 complex (Fig. 1). hydrophilicity and inclusion ability [2, 3]. 200 2. MATERIALS AND METHODS 180 y = 0.75x - 2.5057 ol/L]m 160 R² = 0.9934 2.1. Materials 140 DMH was kindly supplied by Bosnalijek, d. d., H [m 120 B&H and Dexolve™(SBE-β-CD)was 100 generously gifted from CycloLab, Hungary. 80 60 2.2. Phase solubility study 40 Phase solubility study was conductedto 20 determine stability constant (K 0 s) and Concentration of DM complexation efficacy (CE), important for 0 50 100 150 200 250 assessing the binding characteristics of DMH Concentration of SBE-β-CD [mmol/L] and SBE-β-CD.An excess amount of DMH was mixed in aqueous solutions containing Figure 1. Phase solubility diagram of DMH and SBE-β- increasing amounts of SBE-β-CD (4.62 - CD in distil ed water 231.12 mmol/L) using a magnetic stirrer(Witeg, WiseStir MSH-20D) at 25oC for 24h. The The value of Ks was 148.88 M-1and calculated aliquot was filtered and DMH concentration from the equation: was determined at 277 nm (Shimadzu UV-VIS spectrophotometer-1601). Measurements were Complexes with Ksbetween 100 - 5000 M-1 are made in triplicates. suitable for practical applications. Values below 100 M-1 indicate very labile complexes with premature release of the guest and insignificant improvement in solubility. Values of Ks over 255 P56 5000 are specific for very stable complexes vibrations). Spectra of physical mixture (Fig. 2 where the release of the guest from the CD c) showed slight shiftment of some bands to cavity is incomplete or obstructed. different wavelengths implyingpartial To avoid inaccuracy in interpretation of the embedding of DMH into CD cavity. More significant changesin position, intensity and results due to dependence of Ks on smal shape of the band at 1645 cm-1were recorded in changes in S0(intrinsic drug solubility) and spectra of inclusion complexes (Fig. 2 d and e), possible self-association of lipophilic drug pointing to possibledisplacement of water molecules in aqueous media, CE was molecules with DMH molecules in the central determined from the equation: cavity of SBE-β-CD. Absence of DMH specific bands in spectra of physical mixture and complexessuggests possible interaction with The value of CE was 3 which implies formation SBE-β-CD just by blending and also confirms of a relatively stable complex. complex formation. 3.2.FTIR analysis 4. CONCLUSION Changesof characteristic bands inFTIR spectra of DMH and SBE-β-CD in 1:1complexes Inclusion complexation with SBE-β-CD may prepared by kneading and solvent evaporation overcome poor solubility of DMH. The results methodwere analyzedconcerning pure of phase solubility study indicate the formation substances and their physical mixture. of a 1:1 complex and FTIR analysis showed that both solvent evaporation and kneading methods provide successful complexation. 5. REFERENCES 1. Khan, Q., Shah, S. N. H., Arshad, M. S., Usman, F., Khalil, R., et al., Formulation and optimization of dimenhydrinate emulgels for topical delivery using response surface methodology. Pakistan Journal of Pharmaceutical Sciences, 2021. 34(1): 245-255 2. Aiassa, V., Garnero, C., Longhi, M., Zoppi, A. Cyclodextrin Multicomponent Complexes: Pharmaceutical Applications. Pharmaceutics, 2021. 13: 1099 3. Sadaquat, H., Akhtar, M. Comparative effects of β-cyclodextrin, HP-β-cyclodextrin and SBE7-β- cyclodextrin on the solubility and dissolution of docetaxel via inclusion complexation. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 2020. 96: 333-351 Figure 2. FTIR spectra of a) pure DMH; b) pure SBE-β- CD; c) physical mixture; d) inclusion complex prepared by solvent evaporation method; e) inclusion complexprepared by kneading method Specific bands of pure DMH (Fig. 2 a) are at 3059.6 cm-1 (stretching of amino groups), 1644.3 cm-1 (C=O stretching), 1112.9 cm-1 (C=C stretching of the aromatic rings) and 749.3 cm-1 (C-Cl stretching of carbonyl chloride).Specific bands of pure SBE-β-CD (Fig. 2 b) are at 3384.4 cm-1(O-H stretching vibrations), 2931.6 cm-1(asymmetric andsymmetric vibrations of -CH and -CH2), 1640.0 cm-1(d-HOH bending of water molecules at ached to CD), 1153.0 cm-1(C-H and C-O vibrations) and1025.0 cm-1(S=O 256 P57 THE INFLUENCE OF POLYMERIC PRECIPITATION INHIBITORS ON SOLUBILITY CHARACTERISTICS OF CARVEDILOL-LOADED SMEDDS Alenka Zvonar Pobirk1, Mila Kovačević1, Ilja German Ilić1, Mirjana Gašperlin1 1University of Ljubljana, Faculty of Pharmacy, Department of Pharmaceutical Technology, Slovenia 1. INTRODUCTION until homogenous mixture was obtained. In the last decade, researchers are extensively Subsequently, carvedilol was added (in content investigating the self-microemulsifying drug of 120 mg per 1 g of SMEDDS), as wel as PPI delivery systems (SMEDDS), a lipid-based (in concentration range 0-5 % w/w). formulation developed to improve the solubility Carvedilol precipitation from prepared of poorly water-soluble drugs, such as SMEDDS was further investigated for in vitro carvedilol [1]. In some cases, by formulating dissolution properties under non-sink SMEDDS it might not be possible to preserve conditions at pH 6,8. sufficient solubilization capacity upon their 3. RESULTS AND DISCUSSION dispersion, which increases the risk for drug precipitation in the gastro-intestine. Since the 3.1. Determination of supersaturation for in positive pharmacological effect is in correlation vitro carvedilol dissolution testing with the amount of drug absorbed, the addition Drug precipitation typical y occurs during of polymeric precipitation inhibitors (PPIs) supersaturation state formed upon dilution (and found to be valuable to prevent the drug digestion) of formulation in the gastro-intestine. precipitation [2]. Thus, the aim of this study was Thus, it was necessary to mimic this condition to investigate whether the presence of PPI in order to study the influence of PPI on shows effectiveness in inhibition of carvedilol carvedilol precipitation from liquid SMEDDS. precipitation from SMEDDS, and to identify the Different degrees of carvedilol supersaturation optimal type and concentration of PPI. were tested to identify the one, at which its precipitation is the most pronounced and 2. MATERIALS AND METHODS therefore the effect of PPI is the most visible. 2.1. Materials Testing conditions in which carvedilol Carvedilol was used as a model drug. Liquid concentration exceeds saturated solubility in SMEDDS was composed of mixture of dissolution medium for about 3-times, was Capmul® MCM EP/NF (mono-diglyceride of found as optimal to study the influence of PPI medium chain fat y acids), refined castor oil, on precipitation of carvedilol from drug loaded Kol isolv® PEG E 400 and Kol iphor® RH 40 SMEDDS. (polyoxyl 40 hydrogenated castor oil). 3.2. The influence of PPI type and Different polymers (added to liquid SMEDDS) concentration on carvedilol precipitation were used as precipitation inhibitors: povidone from liquid SMEDDS K30 (PVP K30), hypromel ose (Pharmacoat® Al three PPIs tested delayed the precipitation 606) and Soluplus®. of carvedilol after dispersion of SMEDDS in aqueous media. Soluplus® turned out to be the 2.2. Preparation and characterization of most effective, as the supersaturation was liquid SMEDDS containing PPIs maintained for the entire testing period (8 h) Liquid SMEDDS was prepared by heating the when added in 5 % w/w concentration. When components (40 % w/w Kol iphor® RH, 10 % SMEDDS was admixed with the same amount w/w Capmul® MCM EP/NF, 10 % w/w castor of Pharmacoat® 606, carvedilol concentration oil and 40 % w/w PEG 400) at 40°C and stirring remained above 95 % even after 7 h. The worse 257 P57 carvedilol dissolution performance was observed for SMEDDS with addition of 3 % 100 w/w PVP K30, that postponed drug ) precipitation only for 60 min. Additional y, the 80 drug concentration dropped below 50 % after 4 h (Fig. 1.). This was to some extend in contrary 60 to our expectations, as PVP K30 showed good 40 5% drug solubilization ability in our previous 4% research, focusing of solidification of carvedilol Carvedilol released (% 20 3% loaded SMEDDS with swirling fluidized bed 1% liquid SMEDDS pel et coating (3). 0 0 100 200 300 400 time (min) Figure 2. In vitro carvedilol dissolution profiles of 100 ) liquid SMEDDS containing different concentrations of Soluplus®. 80 4. CONCLUSION 60 By incorporating PPI into SMEDDS 40 Soluplus formulation, it was possible to produce high- Carvedilol released (% Pharmacoat 606 20 drug load SMEDDS, with low risk for PVP K30 carvedilol precipitation upon dispersion in liquid SMEDDS 0 aqueous media. With the addition of Soluplus® 0 100 200 300 400 and Pharmacoat® 606 (5 % w/w), carvedilol time (min) concentration was maintained in supersaturated Figure 1. In vitro carvedilol dissolution profile of state for 8 h, in that way preventing the untimely liquid SMEDDS containing different PPIs. precipitation, that would most probably lead to incomplete carvedilol absorption and In addition to the type of PPI, its concentration inadequate therapeutic effect. in the SMEDDS must also be considered in order to develop drug loaded SMEDDS with 5. REFERENCES optimal solubility characteristics. Indeed, 1. Čerpnjak K. et al. Lipid-based systems as a concentration of PPI was found to considerably promising approach for enhancing the correlate with time during which carvedilol was bioavailability of poorly water-soluble drugs. in supersaturated state. Higher Soluplus® Acta Pharmaceutica. 2013 63(4): 427–45. concentrations demonstrated longer 2. Warren D.B. et al. Using polymeric supersaturation periods, as shown in Fig. 2. precipitation inhibitors to improve the absorption of poorly water-soluble drugs: A mechanistic basis for utility. Journal of Drug Targeting. 2010 18(10): 704–31. 3. Mandić J. et al. Solidification of carvedilol loaded SMEDDS by swirling fluidized bed pel et coating. International Journal ofPpharmaceutics, 2019, 566: 89-100. 258 P58 SIMULTANEOUS DETERMINATION OF FIVE NITROSAMINES IN SARTAN DRUG PRODUCTS BY A LC-MS/MS METHOD Timeja Planinšek Parfant1, Anja Hrovat1, Robert Roškar1 1 Department of Biopharmaceutics and Pharmacokinetics, University of Ljubljana, Faculty of Pharmacy, Slovenia 1. INTRODUCTION was used. Al sample solutions and mobile Since 2018, interest in N-nitrosamines (NAs) phase were prepared using the Mil i-Q water increased dramatical y among pharmaceutical obtained through a Mil i-Q A10 Advantage regulatory authorities, industry and researchers water purification system. when they were first detected in angiotensin II 2.2. LC-MS/MS method receptor antagonists’ medicines which caused A Kinetex F5 100 × 2.1 mm, 2.6 µm particle global recalls [1]. NAs are a group of N-nitroso size column (Phenomenex, USA) with gradient compounds and are potent carcinogens in elution at 0.3 mL/min using 0.05% formic acid several animal species and some are classified and methanol were used for the optimal as probable or possible human carcinogens separation on a UHPLC 1290 Infinity coupled based on International Agency for Research on to 6460 Triple Quadrupole mass spectrometer. Cancer (IARC) classification system [2]. To determine the effect of ionization and Therefore, European Medicines Agency (EMA) sample injection volume on the matrix effect, and Food and Drug Administration (FDA) have two methods utilizing both ESI and APCI ion established acceptable daily intake limits source were developed which operated in MRM (8-1300 ng/day) for 18 and 9 NAs, respectively mode (Agilent Technologies, USA), and [1,3]. Due to the lack of analytical methods for different sample injection volumes were used. the simultaneous determination of NAs in pharmaceuticals, our main objective was to 2.3. Sample preparation develop a sufficiently sensitive and selective For the evaluation of optimal NA extraction analytical method for the simultaneous from DPs different solvents were added to quantification of five NAs in sartan drug defined amounts of ground tablets. To reduce products (DPs). the matrix effect, selected solvent was added to various amounts of ground tablets. Samples 2. MATERIALS AND METHODS were then vortexed for 5 minutes, sonicated for 2.1. Reagents and chemicals 30 minutes and centrifuged for 10 minutes at Reference standards of N-Nitrosodiethylamine 16100 g at 25 °C. Prior to LC-MS/MS analysis, (NDEA) and N-Nitrosodimethylamine the samples were filtered through 0.22 µm RC (NDMA) were obtained from Sigma-Aldrich, syringe filters. The developed analytical N-nitroso-N-methyl-aminobutyric acid method was validated according to ICH (NMBA) and N-Nitrosodi sopropylamine guidelines and was successful y applied to real (NDIPA) were obtained from Carbosynth and samples [4]. N-Nitrosoethylisopropylamine (NEIPA) was 3. RESULTS AND DISCUSSION obtained from LGC. DPs were purchased from local pharmacy. Methanol, acetonitrile and The lowest matrix effect and good isopropanol were purchased from Honeywel , chromatographic peak shape was obtained ethanol absolute and formic acid were using methanol in Mil iQ (1:1, v/v) among other purchased from Merck. For LC-MS/MS used solvents for extraction (acetonitrile, analysis, LC-MS-grade methanol (Honeywel ) isopropanol or ethanol in Mil iQ (1:1, v/v), 259 P58 methanol or mixture of acetonitrile and content can be detected (> LOD) or quantified (> methanol 1:1, v/v). Among solvents with LOQ), red (-) - the NAs content cannot be different methanol contents in Mil iQ, the best quantified. results were obtained using 20% methanol in Mil iQ. The addition of formic acid to solvent for extraction did not affect matrix effect or recovery. The sample amount and sample injection volume had a significant influence on the matrix effect when using the ESI ion source compared to the APCI (Fig. 1). 100 4. CONCLUSION ] 80 A highly sensitive and selective analytical 60 method for the determination of five regulated 40 NAs (NDMA, NMBA, NDEA, NDIPA and atrix effect [% 20 NEIPA) in sartan DPs was developed. It was M 0 found out that ion source has a significant effect NDMA NMBA NDEA NEIPA NDIPA on matrix effect and sensitivity of al analytes. ESI APCI The method was ful y validated and was successful y applied to real samples, where al Figure 1. The influence of ion source on matrix met the regulatory requirements for nitrosamine effect. content. The results demonstrated that the Also, the sensitivity for al analytes (except for proposed analytical method is suitable for NMBA) increased when the APCI ion source routine analysis of selected NA in satrans with was used. The most significant improvement in the LOQ within regulatory levels. sensitivity (20-fold) was observed for NDMA. 5. REFERENCES The method was validated according to ICH guidelines and was proved to be linear in the 1. EMA, Questions and answers for marketing concentration range 0.25-200 µg/L. Accuracy authorisation holders/applicants on the CHMP Opinion for the Article 5(3) of Regulation (EC) and precision were within acceptable limits No 726/2004 referral on nitrosamine impurities (< 5%) and suitable selectivity was achieved. in human medicinal products, The method was successful y applied to real EMA/409815/2020, 2022. samples and tested for the presence of NAs 2. List of Classifications – IARC Monographs on the Identification of Carcinogenic Hazards to impurities in six selected sartan-containing Humans.https://monographs.iarc.fr/list-of-class DPs. The NA content in al analyzed DPs was ifications (accessed 09 Jun 2022). below the detection limit. The developed 3. FDA, Guidance for Industry. Control of Nitrosamine Impurities in Human Drugs, FDA- analytical method is suitable for the detection of 2020-D-1530, 2021. al five NAs in selected DPs at the regulatory 4. ICH Q2 (R1), 2005. levels. The limit of quantification is achieved in al selected DPs, except for NDEA and NEIPA in some samples (Table 1). The required LOQs ACKNOWLEDGMENT for NDEA and NEIPA in this samples could be This research was funded by Slovenian reached by further method optimization (e.g. Research Agency (ARRS), grant number increasing the amount of ground tablets). [P1-0189]. Table 1. Limit of detection (LOD) and limit of quantification (LOQ) according to regulatory levels for the NAs in DP. Green (+) - the NA 260 P60 PREDICTION OF ENVIRONMENTAL MICROBIAL DEGRADATION OF AZITHROMYCIN IN SOIL AND WATER AND METABOLISM IN HUMANS Tin Takač1, Milena Jadrijević-Mladar Takač2, Tomislav Jednačak3 1University of Zagreb, Faculty of Science, Doctoral Study in Chemistry, Zagreb, Croatia; 2University of Zagreb, Zagreb, Croatia; 3University of Zagreb, Faculty of Science, Zagreb, Croatia 1. INTRODUCTION it is of great importance to study their potential Several contaminants of emerging concern degradation pathways. (CECs), including macrolide antibiotics, enter aquatic compartments, such as surface water, 2. MATERIALS AND METHODS groundwater and even drinking water at 2.1. Materials concentrations ranging from a few ng L-1 to The macrolide antibiotic azithromycin was used several μg L-1 1]. As conventional wastewater in this study. treatment plants are not designed to remove complex organic compounds, such as pharmaceuticals, their metabolites, and 2.2. Methods potential transformation products, CECs enter Using BioTransformer 3, a web server software the environment and can have adverse effects available at www.biotransformer.ca 3] the on living organisms and plants. Among the aerobic and anaerobic microbial degradation of different classes of pharmaceuticals found in azithromycin in the environment in soil and the environment, special at ention has been paid water was performed and the predicted to macrolide antibiotics, such as azithromycin, biotransformation metabolites were compared clarithromycin, and erythromycin, which are with the predicted metabolites of azithromycin in the most discussed because of their potential to humans. contribute to the development of antimicrobial resistance (AMR) 2]. To date most studies have 3. RESULTS AND DISCUSSION focused on the parent compounds, and lit le at ention has been paid to the 3.1. Predicted aerobic and anaerobic intermediates/metabolites they produce. Drugs microbial degradation of azithromycin in the excreted by humans and/or animals may be environment. present in wastewater treatment plant effluents Several aerobic and anaerobic at higher concentrations than in their respective intermediates/metabolites were predicted for influents, because they are excreted as the azithromycin molecule (Table 1, Figure 1) conjugates, which are broken down in most of which are formed by oxidation of the wastewater treatment plants and general y hydroxyl group at positions C2’ (1), C11 (2), metabolized during biological treatment, C4’’ (3), or after hydrolysis of the glycoside increasing the concentration of the parent bonds and removal of the sugar moieties, by compound at the output of the treatment plants. oxidation of the formed hydroxyl groups to Since there is a lack of data on the chronic ketone moieties, C3 (8), C5 (7), C1’ (4) and C1” toxicity of such compounds, and (11) or by reduction of formed ketones to the ecotoxicological data for mixtures of drugs, secondary hydroxyl group at positions C3 (10), their metabolites, and transformation products, C5 (5), C1’ (6) and C1” (9). In addition to the oxidation/reduction reactions, hydrolysis of the 261 P60 lactone bond was also observed, 13: C1(=O)– (5) and the charged glucuronide R–N+(CH3)2– OH and C13–OH, as wel as the formation of Gluc (6) were predicted (Figure 2). C3” (12) metabolites after O-dealkylation. 9a 9 2: C6-OGluc 3: C11-OGluc N 8 HO 10 7 OH 5' 9a 9 4: C12-OGlucHO 11 6 O 4' 2: C11=O N 8 12 HO 5 13 1' 3' 10 7 OH HO O 4 O 2' N 11 5' 6 O 4' 14 12 1 3 OH O 2 O 6: N+'-C2Gluc 5 1: C2'-OGluc 13 1' 3' O 4 O 2' N 1" 14 O 2" 5: C5-OH 1 3 OH 7: C5=O O 2 1: C2'=O 13: C1(=O)-OH; O 4: C1'=O 5" 3" C13-OH 6: C1'-OH 4" O 9: C1''-OH 1" 8: C3=O 11: C1' =O O 2" OH 5: C4' -OGluc 10: C3-OH Figure 2. Predicted conjugation metabolites of 5" 3" 4" O azithromycin with glucuronic acid (www.biotransformer.ca) >3] 3: C4' =O OH 12:C3'-OH 4. CONCLUSION Figure 1. Predicted environmental aerobic and anaerobic microbial degradation of azithromycin The predicted biotransformation of in soil and water (www.biotransformer.ca) >3] azithromycin either by microbial degradation in the environment or by metabolism in humans by CYP enzymes or glucuronic acid conjugation Table 1. List of predicted microbial aerobic and generates many metabolites that may increase anaerobic metabolites of azithromycin in the the environmental toxicity of this antibiotic and environment. its contribution to antimicrobial resistance (AMR). No MF MW AlogP 1 C 5. REFERENCES 38H7ON2O12 746.49 2.33 2 C38H70N2O12 746.49 2.33 3 C 1. Barbosa, M. O., et al., Occurrence and removal 38H70N2O12 746.49 2.33 4 C of organic micropollutants: An overview of the 8H15NO3 173.11 0.02 5 C watch list of EU Decision 2015/495. 30H57NO10 591.40 1.69 Water 6 C8H17NO3 175.12 -0.37 Research, 2016, 94: 257–279. 7 C30H55NO10 589.38 1.92 2. Kümmerer, K., Antibiotics in the aquatic 8 C30H56N2O9 588.40 1.79 environment — A review — Part I. 9 C8H16O4 176.11 0.25 Chemosphere 2009, 75(4): 417–434. 10 C30H58N2O9 590.41 1.56 3. Djoumbou-Feunang, Y., et al., BioTransformer: 11 C8H14O4 174.09 0.08 A comprehensive computational tool for small 12 C37H70N2O12 734.49 1.67 molecule metabolism prediction and metabolite 13 [C38H73N2O13]- 765.51 0.79 identification. Journal Cheminformatics 2019, MF –molecular formula of formed metabolite; 11(2): 1–25. MW - molecular weight; AlogP - lipophilicity 3.2. Predicted metabolism of azithromycin in humans The predicted metabolites formed by CYP enzymes in humans are similar to the predicted oxidative/reductive degradation products in the environment, but also include N-dealkylated metabolites. In human metabolism, the degradation of the lactone ring was not predicted. Conjugation with glucuronic acid at positions C2’ (1), C6 (2) C11 (3), C12 (4), C4” 262 P61 A FAST HPLC-DAD METHOD FOR THE SIMULTANEOUS DETERMINATION OF ALL MAIN WATER-SOLUBLE VITAMINS IN FOODS AND SUPPLEMENTS Žane Temova Rakuša, Robert Roškar Department of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, University of Ljubljana, Slovenia 1. INTRODUCTION 2.1. Chemicals and reagents Water-soluble vitamins (WSV) are essential WSV standards: L-ascorbic acid (C, 99.4%), nutrients and common ingredients in fortified thiamin hydrochloride (B1, 99.9%), riboflavin foods (FF), food supplements (FS), and (B2, 100.0%), nicotinamide (B3, 99.9%), medicines. One of the main issues in the calcium-D-pantothenate (B5, 99.0%), formulation, production, and quality assurance pyridoxine hydrochloride (B6, 100.0%), and of WSV products is their general instability, cyanocobalamin (B12, 99.8%) were purchased leading to degradation under various production from Sigma-Aldrich (Germany). D-biotin (B7, and storage conditions, which manufacturers 100.6%), MTHFA (98.1%), and folic acid (B9, typical y conceal with overages [1]. Aware of 97.8%) were purchased from Carbosynth (UK). this, the European Commission published HPLC grade methanol was purchased from guidance on the acceptable overages and Sigma-Aldrich (Germany) and sodium tolerances for vitamin contents in relation to the dihydrogen phosphate monohydrate from labeled values: 80-150% for FS and 65-150% Merck (Germany). Ultra-pure water was for FF [2]. Despite the wide acceptance limits, obtained through a Mil i-Q purification system a growing number of studies reveal WSV A10 Advantage (Mil ipore Corporation, USA). contents outside these intervals and even above 2.2. HPLC-DAD analysis their tolerable upper intake levels in FF and FS A chromatographic system Infinity 1290 [1,3]. Thus, quality control of these loosely (Agilent Technologies, USA), equipped with a regulated products (FF and FS) using proper diode array detector (DAD) and EZChrom analytical methodology is crucial in ensuring acquisition system was used for the analysis. An their quality, safety, and efficacy. Though, their XSelect CSH C18 150×4.6 mm, 3.5 µm column analytics is complicated due to different WSV (Waters Corporation, USA) at 40°C and a amounts in FF and FS (e.g., up to 20,000 fold mobile phase consisting of solvent A: 25 mM differences between C and B12), lack of NaH2PO4×H2O, pH = 4.0, and solvent B: chromophores (B5 and B7), limited stability (C methanol were used. Initial isocratic elution and B9), and water solubility (B2, B9) [1]. (99% A and 1% B), fol owed by gradient Despite the recent progress in WSV analytics, elution (up to 35% B) at a flow rate of 1 mL/ enabling the simultaneous analysis of the main min was utilized for the separation of al WSV in a single run [1], with the development evaluated WSV in a total run time of 15 min. of the food and pharmaceutics industry, the new The injection volume was 5 μL during finding and use of new vitamin forms (e.g., 5- validation and was adjusted according to WSV methyl-tetrahydrofolic acid (MTHFA), there is contents in FF and FS (from 0.2 to 20 μL). scope for improvement of the current WSV Detection was carried out at different analytics. Thus, we aimed to develop a fast and characteristic wavelengths for the individual simple method for the simultaneous analysis of WSV (210, 245, 260, 287, 362, and 445 nm). WSV in their most common forms, including the active folate form MTHFA and the WSV, 2.3. Method validation typical y determined individual y because of The method was validated fol owing the ICH their low concentrations (B7 and B12). guidelines Q2(R1) [4] in terms of specificity, linearity, accuracy, precision, limit of detection 2. MATERIALS AND METHODS (LOD), limit of quantification (LOQ), sample stability, and recovery. The concentration ranges were adjusted to the WSV recommended 263 P61 daily amounts (RDA). Thus, 100% RDA selected concentration ranges, precision, represents the RDA dissolved in 200 mL, except accuracy, recovery, and sample stability. for B7 and B12, where due to low RDAs higher concentrations were used. Seven calibration 3.3. Method application standards containing 5-400% RDA of the WSV, The obtained results for the WSV assay (Table and three quality control samples (low, 1) confirm the applicability of the method and medium, and high) were prepared daily, on each emphasize the importance of the quality control of the three validation days. The samples were of FF and FS, as al products, except FF5 prepared in 0.1 mM EDTA (0.1 mM EDTA contained an inappropriate amount of at least containing 1 mM NaOH for B2, B7, B9, and one WSV considering the European MTHFA). Commission acceptable tolerances. 2.3. Method application Table 1. WSV assay in the tested FF and FS. The method was applied to assay WSV content Product C B1 B2 B3 B5 B6 B7 B9 MTHFA B12 FF 1 in 10 commercial FF (vitamin waters) and 10 FS FF 2 (capsules (FS1) and tablets (FS2-9)). The tested FF 3 FF 4 FF were directly injected into the UHPLC FF 5 FF 6 system. FS samples were prepared by a FF 7 previously developed extraction procedure [1]. FF 8 FF 9 FF 10 3. RESULTS AND DISCUSSION FS 1 FS 2 3.1. Method development FS 3 FS 4 Our main aim was to develop the first so far FS 5 FS 6 reported HPLC-DAD method for the FS 7 simultaneous analysis of al main WSV, FS 8 FS 9 including the newer and active folate form FS 10 MTHFA, as well as B7 and B12, which usually Green – contents within, red – above, and orange, below the require individual analysis due to their low acceptable tolerances, blue – contents below the LOD. amounts in FF and FS. As WSV are typical y 4. CONCLUSION found in combinations or al together in FF and FS, by the development of this method, we The developed method represents a step provided a tool for their fast and simple analysis forward in WSV analysis, enabling the analysis using common systems (HPLC-DAD). The of al WSV with single sample preparation and developed method resulted in symmetric and chromatographic run in a favourable analysis separated peaks (Fig.1) and significantly shorter time of 15 min. run time compared to other similar published 5. REFERENCES methods for the analysis of less WSV. 1. Temova Rakuša Ž., et. al., Comprehensive Approach for the Simultaneous Analysis of All Main Water-Soluble Vitamins in Multivitamin Preparations by a Stability-Indicating HPLC-DAD Method. Food Chemistry, 2021. 337: 127768. 2. European Commission Set ing of Tolerances for Nutrient Values Declared on a Label Guidance For Food Supplements, 2014. 3. Andrews K.W., et al. Analytical Ingredient Content and Variability of Adult Figure 1. Chromatogram of a standard mixture Multivitamin/Mineral Products: National Estimates of WSV at 210 nm, representing their for the Dietary Supplement Ingredient Database. chromatographic separation. The American Journal of Clinical Nutrition, 2017. 105:526–539. 3.2. Method validation 4. ICH Harmonised Tripartite Guideline Validation The method was successful y validated of Analytical Procedures: Text and Methodology according to ICH guidelines Q2(R1) [4] Q2(R1). 2005. proving its selectivity, linearity over the 264 P62 NATURAL APOCAROTENOIDS AND THEIR SEMISYNTHETIC GLYCOPEPTIDE CONJUGATES AGAINST SARS-COV-2 Ilona Bereczki,1,2 Henrietta Papp,2,3 Veronika Nagy,4 Attila Agócs,4 Ferenc Jakab,2,3 Pál Herczegh,1 Anikó Borbás1,2 1Department of Pharmaceutical Chemistry, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary 2National Laboratory of Virology, Szentágothai Research Centre, Ifjúság útja 20, H-7624 Pécs, Hungary 3Institute of Biology, Faculty of Sciences, University of Pécs, Ifjúság útja 6, H-7624 Pécs, Hungary 4Department of Biochemistry and Medical Chemistry, Medical School, University of Pécs, Szigeti u. 12, H-7624 Pécs, Hungary 1. INTRODUCTION The development of effective antiviral drugs against SARS-CoV-2 is one of the most important tasks today. The clinical y used glycopeptide antibiotic, teicoplanin, emerged as a potential antiviral [1], and its efficacy can be improved with lipophilic modifications [2], but unfortunately their cytotoxicity is also elevated. Apocarotenoids are produced natural y in plants by oxidative cleavage of carotenoids. They are not toxic hydrophobic substances with beneficial biological effects. We planned to synthesize new types of lipophilic glycopeptide antibiotic derivatives equipped with different apocarotenoid side chains. Al conjugates effectively inhibited SARS-CoV-2 replication and interestingly, bixin also exerted remarkable a Figure 2. Apocarotenoid-glycopeptide nti-SARS-CoV-2 activity on its own. 2. MATERIALS AND METHODS conjugates. 2.1.Synthesis of glycopeptide derivatives From apocarotenoids (1a, 1b and 1c) active esters were prepared (2a, 2b and 2c) and the N- For the synthesis of glycopeptide conjugates, terminal primary amino groups of glycopeptide we used bixin (1a), crocetin monomethyl ester derivatives were acylated by these active esters (1b) and β-apo-8’-carotenoic acid (1c) as to produce apocarotenoid derivatives. 2.2.Antiviral evaluations Antiviral activity of the semisynthetic derivatives as wel as the starting glycopeptides and apocarotenoids was evaluated in Vero E6 cel s using three orthogonal assays. The one order of magnitude difference between the EC 50 values can be explained by the four times lower Figure 1. Preparaition of the active esters from multiplicity of infection and the removal of the apocarotenoids. viral inoculum after a half hour incubation in the RNA reduction assay. apocarotenoids (Figure 1.) and teicoplanin, To elucidate the mechanism of antiviral action teicoplanin pseudoaglycone and ristocetin of the active derivatives, human cathepsin L and aglycone as glycopeptides. B (required for the viral infection of SARS- 265 P62 CoV-2) and viral 3CLPro inhibitory assays antibiotic teicoplanin and ristocetin. Several were performed. derivatives have been synthesized, which showed remarkable antiviral activity and were Table 1. Antiviral effects of the glycopeptide completely devoid of cytotoxicity. However, conjugates and apocarotenoids. the anti-SARS-CoV-2 activity of the natural food colorant bixin seems to be the most Compound SARS- SARS- SARS- CC50 CoV-2 CoV-2 CoV-2 interesting and promising result. (µM) RNA CPE, IFA, 5. REFERENCES reduction EC50 EC50 (µM) (µM) EC50 (µM) 1. Zhou, N., et al., Glycopeptide antibiotics potently inhibit cathepsin L in the late 3a 5.9 91±88 74±20 >100 endosome/lysosome and block the entry of Ebola virus, Middle East respiratory syndrome 3b 5.2 19 ±1.8 8.8±1.4 >100 coronavirus (MERS-CoV), and severe acute 3c 4.4 56±10 24±5.3 >100 respiratory syndrome coronavirus (SARS-CoV). Journal of Biologycal chemistry, 2016, 291(17), 4 1.8 56 ±6.2 51±9.8 >100 9218–9232. 2. Szűcs, Z., et al., Structure-activity relationship 5 6.7 n.d. n.d. n.d. studies of lipophilic teicoplanin pseudoaglycon Teicoplanin not active at n.d. n.d n.d. derivatives as new anti-influenza virus agents. ψaglycone 50 µM European Journal of Medicinal Chemistry, 2018, 157, 1017–1030. Teicoplanin 5.6 16 ±1.7 18±5.4 >100 ACKNOWLEDGMENT Ristocetin not active at n.d. n.d. n.d. aglycone 50 µM This work was supported by the European 1a 5.9 14±3.5 28±8.8 >100 Regional Development Fund under the projects GINOP-2.3.2-15-2016-00044, and GINOP- 1b not n.d. n.d. n.d. 2.3.4-15-2020-00008. This research was also active at 50 funded by the National Research, Development µM and Innovation Office of Hungary (K 131493). Viral RNA reduction assay was supported by 1c 15 14±2.2 31±7.9 >100 the European Union, and co-financed by the European Social Fund: Comprehensive Development for Implementing Smart 3. RESULTS AND DISCUSSION Specialization Strategies at the University of The semisynthetic glycopeptides and native Pécs (EFOP-3.6.1.-16-2016-00004), and by the teicoplanin inhibited the viral reproduction with Ministry for Innovation and Technology of similarly high efficacy, the EC Hungary (TUDFO/47138/2019-ITM). Project 50 values were in the low micromolar range in the RNA reduction no. TKP2021-NVA-07 and TKP2021-EGA-13 assay and in the ten-micromolar range in the have been implemented with the support CPE-based and immunofluorescent assays. provided from the National Research, Although, the most interesting finding of this Development and Innovation Fund of Hungary, study is the remarkable, unexpected antiviral financed under the TKP2021-NVA and activity of two apocarotenoids, bixin (1a), and TKP2021-EGA funding schemes. β-apo-8’-carotenoic acid (1c). The activity of bixin is based on cathepsin L inhibition while conjugates of teicoplanin pseudoaglycone with apocarotenoids have a combined mechanism of action based on cathepsin and 3CLPro inhibition. 4. CONCLUSION The aim of this work was to study the effect of lipophilic carotenoid side chains on the anti- SARS-CoV-2 activity of the glycopeptide 266 P63 DESIGN, SYNTHESIS AND EVALUATION OF NOVEL BChE/p38α MAPK DUAL INHIBITORS FOR THE TREATMENT OF ALZHEIMER'S DISEASE Svit Ferjančič Benetik1; Boris Markoja1; Matic Proj1; Damijan Knez1; Stanislav Gobec1; Aleš Obreza1; Urban Košak1 1University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 rLjubljana, Slovenia Correspondence: ales.obreza@ffa.uni-lj.si 1. INTRODUCTION the driving force in AD progression. Aβ plaques Alzheimer's disease (AD) is a progressive stimulate microglial p38α MAP kinase which then neurodegenerative disorder and represents a major up-regulates proinflammatory cytokines such as cause of dementia leading to disability and death. It TNF-α and IL-1β. The latter then activates p38α is estimated that around 50 mil ion people MAPK in astrocytes which leads to a chronic worldwide suffer from AD or other dementias with inflammatory state and excitotoxicity. Additional y, the number expected to reach 75 mil ion by 2030 [1]. p38α MAPK can phosphorylate the neurotoxic tau protein [5]. We intend to combine the cholinergic There are only four marketed smal -molecule drugs and inflammation hypothesis by developing dual for the treatment of Alzheimer's disease – donepezil, BChE/p38α MAPK inhibitors. galantamine, rivastigmine and memantine which only help to temporarily al eviate symptoms while 2. RESULTS AND DISCUSSION the disease progresses. The recently approved monoclonal antibody aducanumab was designed to First, a library of smal molecules with confirmed address the cause of AD by clearing off amyloid β activity against p38α MAPK was prepared using (Aβ) aggregates but clinical trials showed no ChEMBL and PDB (in ChEMBL activity treshold of convincing evidence of reduced cognitive decline in Kd/IC50 < 50 μM was accounted for). Of the 5490 patients. Design of novel effective disease- compounds 172 were commercial y available. These modifying drugs in this field therefore presents a were further divided into 30 clusters according to major chal enge in medicinal chemistry. molecular fingerprint and docked into the BChE active site gorge. According to docking results 8 best A multitude of hypotheses must be taken into smal molecules were purchased and evaluated in vitro against BChE by the method of El man. account when explaining AD pathology - the most popular being the cholinergic, Aβ toxicity, tau and Of the eight compounds, very promising activity inflammation hypothesis [2]. The cholinergic against BChE was exhibited by ARRY-371797 hypothesis states that forebrain cholinergic neuron (Figure 1) a p38α MAPK inhibitor of Pfizer Inc. loss is characteristic of AD, so by inhibiting the This molecule was then subjected to further hydrolytic action of cholinesterases optimization. [acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)], we may augment the activity of surviving cholinergic neurons [3]. This is the principle idea behind donepezil, galantamine and rivastigmine as wel as the in vivo active piperidine-based selective BChE inhibitor developed previously by our research group [4]. Since cholinesterase inhibitors alone do not directly alter the pathophysiological processes in AD we aim to further exploit the possible drug targets in AD to Figure 9. Structure of p38α MAPK inhibitor ARRY- halt disease progression by various mechanisms. 371797 According to one theory inflammation response is 267 P63 3. CONCLUSION We started the synthesis of analogues by substituting the indazole core with various azines, bicyclic nitrogen heterocycles and 4- phenyl azines. The analogues with switched positions of the amide and ether were also synthesized. Al synthesized compounds were initial y evaluated for their in vitro activity against p38α MAPK and BChE with ADP-Glo kinase assay and El man's method respectively. 4. REFERENCES 1. Masters, CL., et al., Alzheimer’s disease. Nature Reviews, Disease Primers, 2015. 1: 15056. 2. Kheiri, G., et al., Role of p38/MAPKs in Alzheimer’s disease implications for amyloid beta toxicity targeted therapy. Reviews in the Neurosciences, 2019. 30(1): 9–30. 3. Darvesh, S., et al., The biology of butyrylcholinesterase. Nature reviews, Neuroscience, 2003. 4: 131–8. 4. Košak, U., et al., Development of an in-vivo active reversible butyrylcholinesterase inhibitor. Scientific Reports, 2016. 6(1): 39495. 5. Munoz, L., et al., Targeting p38 MAPK pathway for the treatment of Alzheimer’s disease. Neuropharmacology, 2010. 58(3): 561–568. ACKNOWLEDGMENT The authors acknowledge the financial support from the Slovenian Research Agency. 268 P64 COUNTERING DIVERSION OF PHARMACEUTICAL-BASED COMPOUNDS ALONG THE CHEMICAL SUPPLY CHAIN: A WORKSHOP Yavana Ganesh1, Jonathan E. Forman1, Kabrena E. Rodda1, John R. Cort2, Ellen M. Wynkoop1 1National Security Directorate, Pacific Northwest National Laboratory, United States of America 2Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, United States of America 1. INTRODUCTION countries that play a role in the global trade of Pharmaceuticals are chemical substances pharmaceutical chemicals. designed to act on physiological processes. 2.2. Method Despite legitimate uses, there is potential to PNNL instructors used the Zoom and misuse pharmaceuticals for harmful purposes. Mentimeter platforms to facilitate interactive State and non-State actors can exploit legitimate presentations and discussions with webinar chemical supply chains to acquire participants. pharmaceutical compounds, chemicals, and precursors. For example, in October 2002, Russian security forces used a pharmaceutical- 3. RESULTS AND DISCUSSION based agent (PBA), suspected to be a fentanyl 3.1. Day one derivative, to end a hostage situation in the Dubrovka Theater Incident in Moscow, The first day began with an interactive resulting in the death of 170 people and questionnaire to identify participants most demonstrating the potential of weaponizing pressing concerns with pharmaceutical-based these substances. In 2021, to address the threat agents and supply chain diversion. This was of pharmaceutical-based agents, Pacific fol owed by presentations that discussed Northwest National Laboratory developed a pharmaceutical-based agents with a focus on webinar series to raise awareness for fentanyl as an archetypal PBA, legitimate uses international partners on Countering Diversion of pharmaceuticals in health, medicine, and of Pharmaceutical Based Compounds Along the other sectors. The day concluded with Chemical Supply Chain. presentations discussing the growing threat of PBAs as il icit drugs, fentanyl as an incapacitating agent, and PBAs as chemical 2. MATERIALS AND METHODS weapons. 2.1. Materials 3.2. Day two Subject mat er experts at Pacific Northwest The second day began with an activity and National Laboratory developed a set of training discussion on identifying threats from PBAs in modules covering the growing threat of the participant country. This was fol owed by pharmaceutical-based compounds, a Novichok presentations discussing fentanyl and central- agent case study, chemical weapons and Syria, nervous system-acting agents within the context and emerging threats in the pharmaceutical of the Chemical Weapons Convention, and a sector. These modules were delivered through case study of Novichok poisoning. The day the webinar series to government officials, concluded with a presentation on improving chemical producers, distributors, academic supply chain security (Figure 1), where researchers, and other stakeholders in participants learned to recognize supply chain pharmaceutical and chemical supply chains in diversions through red-flag indicators and “know your customer” best practices. 269 P64 ACKNOWLEDGMENT I want to thank Dr. Michael Jones for his support and encouragement of the webinar series and Dr. Kristin Omberg of Pacific Northwest National Laboratory for reviewing and providing constructive feedback in the Figure 1. A generic pharmaceutical supply development of this poster presentation. chain [1]. 3.3. Day three In the opening of day 3, participants applied knowledge from the first two days to identify supply chain security threats associated with a fictitious scenario. This was fol owed by presentations discussing emerging threats in the pharmaceutical sector, such as clandestine medicinal chemistry, custom synthesis, and novel countermeasures, and the roles of government regulators and industry in preventing the proliferation of PBAs. The day concluded with a roundtable discussion on identifying country-specific pharmaceutical supply chain security chal enges. 3.4. Day four The final day opens with a case study focused on the intersections of pharmaceutical-based production and chemical weapon-related activities. The day concludes with participants identifying actionable ways to address PBA- related chal enges identified on day three. 4. CONCLUSION The webinar series provided a forum for sharing perspectives and insights amongst stakeholders along the chemical supply chain. Participants were introduced to chemical security and through discussion identified specific chal enges in their countries and mitigation tactics to counter diversion and proliferation. 5. REFERENCES 1. Gedan, M., Taylor, K, Covid-19 and pharma supply chain resilence. Infosys Knowledge Institue, 2020. 270 P65 DESIGN OF NEW POTENT AND SELECTIVE THIOPHENE-BASED KV1.3 INHIBITORS AND THEIR POTENTIAL FOR ANTICANCER ACTIVITY Špela Gubič 1, Louise Antonia Hendrickx 2, Xiaoyi Shi 3, Žan Toplak 1, Kenny M. Van Theemsche 4,5, Ernesto Lopes Pinheiro-Junior 2, Steve Peigneur 2, Alain J. Labro 4,5, Luis A. Pardo 3, Jan Tytgat 2, Tihomir Tomašič 1 and Lucija Peterlin Mašič 1,* 1Faculty of Pharmacy, Aškerčeva 7, University of Ljubljana, 1000 Ljubljana, Slovenia; 2Campus Gasthuisberg, University of Leuven, Toxicology and Pharmacology, Onderwijs en Navorsing 2, Herestraat 49, 3000 Leuven, Belgium 3AG Oncophysiology, Max-Planck Institute for Multidisciplinary Sciences, Hermann-Rein-Str. 3, 37075 Göttingen, Germany 4Laboratory for Molecular, Cellular and Network Excitability, Department of Biomedical Sciences, University of Antwerp, 2610 Wilrijk, Belgium 5Department of Basic and Applied Medical Sciences, Ghent University, Corneel Heymanslaan 10 (Entrance 36), 9000 Ghent, Belgium 1. INTRODUCTION family channels or more distantly related The K channels. V1.3 channel has only recently emerged as a molecular target in cancer therapy. Cancer 2. MATERIALS AND METHODS cel s display mutations enabling extensive proliferation and resistance to apoptosis. K 2.1. V1.3 Apoptosis and Proliferation Assays channel activity has been found to be involved For cel proliferation assays, cel s were seeded in cel proliferation, migration, and invasion, at a density of 10,000 cel s/wel in 96-wel flat which are among the most important processes bot om culture plates (Corning, Kaiseslautern, in cancer progression. Moreover, Germany) and proliferation was measured overexpression of KV1.3 enhances tumorigenic through culture confluency using an IncuCyte processes and promotes tumor progression [1– device (Sartorius, Göt ingen, Germany). Every 2]. The use of smal -molecule KV1.3 inhibitors hour, two phase contrast images per wel were could selectively suppress the proliferation of acquired, and a confluency mask was generated cancer cel s, thus providing a new potential by training the analysis algorithm using therapeutic approach [2]. Although KV1.3 has representative images. Every image was then been recognized as a tumor marker in cancer analyzed using the obtained parameters to tissues with predominantly higher KV1.3 determine culture confluency. Treatments were expression, a clear pat ern of altered KV1.3 then added when cel s reached a confluency of expression in cancer cel s compared with ~30% (Panc-1) or ~55% (Colo-357), and healthy cel s has not yet been found. The type proliferation was determined for the fol owing and stage of disease also influence KV1.3 60 h. Proliferation is reported as confluency expression. Up-regulated KV1.3 expression was increase with respect to the start of the detected in breast, colon, and prostate tumors, in treatment. smooth muscle and skeletal muscle cancers, and in mature neoplastic B cel s in chronic Spheroids were cultured in round bot om ultra- lymphocytic leukemia [3]. low at achment 96-wel plates (Corning). The optimal seeding densities were empirical y In previous work, we identified two new KV1.3 determined (8000 cel s/wel for Colo-357 or inhibitors by virtual screening based on a 3D 10,000 cel s/wel for Panc-1 spheroids. The similarity search using the KV1.3 inhibitor PAC cel s were suspended in 2% Matrigel (Corning), as a query [4]. Hit compound (Figure 1) with an centrifuged at 1000× g for 10 min and the IC50 of 17.4 µM (applied ex vivo in Xenopus spheroids were al owed to form in the incubator. oocytes expressing KV1.3) was a weaker KV1.3 Once the spheroids were formed, the treatments inhibitor. However, it exhibited a good indicated together with 8.9 µM cycloheximide selectivity profile and did not affect other KV1.x (as apoptosis sensitizer) were added and 271 P65 apoptosis was determined by live imaging in the PAP-1 0.78 ± 0.01 2.67 ± 0.300.37 ± 0.02 Incucyte system in real time for approximately 14 0.57 ± 0.36 1.03 ± 0.031.33 ± 0.20 60 h using Caspase-3/7 green reagent 37 3.96 ± 0.47 1.97 ± 0.141.35 ± 0.04 (Sartorius) as a reporter of apoptosis, which 43 0.59 ± 0.15 1.20 ± 0.021.02 ± 0.07 crosses the cel membrane and is cleaved by the 44 0.47 ± 0.02 1.99 ± 0.610.95 ± 0.24 activated Caspase-3/7, resulting in the fluorescent staining of nuclear DNA. Apoptosis 3.3. Effects on the Growth of Cell Lines in 2D on Panc-1 tumor spheroids is presented as and 3D Cell Cultures integrated green fluorescence in the whole KV1.3 is associated with the control of cel spheroid. proliferation in various cancer cel types. Therefore, we investigated the effects of 3. RESULTS AND DISCUSSION compounds 14, 37, 43 and 44 on the proliferation of two pancreatic cancer cel lines, 3.1. Design and Chemistry Panc-1 and Colo-357, which have been reported Six different types of cyclopentane-, to overexpress K cyclohexane- and tetrahydropyran-based K V1.3. The compounds were V1.3 tested at a concentration of 100 µM, which is inhibitors were designed and synthesized with the maximal concentration we can achieve the aim of increasing potency on KV1.3, and while maintaining a low concentration of selectivity against other members of KV1 family vehicle (1% DMSO, which was also added to of the hit compounds 4 (TVS-06) and 5 (TVS-the control). Proliferation was determined as 12), and to investigate structure-activity confluence of the culture using live cel imaging relationships (SAR) important for KV1.3 over a 72 h period. For Panc-1 cel s (Fig. 1A,C), inhibition. compound 44 caused moderate growth 3.2. IC inhibition, whereas 14, 43, and especial y 37 50 Determinations of the Most Potent K caused strong inhibition. However, the growth V1.3 Inhibitors The most potent compounds (14, 37, 43 and 44) of Colo-357 cel s (Fig. 1B,D) was not and the reference compound PAP-1 were tested significantly affected by any of the compounds. for KV1.3 inhibition with an additional To test the ability of the compounds to inhibit independent method of manual patch-clamp tumour progression in a more predictive system, procedures on Ltk− cells (Table 1). The aim was we performed experiments on tumour spheroids to demonstrate the inhibition of KV1.3 in a of the pancreatic cancer cel lines used for 2D mammalian cel line and to have a direct com- culture. In Panc-1 spheroids (Fig. 1E), the parison with the positive control PAP-1, which compounds that effectively reduced was previously tested in L929 cel s and human proliferation in 2D culture (14, 37, 43, and 44) T-cel s (IC50 of 2 nM). Interestingly, the did not induce detectable levels of apoptosis, reference compound PAP-1 had an IC50 value of whereas the reference compound PAP-1 did. In 780 nM (manual voltage clamp on oocytes) and Colo-357 cel spheroids, significant induction 370 nM (manual patch clamp on Ltk− cells), of apoptosis was observed in the presence of the which is a much lower potency compared to hit compound 4 (Fig. 6F). None of the other literature data (IC50 of 2 nM, L929 cel s, manual compounds tested differed significantly from whole-cel patch-clamp). The best compound of the control. The degree of apoptosis induction Types I- VI had comparable potency on oocytes was dose-dependent, although we could not use (Table 1, manual voltage-clamp) and Ltk− cells concentrations higher than 100 µM and (manual patch-clamp) of 470 nM and 950 nM, therefore cannot determine the IC respectively. 50 for induction of apoptosis. The level of apoptosis Table 1. Comparison of Kv1.3 IC achieved by 100 µM of hit compound 4 was 50 values for compounds 14, 37, 43, 44, and PAP-1 obtained similar to that achieved by 50 µM PAP-1 (1). with HiClamp and manual voltage-clamp on Xenopus laevis oocytes and with manual patch- clamp on the Ltk− cel -line. IC50 Compou IC50 (Manual (HiClamp IC50 (Manual nd ID Voltage-Clamp Patch-Clamp Oocytes) [µM] Oocytes) [µM] Ltk−) [µM] 272 P65 the extent of apoptosis achieved by 100 µM of hit compound was comparable to that achieved by 50 µM PAP-1. Based on the efficacy data in PDAC cel lines and Colo-357 tumour spheroids, we can assume that newly developed KV1.3 inhibitors do not reach the mitochondrial KV1.3 channels required for the induction of apoptosis. There is an opportunity to further develop the new structural class of potent and selective KV1.3 inhibitors into mitochondrial KV1.3 inhibitors by adding mitochondrial targeting moieties. 5. REFERENCES 1. Pérez-García, M.T.; Cidad, P.; López-López, J.R. The Secret Life of Ion Channels: Kv1.3 Potassium Channels and Proliferation. Am. J. Physiol.-Cel Physiol. 2018, 314, C27–C42. https://doi.org/10.1152/ajpcell.00136.2017. 2. Teisseyre, A.; Palko-Labuz, A.; Sroda- Pomianek, K.; Michalak, K. Voltage-Gated Potassium Channel Kv1.3 as a Target in Therapy of Cancer. Front. Oncol. 2019, 9, 933. https://doi.org/10.3389/fonc.2019.00933. Figure 1. Effects on pancreatic cancer cel lines 3. Comes, N.; Bielanska, J.; Val ejo-Gracia, A.; in 2D and 3D cel cultures. (A) Growth curves Serrano-Albarrás, A.; Marruecos, L.; Gómez, (culture confluence measured through phase D.; Soler, C.; Condom, E.; Ramón y Cajal, S.; contrast imaging) of Panc-1 cel s in the presence Hernández-Losa, J.; et al. The Voltage- of the indicated compounds (100 µM) or the Dependent K+ Channels Kv1.3 and Kv1.5 in Human Cancer. Front. Physiol. 2013, 4, 283. vehicle (DMSO, black symbols). Mean ± S.E.M, https://doi.org/10.3389/fphys.2013.00283. N = 9. (B) The equivalent experiment on Colo- 4. Hendrickx, L.A.; Dobričić, V.; Toplak, Ž.; 357 cel s revealed that the growth inhibition was Peigneur, S.; Mašič, L.P.; Tomašič, T.; Tytgat, cel -type specific. Mean ± S.E.M, N = 15. The J. Design and Characterization of a Novel inhibition 24 h after the start of treatment is Structural Class of Kv1.3 Inhibitors. Bioorganic quantified in (C, D) for the data presented in Chem. 2020, 98, 103746. (A, B) respectively. (E) Apoptosis measured as https://doi.org/10.1016/j.bioorg.2020.103746. caspase 3/7 activity on Panc-1 tumor spheroids 5. Gubič, Š.; Hendrickx, L.A.; Shi, X.; Toplak, Ž.; in the presence of the indicated compounds. (F) Možina, Š.; Theemsche, K.M.V.; Pinheiro- Apoptosis in Colo-357 tumor spheroids ( p < Junior, E.L.; Peigneur, S.; Labro, A.J.; Pardo, L.A.; Tytgat, J.; Tomašič, T.; Mašič, L.P. 0.001, Mean ± S.E.M, N = 4). Design of New Potent and Selective Thiophene- 4. CONCLUSION Based KV1.3 Inhibitors and Their Potential for Anticancer Activity. Cancers 2022, 14, 2595. To discover a novel structural class of K https://doi.org/10.3390/cancers14112595 V1.3 inhibitors overexpressed in many different tumour types, we used a structural optimization approach and successful y prepared novel potent and selective thiophene-based KV1.3 inhibitors [8]. We identified the potent and appropriately selective nanomolar KV1.3 inhibitor 44 (Figure 1), which contains 3- thiophene and tetrahydropyran scaffolds. We demonstrated the inhibition of the Panc-1 cancer cel line proliferation by the new KV1.3 inhibitors. Hit compound induced significant apoptosis in Colo-357 tumour spheroids, and 273 P66 SYNTHESIS AND TRANSFORMATION OF AZA-KYNURENIC ACID DERIVATIVES Bálint Lőrinczi1, Zsófia Sánta1, István Szatmári1 1Institute of Pharmaceutical Chemistry, Interdisciplinary Excellence Center, University of Szeged and MTA-SZTE Stereochemistry Research Group, Hungarian Academy of Sciences Eötvös u. 6, H-6720 Szeged, Hungary 1. INTRODUCTION as the lock and TMS as internal standard (1H). Based on a 2019 report from WHO 55% of Melting points were determined on a Hinotek deaths worldwide are accounted for 10 diseases. X-4 melting point apparatus. Merck Kieselgel The top two diseases are connected to ischaemic 60F254 plates were used for TLC. CEM conditions and neurodegenerative diseases take Discover SP microwave reactor was used for number 7 on this list with lung cancer at number microwave treatment. 6. 2.1.Materials In the past few years the University of Szeged focused aimed toward the treatment of these Starting materials in reagent grade were conditions. In one of the stil in progress purchased from Sigma-Aldrich. research the main aim is on the derivatives of kynurenic acidy (KYNA). In the last two 2.2.Methods decades it has been proven that loss of KYNA General procedure for the synthesis of 6- and can be connected to neurodegenerative 7-chloro-aza-KYNAs (3b,c) conditions such as Parkinson’s or Huntington’s In a 50 mL round bot om flask 5 mmol disease. It has also been shown that it can also antranilic acid derivatives with 6 mmol of contribute to al eviate the symptoms of diethyl-oxalate was dissolved in 30 mL EtOH ischaemic conditions [1,2]. However, the direct and refluxed for 8 hours. After evaporation of medical usage of KYNA is inhibited because of the solvent EtOAc was used for crystal ization. its poor penetration through the blood-brain- barrier (BBB). There have already been several General procedure for the synthesis of 5- and at empts trying to solve this problem, with the 8-chloro-aza-KYNAs (6a,b) synthesis of derivatives through the alteration of In a 35 mL sized pressure-resistant vessel 0,25 the quinoline structure. mmol antranilic acid derivative and 5 mL of Derivatives of KYNA based on the diethyl-oxalate was measured and treated at 110 functionalization of C–3 of the KYNA structure °C for 3 h. The crystal ized product was filtered are get ing abundant [3]; however, there has and washed with 2 x 15 mL Et2O. only be been a few examples of derivatives General procedure for the amidation of chloro- bearing an additional nitrogen in position 3 aza-KYNAs (7a-h) (aza-kynurenic acid, aza-KYNA). In a round bot om flask 0,25 mmol from compounds 3a-c, 6a and 5 mmol from amines We aimed to created derivatives of this special was dissolved in 20 mL EtOH and refluxed for aza-KYNA bearing a cationic center that is 6 h. After evaporation of the solvent, the believed to promote the biological effect [4,5] products were crystal ized from 5 mL Et2O. of KYNA through bet er water solubility and blood-brain-barrier penetration. 3. RESULTS AND DISCUSSION 2. MATERIALS AND METHODS 3.1. Aza-kynurenic acid and its 6- and The 1H-NMR spectra were recorded in 7-chloro derivatives DMSO-d6 solutions in 5 mm tubes at room In case of the unsubstituted aza-KYNA (3a) and temperature (RT), on a Bruker DRX-500 its 6- and 7-chloro derivatives (3b,c) the spectrometer (Bruker Biospin, Karlsruhe, synthetic procedure described in literature Baden Würt emberg, Germany) at 500 (1H) yielded low amounts and was optimized using MHz, with the deuterium signal of the solvent acid catalysis (Scheme 1). 274 P66 Scheme 3. Direct amidation of the synthesized esters. 4. CONCLUSION The synthesis of chloro-substituted aza-KYNA derivatives have successful y been optimized and in case of 5- and 8-chloro-aza-KYNA, a new procedure was developed for their synthesis. As a continuation of the previous KYNA based work, special amide derivatives of these compounds have also been synthesized with possible BBB penetrating and CNS effects. Scheme 1. Synthesis of unsubstituted, 6- and 7-chloro-aza-KYNA derivatives. 5. REFERENCES 3.2. 5- And 8-chloro-aza-KYNA derivatives 1. Fülöp F., Szatmári I., Vámos E., Zádori D., For the synthesis of 5- and 8-chloro-aza-KYNA Toldi J., Vécsei L. Syntheses, transformations and pharmaceutical applications of kynurenic derivatives (6a,b) the needed antranilic acid acid derivatives Curr. Med. Chem. 2009, 16 derivatives (5a,b) had to be synthesized. The 4828 . best results were achieved starting from 4a,b. 2. Gel ért L.; Fuzik J; Göblös A.; Sárközi K.; However, the ring-closure had to be further Marosi M.; Kis Z.; Farkas T.; Szatmári I.; Fülöp altered and a microwave assisted procedure was F.; Vécsei L.; Toldi J. Memantine and developed (Scheme 2). Kynurenic Acid: Current Neuropharmacological Aspects Eur. J. Pharmacol. 2011, 667, 182. 3. Molnár, K.; Lőrinczi, B.; Fazakas, C.; Szatmári, I.; Fülöp, F.; Kmetykó, N.; Berkecz, R.; Ilisz, I.; Krizbai, A. I.; Wilhelm, I.; Vécsei, L. SZR-104, a novel kynurenic acid analogue with high permeability through the blood–brain barrier. Pharmaceutics 2021, 13, 61. 4. Zádori D.; Veres G.; Szalárdy L.; Klivényi P.; Toldi J.; Vécsei L. Glutamatergic dysfunctioning in Alzheimer’s disease and related therapeutic targets J. Alzheimers Dis. 2014, 42, 177. Scheme 2. Synthesis of 5- and 8-chloro-aza- 5. Greco R.; Demartini C.; Zanaboni A. M.; Redavide E.; Pampalone S.; Toldi J.; Fülöp F.; KYNA derivatives. Blandini F.; Nappi G.; Sandrini G.; Vécsei L.; 3.2. Amidation of the aza-KYNA esters Tassorel i C. Effects of kynurenic acid analogue After achieving the synthesis of the esters the 1 (KYNA-A1) in nitroglycerin-induced hyperalgesia: Targets and anti-migraine amidation of the compounds was carried out mechanisms Cephalalgia 2017, 13, 1272 . utilizing direct amidation with the use of N1,N1-dimethylethane-1,2-diamine and ACKNOWLEDGMENT 2-(pyrrolidin-1-yl)ethanamine (Scheme 3). These amines contain the relevant cationic The authors' thanks are due to the Hungarian centers that are presumably needed for bet er Research Foundation (OTKA No. K-138871) biological activity. and the Ministry of Human Capacities, Hungary grant, TKP-2021-EGA-32. and to the Gedeon Richter Plc. Centenarial Foundation. 275 P67 SYNTHESIS OF NEW CARBAMATE-TYPE HARMICINES Marina Marinović, Zrinka Rajić Department of Medicinal Chemitry, University of Zagreb Faculty of Pharmacy and Biochemistry, Croatia 1. INTRODUCTION and ful y characterized by standard methods Harmine is a naturally occurring β-carboline (IR, 1H- and 13C-NMR, and MS). alkaloid that possesses a broad range of 2.1. Synthesis of amine 1 biological activities, including antitumor and Alkylation of harmine at the N-9 position with antimalarial [1]. We designed and prepared 2-(Boc-amino)ethyl bromide in the presence of several series of hybrid compounds in which Cs2CO3 in anhydrous DMF fol owed by the harmine was covalently linked to other removal of the Boc protecting group under biological y active compounds: cinnamic acid acidic conditions (HCl/MeOH) resulted in the derivatives (CADs, harmicines), quinolines preparation of the primary amine 1. (harmiquins), coumarins (harmirins) and ferrocene (harmicens), with the aim of 2.2. Synthesis of cinnamyl alcohols (2a-g) improving its biological activities and Cinnamyl alcohols (2a-g) were prepared from overcoming the growing resistance to malaria the corresponding CADs. In the first step, CAD and cancer chemotherapy [2,3]. The linker was converted to acyl chloride by the means of between the two moieties was either an amide thionyl chloride and a catalytic amount of bond or triazole ring. Our research showed that anhydrous DMF in anhydrous toluene. Acyl hybridization is a valuable strategy for chloride was further reduced to the obtaining compounds with enhanced activities. corresponding alcohol by sodium borohydride Encouraged by those results, we decided to in anhydrous ether with the dropwise addition prepare novel carbamate-type harmicines at the of anhydrous MeOH. This method enabled N-9 position of harmine’s β-carboline core. avoiding the use of stronger reducens, such as lithium aluminum hydride and partial reduction 2. MATERIALS AND METHODS of the CAD double bond. The targeted carbamates 4a-g were obtained by 2.3. Synthesis of carbamate-type harmicines the reaction of amine 1 and cinnamyl alcohols (4a-g) 2a-g under mild reaction conditions. Cinnamyl Carbamate-type harmicines were synthesized alcohols 2a-g were prepared from the by a straightforward two-step procedure. corresponding CADs, whereas amine 1 was Firstly, the reaction of cinnamyl alcohols (2a-g) prepared from harmine. To investigate the with 1,1’-carbonldi midazole (CDI) in effect of substituents at the CADs benzene ring anhydrous ether afforded alkoxy- on harmicines biological activity, we chose carbonylimidazoles (3a-g) which consequently seven CADs with different substitution pat ern reacted with amine 1 in anhydrous DMF to yield ( o-Cl, m-Cl, m-OCF carbamates 4a-g. 3, p-Br, p-CH3, p-CF3, p- NO 2). Reactions which require anhydrous conditions 3. RESULTS AND DISCUSSION were performed under argon atmosphere in anhydrous solvents. Al reagents and solvents Here we present the synthesis of seven novel were purchased from commercial sources. The carbamate-type harmicines, harmine and CADs title compounds 4a-g were purified by column hybrids, synthesized at N-9 position of the chromatography, triturated with diethyl ether harmine’s β-carboline core. 276 P67 3.1. Synthesis of amine 1 5. REFERENCES Primary amine 1 was obtained by the alkylation 1. Javeed, M., et al., Harmine and its derivatives: of harmine at the N-9 position and consequently Biological activities and therapeutic potential in the removal of the Boc protecting group. The human diseases. Bangladesh Journal of synthetic route is depicted in Fig. 1. Pharmacology, 2018. 13: 203-213. 2. Marinović, M., et al., Further investigation of harmicines as novel antiplasmodial agents:Synthesis, structure-activity relationship and insight into the mechanism of action. European Journal of Medicinal Chemistry, Figure 1. Synthetic pathway to amine 1 2021. 224: 113687. 3. Pavić, K., et al., Synthesis and Biological 3.2. Synthesis of cinnamyl alcohols (2a-g) Evaluation of Harmirins, Novel Harmine– The synthetic route for the preparation of Coumarin Hybrids as Potential Anticancer cinnamyl alcohols 2a-g from acyl chlorides is Agents. Molecules, 2021. 26: 6490. depicted in Fig. 2. ACKNOWLEDGMENT This work was ful y supported by the Croatian Science Foundation under the project number UIP-2017-05-5160 and by the Young Figure 2. Synthetic pathway to cinnamyl researcher’s career development project – alcohols 2a-g training of doctoral students of the Croatian Science Foundation founded by the European 3.3. Synthesis of carbamate-type harmicines Union from the European Social Fund. 4a-g Carbamate-type harmicines were prepared by the reaction of amine 1 and cinnamyl alcohols 2a-g in good to moderate yields (34‒49%). The synthetic route and structures of novel harmicines 4a-g are depicted in Fig. 3. Figure 3. Synthetic pathway to carbamate-type harmicines 4a-g 4. CONCLUSION Seven novel carbamate-type harmicines 4a-g were successful y synthesized and characterized. Evaluation of harmicines antimalarial activity, as wel as cytotoxicity against human cell lines, is in progress. 277 P68 DISCOVERY OF INHIBITORS OF Hv1 PROTON CHANNELS AS POTENTIAL ANTICANCER DRUGS Martina Piga1; Zoltán Varga2; Ádám Fehér2; Ferenc Papp2; Eva Korpos Pintye-Gyuri2; Tihomir Tomašič1; Nace Zidar1 1University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia 2University of Debrecen, Faculty of Medicine, Egyetem tér 1. H-4032 Debrecen, Hungary Correspondence: nace.zidar@ffa.uni-lj.si 1. INTRODUCTION (2GBI), on the voltage-sensing domain [4]. A Voltage-gated proton channels (Hv1) are series of molecules was selected to be tested by proton-selective voltage-dependent channels patch-clamp electrophysiology on Hv1 that have been found in various mammalian channels. A smal series of analogues was cel s as wel as in cancer cel s. They play an prepared by organic synthetic procedures and important role in many signaling pathways by their identity and purity were evaluated by regulating the intracel ular pH and preventing different spectroscopic and chromatographic intracel ular acidification [1]. In physiological techniques. conditions, at the resting membrane potential, the channels are closed; however, when various 3. RESULTS AND DISCUSSION pathological conditions occur, these channels Virtual screening results were evaluated, and a can open even at the resting membrane series of most promising hits were selected for potential. In this acidic microenvironment, biological evaluation on Hv1 channels. Four tumor cel s can adapt extremely wel , while hits, NZ-10-2, NZ-49, NZ-58 and NZ-63-2 immune cel s functions are impaired. were found to have an effect on proton currents The aim of our work is to discover and evaluate (more than 50% block at 50 µM). The a series of new Hv1 inhibitors. At present, there compound that showed the greatest effect, are no selective inhibitors specific for Hv1 inhibiting the Hv1 current by more than 90% at proton channels. A selective Hv1 inhibitor 100 mV, was NZ-58 (Fig.1; Fig.2). Results would al ow us to modulate the acidic tumor obtained using the patch-clamp technique in the microenvironment and to study how this affects whole-cel configuration in Chinese Hamster tumor cel functions and tumor growth. A Ovary (CHO) cel s transfected with hHv1, show neutral tumor microenvironment would support that binding of NZ-58 causes a rightward shift the tumor suppressive function of immune cel s. of the opening threshold potential, similar to Hv1 inhibitors could be used as therapeutics in that observed with the application of acidic the treatment of cancer or serve as adjuvants for extracel ular solution. This suggests that NZ-58 other existing therapies. binding modifies the opening of Hv1, most likely by hindering the movement of the 2. MATERIALS AND METHODS voltage-sensing domain, al owing Hv1 to open An open structure of the human Hv1 channel only at more positive voltages. However, in a was used to perform virtual screening (VS) of certain membrane potential range, binding of an in-house library of compounds and selected NZ-58 appears to have an inhibitory effect, but known Hv1 inhibitors [2, 3]. Compounds were in a membrane potential-dependent manner. On docked to the binding site of guanidine the other hand, this compound has shown low derivatives, such as 2-guanidinobenzimidazole selectivity because it also acts on voltage-gated sodium and potassium channels. For these 278 P68 molecules, dose-response tests, selectivity screening and viability assays would be the next steps and new molecules wil be synthesized by systematical y modifying key functional groups. Figure 12. General structure of the new synthetized compounds - Hy=heterocycle. 4. CONCLUSION By bringing together the knowledge and the results from ligand- and structure-based drug design, biophysical and pharmacological characterization, and medicinal chemistry methods, we have obtained a valid and solid Figure 10. starting point for the development of potential The effect of NZ-58 on Hv1 proton currents. Hv1inhibitors. The selected promising hits wil be used for further hit-to-lead optimization to obtain molecules with improved inhibitory potency, bet er hHV1 channel selectivity, and desired physicochemical properties. 5. REFERENCES 1. DeCoursey, T.E., Voltage-gated proton channels: molecular biology, physiology, and pathophysiology of the H(V) family. Physiol Rev, 2013. 93(2): p. 599-652. 2. Geragotelis, A.D., et al., Voltage-dependent structural models of the human Hv1 proton Figure 11. Effects of NZ analogues - remaining Hv1 channel from long-timescale molecular current fraction measured at +100 mV in the presence of dynamics simulations. 50 µM of the compounds. Proc Natl Acad Sci U S A, 2020. 117(24): p. 13490-13498. In addition, three analogues of the recently 3. Zhao, C., et al., HIFs: New arginine mimic discovered Hv1 inhibitors, so-cal ed HIF (Hv1 inhibitors of the Hv1 channel with improved Inhibitor Flexible) compounds were synthetized VSD-ligand interactions. J Gen Physiol, 2021. [3]. The general structure of these analogues 153(9). comprises an aminoimidazole ring and an 4. Hong, L., I.H. Kim, and F. Tombola, Molecular determinants of Hv1 proton channel inhibition by aromatic or aliphatic ring connected by flexible guanidine derivatives. Proc Natl Acad Sci U S A, linkers (Fig.3). We wanted to investigate the 2014. 111(27): p. 9971-6. effects of introducing an aromatic or aliphatic nitrogen-containing heterocycle on ligand- ACKNOWLEDGMENT channel interactions. By introducing an amide The authors acknowledge the financial support bond and increasing the distance between the from the Slovenian Research agency. two cycles of the molecules, we wanted to bet er explore the 2GBI proposed binding-pocket. 279 P69 IN SILICO DISCOVERY AND OPTIMISATION OF VOLTAGE-GATED POTASSIUM CHANNEL KV10.1 INHIBITORS WITH ANTIPROLIFERATIVE ACTIVITY Žan Toplak1, Louise Antonia Hendrickx2, Špela Gubič1, Jan Tytgat2, Luis A. Pardo3, Lucija Peterlin Mašič1, Tihomir Tomašič1 1University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana; Slovenia 2University of Leuven, Toxicology and Pharmacology, Campus Gasthuisberg, Onderwijs en Navorsing 2, Herestraat 49, PO Box 922, 3000 Leuven, Belgium 3AG Oncophysiology, Max-Planck Institute for Experimental Medicine, Hermann-Rein-Str. 3, 37075 Göttingen, Germany 1. INTRODUCTION Anticancer activity was determined using 2D The voltage-gated potassium channel K and 3D cel based models. Antiproliferative V10.1 (Eag1) is a member of the ether-à-go-go family, activity and target selectivity were evaluated on which also includes the erg (eag-related gene, MCF-7, Colo-357, and Panc1 cells lines. KV11) subfamily. In healthy tissues, KV10.1 is 3. RESULTS AND DISCUSSION almost undetectable outside the central nervous system, although it is highly expressed in over We utilized the ligand-based drug discovery 70% of human cancers, regardless of tumour methodology using 3D pharmacophore type. On this basis, K model ing (Fig. 1) and medicinal chemistry V10.1 is considered a nearly universal tumour marker and represents approaches to prepare a novel structural class of a promising new target for new anticancer drug KV10.1 inhibitors. discovery. The channel is involved in cel cycle control and cel proliferation, migration, angiogenesis, and resistance to hypoxia. However, to date, no KV10.1-specific smal - molecule inhibitors have been reported, as most of them also inhibit voltage-gated sodium channels or, most importantly, the cardiac hERG channel, thus posing the risk of cardiac side effects [1]. 2. MATERIALS AND METHODS Figure 1. Pharmacophore-based virtual 2.1. In silico methods and organic synthesis screening hit discovery and optimisation of novel Homology model ing (Model er9.21) was used voltage-gated potassium channel Kv10.1 to prepare open conformation of the channel inhibitors that have antiproliferative activity on used for virtual screening (LigandScout, cells. OpenEye software) and molecular dynamics Ligand-based pharmacophore was build based (NAMD). Structure and ligand-based one the known purpurealidin series of pharmacophore model ing (LigandScout) were compounds. This pharmacophore model was used for virtual screening and molecular evaluated and used for virtual screening and dynamics. identified 18 virtual screening hits. The virtual 2.3. In vitro methods screening hit compound ZVS-08 (Fig. 1) Electrophysiological recordings using whole- exhibited an IC50 value of 3.70 μM against cel patch clamp method were performed to KV10.1 and inhibited the channel in a voltage- evaluate compound potency, selectivity, dependent manner consistent with the action of kinetics, and mode of inhibition. a gating modifier. Structural optimization resulted in the most potent KV10.1 inhibitor of 280 P69 the series with an IC50 value of 740 nM, which 2) Toplak, Ž.; Hendrickx, L. A.; Gubič, Š.; Možina, was potent on the MCF-7 cel line expressing Š.; Žegura, B.; Štern, A.; Novak, M.; Shi, X.; high K Peigneur, S.; Tytgat, J.; Tomašič, T.; Pardo, L. A.; V10.1 levels and low hERG levels, induced significant apoptosis in tumour Mašič, L. P. 3D Pharmacophore-Based Discovery of spheroids of Colo-357 cel s, and was not Novel KV10.1 Inhibitors with Antiproliferative mutagenic [2]. We have also developed a Activity. Cancers. 2021, 13 (6), 1244. structure-based pharmacophore model from 3) Toplak, Ž.; Merzel, F.; Pardo, L. A.; Peterlin molecular dynamics simulations of KV10.1 pore Mašič, L.; Tomašič, T. Molecular Dynamics- blockers (Fig. 2). Our data suggests that Derived Pharmacophore Model Explaining the targeting the K Nonselective Aspect of KV10.1 Pore Blockers. V10.1 channel pore is also likely to result in undesired hERG inhibition, and International Journal of Molecular Sciences. 2021, other potential binding sites should be explored 22 (16), 8999. to develop true KV10.1-selective inhibitors as 4) Caval i, A.; Poluzzi, E.; De Ponti, F.; Recanatini, new anticancer agents [3]. M. Toward a Pharmacophore for Drugs Inducing the Long QT Syndrome:  Insights from a CoMFA Study of HERG K+ Channel Blockers. Journal of Medicinal Chemistry. 2002, 45, 3844–3853. ACKNOWLEDGMENT We thank OpenEye Scientific Software, Santa Fe, NM, USA, for free academic licenses for the use of their software. Luis A. Pardo thanks the expert technical assistance of V. Díaz. Jan Tytgat was supported by grants G0E7120N, GOC2319N and GOA4919N (FWO‐ Figure 2. A) Structure-based pharmacophore model of K Vlaanderen), and grant CELSA/17/ 047 (KU v10.1 pore blockers, developed by molecular dynamics simulations. B) Similarity Leuven). Steve Peigneur was supported by of our present model is seen by the hERG grant PDM/19/164 (KU Leuven). Lucija Peterlin‐Mašič was supported by grants J1‐ pharmacophore model described by Caval i et al 9192, N1‐0098, P1‐0208 (ARRS), and gran [4]. t CELSA 005‐1/2017 (University of Ljubljana). 4. CONCLUSION Luis A. Pardo acknowledges the support of the Max‐Planck Society, and the Göttingen In this work, we used an innovative ligand- Graduiertenschule für Neurowissenschaften, based pharmacophore model ing approach to Biophysik und Molekulare Biowissenschaften create 3D pharmacophore models in (GGNB) (RA). This project has received combination with molecular dynamics to funding from the from the Eur Union through discover and evaluate hEAG1 inhibitors. The Horizon 2020 research and innovation program obtained results represent a good foundation for under the Marie Skłodowska‐Curie grant further development of hEAG1 inhibitors with agreement No. 813834‐PHIONIC‐H2020‐ antiproliferative activity and represents an MSCA‐ITN‐2018. Open access funding original contribution to the science in the field enabled and organized by Projekt DEAL. of anticancer drug discovery. 5. REFERENCES 1) Toplak, Ž.; Hendrickx, L. A.; Abdelaziz, R.; Shi, X.; Peigneur, S.; Tomašič, T.; Tytgat, J.; Peterlin-Mašič, L.; Pardo, L. A. Overcoming Challenges of HERG Potassium Channel Liability through Rational Design: Eag1 Inhibitors for Cancer Treatment. Medicinal Research Reviews. 2022, 42 (1), 183–226. 281 P70 DIFFERENTIATION OF SH-SY5Y CELLS INTO SPECIFIC NEURONAL PHENOTYPES: IN VITRO MODEL OF NEURODEGENERATION Selena Pavšič1, Janko Kos1,2, Anja Pišlar1 1Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, Slovenia 2Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia 1. INTRODUCTION markers (NeuN, GAP-43) and phenotypical y The human neuroblastoma SH-SY5Y cel line characteristic, TH for dopaminergic neurons is the most frequently used in vitro cel model and ChAT for cholinergic neurons. for neurodegeneration research. Retinoic acid (RA) in a combination with brain-derived 2.4 Cytotoxicity and Neuroprotective Assay neurotrophic factor (BDNF) induces in Differentiated Cells differentiation toward a cholinergic neuronal The cytotoxic effect of 6-OHDA was tested on phenotype, whereas treatment with RA differentiated SH-SY5Y cel s to determine the fol owed by phorbol 12-myristate 13-acetate susceptibility of the different neuronal- like (PMA) results in dopaminergic neuronal phenotypes to neuronal death. After a 7-day phenotype - both the most affected neuron differentiation period and 24 h exposure to 100 subsets in Alzheimer’s and Parkinson’s disease, µM 6-OHDA, cel viability was performed with respectively [1, 2]. Differentiated neuron- like 7AAD staining using flow cytometry. In the cel s could provide an effective tool for drug case of neuroprotection assay, on day 7, screening, which assesses sustainable therapies differentiated SH-SY5Y cel s were pretreated for neurodegenerative disorders [3, 4]. with 10 μM rasagiline or safinamide for 1 h followed by 24 h treatment with 100 μM 6- 2. MATERIALS AND METHODS OHDA and assessed by flow cytometry. 2.1 Maintenance and Differentiation of SH- SY5Y Cells into Specific Neuronal 3. RESULTS AND DISCUSSION Phenotype 3.1 Differentiation Protocol Increased Human SH-SY5Y cel line was cultured in Neurite Growth DMEM/F12 containing 10% foetal bovine The formation of a robust neuritic network is serum (FBS) and 1 % penicil in/streptomycin indicating a switch to a neuronal phenotype (P/S) in a humidified incubator at 37 °C with resembling a highly connected synaptic 5% CO network. When compared to RA treatment, 2. The cel medium was replaced every 3 days and cel s were subcultured once they BDNF-treated cel s exhibited longer neurites, reached 80% confluence. After 24 h of plating while PMA-treated cel s expressed more the cel s on a col agen-coated plate, branched neurites (Fig 1). differentiation was initiated by lowering FBS in the culture medium to 2% and 0,25 % P/S and supplementing with 10 μM RA for 7 days. This treatment was replaced every 3 days. To induce a specific neuronal phenotype, on the fourth day of differentiation RA was supplemented with 80 nM PMA or 50 ng/mL BDNF. 2.2 Morphological observation The morphology of cel s was visualized every day during differentiation protocol using a motorized inverted microscope EVOS Cel Imaging System. Figure 1. Morphology of SH-SY5Y cel s on day 2.3 Characterisation of Neuronal Phenotype The flow cytometry method was used to define 7 of the differentiation protocol. White arrows the SH-SY5Y cel differentiation into the are indicating neurites. individual neuronal phenotype by using general 282 P70 3.2 Differentiation Protocol Induced a Shift the SH-SY5Y cel line as a useful in vitro model into a Specific Neuronal Phenotype for neurodegenerative diseases. Moreover, the We observed higher expression of increased susceptibility to the neurotoxin 6- dopaminergic marker (TH) in RA/PMA-treated OHDA shows the formation of more neuron- cel s and cholinergic marker (ChAT) in like cel s suitable for model ing PD and AD. RA/BDNF-treated cel s (Fig. 2). Differentiation The monoamine oxidase inhibitors showed towards the cholinergic phenotype using the promising neuroprotective effects, however, ChAT marker cannot be confirmed in any other more biological replicates should be performed group of differentiated cel s, while the to confirm the applicability of the used in vitro expression level of TH is slightly increased in model for drug discovery. the treatment of RA and RA/BDNF cells. 5. REFERENCES 1. Hong-rong, X., et al. SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson’s disease. Chinese Medical Journal, 2010. 123(8): 1086-1092. 2. Kovalevich, J. and Langford, D., Considerations for the Use of SH-SY5Y Neuroblastoma Cells in Neurobiology . Neuronal Cel Culture, 2013. 1078: 9–21. Figure 2. Relative expression of neuronal 3. Cetin, S., et al., Cell-based models for markers as the mean value of fluorescence Alzheimer’s and Parkinson’s disease: at the intensity normalized to the control. interface of biology and drug discovery. Biomedicine & Pharmacotherapy, 2022. 149. 3.3 Neuroprotective Effect of 6-OHDA- [4] Slanzi, A. et al., In vitro Models of Mediated Neurotoxicity by Rasagiline and Neurodegenerative Diseases. Frontiers in Cel and Safinamide Developmental Biology, 2020. 328. The cytotoxic effect of 6-OHDA was ACKNOWLEDGMENT significantly higher in RA/PMA and RA/BDNF We thank master’s students Baldwin De Man differentiated cel s, proposing SH-SY5Y cel and Nika Marin for their contribution to the differentiation as a suitable model for research while completing their master’s thesis. neurodegeneration (Fig. 3A). Moreover, the addition of inhibitors of monoamine oxidase, rasagiline, and safinamide, showed a significant positive effect on survivability in a neurotoxic environment induced by 6-OHDA compared to the control, indicating their neuroprotective effect (Fig. 3B). Figure 3. (A) 6-OHDA-induced toxicity in differentiated SH-SY5Y cel s. (B) The effect of monoamine oxidase inhibitors on 6-OHDA- induced toxicity. 4. CONCLUSION Our differentiation protocol revealed positive morphological and biochemical differentiation towards specific neuronal phenotype, indicating 283 P71 ENGINEERING LACTOCOCCUS LACTIS TO INHIBIT INTESTINAL INFLAMMATION THROUGH SMALL PROTEIN BLOCKERS OF IL-23/IL-17 AXIS USING NOVEL BGLBRICK ASSEMBLY METHOD Tina Vida Plavec1,2, Aleš Berlec1,2 1Department of Biotechnology, Jožef Stefan Institute, Slovenia 2University of Ljubljana, Faculty of Pharmacy, Slovenia 1. INTRODUCTION constructs and investigated the secretion of Lactococcus lactis is a food-grade model lactic ABD-antagonists specific for human IL-17 acid bacterium (LAB), that is widely used as a receptor A (IL-17R) cal ed ARS-ligands. We cel factory for recombinant protein expression also displayed the IL-17 binding Fynomer on and is a promising vector for in vivo delivery of the surface of L. lactis. Similar to the co-bioactive proteins. Genetical y engineered LAB expression of IL-23-neutralizing ILP/REX have already been proposed for the therapy of proteins, we simultaneously expressed the intestinal inflammation by interfering with pro- ARS-ligand of IL-17R and the IL-17 binding inflammatory cytokines and inhibiting the pro- Fynomer to achieve potential synergy in inflammatory cascade [1,2]. Novel genetic preventing IL-17 signaling. The expression and engineering techniques offer the possibility of function of each protein was evaluated and simultaneous multiple protein expression and confirmed by western blot ing, flow cytometry, the advantage of incorporating the desired fluorescent microscopy and ELISA assay. We modifications with good efficiency and using established a cel model based on THP-1 cel s straightforward procedures. In this study, we for IL-23 secretion to study bacterial binding of constructed a new pNBBX plasmid based on the secreted interleukins. modified BglBrick system and used it to 2. MATERIALS AND METHODS assemble multiple gene casset es and achieve control ed simultaneous expression of multiple 2.1. Molecular cloning model proteins [3]. We demonstrated pNBBX Plasmid pNZ8148 was modified by inserting to be a valuable tool for faster and more restriction enzyme recognition sites NheI and efficient genetic engineering in L. lactis. We BglII upstream of the nisin promoter, and used the pNBBX plasmid to engineer L. lactis restriction enzyme recognition sites BclI and to express proteins capable of inhibiting the IL- XhoI downstream of the transcription 23/IL-17 pro-inflammatory axis, which has terminator (TT), resulting in plasmid pNBBX been shown to have a major impact on [3]. exacerbating intestinal inflammation. Our aim 2.2. Flow cytometry was to simultaneously target IL-23 and IL-17, as well as their receptors, to potentially achieve Flow cytometry was used to assess surface a synergistic effect. IL-23 is the first cytokine of display and function of proteins interfering with the IL-23/Th17 axis. For its inhibition, we used IL-23/IL17 axis. Samples were analyzed with a previously prepared albumin binding domain flow cytometer (FACS Calibur; Becton (ABD)-derived antagonists of IL-23 receptor Dickinson) using excitation at 488 nm and (REX-ligands) and ILP317 protein against the emission at 530 nm. p19 subunit of IL-23, which inhibits binding of the IL-23 to its cognate cel -surface receptor. 2.3. Cytokine binding assay IL-23 stimulation of Th17 cel s results in the Fynomer-displaying L. lactis were resuspended production of IL-17. Therefore, to inhibit IL- in incubation buffer (PBS, 0.05% Tween and 17-mediated signaling, we made genetic 0.1% BSA) containing IL-17A (300 pg/mL). 284 P71 Cel s were removed after 2 h incubation and the however, even 10-fold lower bacterial cel concentrations of the remaining cytokines in the number could stil remove up to 83 % of IL-17A supernatant were determined according to the (Fig. 3B). manufacturer’s instructions (Human IL-17A ELISA development kit (HRP; Mabtech). 3. RESULTS AND DISCUSSION 3.1. BglBrick plasmid construction We constructed the pNBBX plasmid (Fig. 1) by inserting four additional restriction sites into pNZ8148. For multiple gene casset e pNBBX, Figure 2. ELISA-determined IL-17A that plasmids gene casset es were inserted upstream remained in the solution after incubation of IL- of the first casset e using the restriction enzyme 17A with 2×109 CFU/mL (A) or 2×108 CFU/mL pairs NheI/BglI and NheI/BclI, or downstream (B) recombinant L. lactis cel s that displayed IL-using the restriction enzyme pairs XhoI/BglI 17A-binding Fynomer (mFyn) on their surface in and XhoI/BclI. comparison to pNBBX empty plasmid control cells. 4. CONCLUSION We used the recently constructed pNBBX plasmid to demonstrate its applicability and to interfere with the IL-23/IL-17 inflammatory pathway through simultaneous expression of IL-23 and IL-17 binders, as wel as of their corresponding receptor antagonists in L. lactis. Final y, we tested the recombinant L. lactis in Figure 1. Preparation of BglBrick plasmid human cel culture model of inflammation. pNBBX (A). Example of upstream BglBrick cloning of a gene casset e using NheI/BglII for 5. REFERENCES the backbone and NheI/BclI for the insert and 1. Škrlec, K., et al., p19-Targeting ILP protein exploiting BglII/BclI complementarity resulting blockers of IL-23/Th-17 pro-inflammatory axis in a scar (B). GOI, gene of interest, e.g. REX009, displayed on engineered bacteria of food origin. REX125, ARS14, ARS19, ILP317, Fynomer; International journal of molecular sciences, 2018. PnisA, nisin promoter; TT, transcription 19(7):1933. terminator; MCS, multiple cloning site. 2. Plavec, TV., et al., Engineered Lactococcus lactis secreting IL-23 receptor-targeted REX protein 3.2. Concomitant expression and surface blockers for modulation of IL-23/Th17-mediated display inflammation. Microorganisms, 2019. 7(5):152. ILP and Fynomer binders were genetical y 3. Plavec, TV., et al., Introduction of modified label ed with Myc tag, and REX and ARS BglBrick system in Lactococcus lactis for ligands with Flag tag to detect and monitor the straightforward assembly of multiple gene cassettes. expression level of an individual recombinant Frontiers in bioengineering and biotechnology, protein by western blot ing, flow cytometry and 2021. 9:797521. fluorescence microscopy. 3.3. IL-17A binding assay ACKNOWLEDGMENT The binding of IL-17A by recombinant This study was funded by the Slovenian Fynomer-displaying L. lactis was evaluated by Research Agency (grant numbers P4-0127, N3- ELISA. 2×109 recombinant bacteria could 0184, J4-9327). remove up to 97 % of IL-17A from the solution (Fig. 3A). Binding depended on the cel number; 285 P72 UTILISATION OF LYMPHOBLASTOID CELL LINES AS IN VITRO MODELS OF OVER-ACTIVATED IMMUNE RESPONSE FOR DRUG REPURPOSING Luka Hiti1, Tijana Markovič1, Irena Mlinarič-Raščan1 1University of Ljubljana, Faculty of Pharmacy, Slovenia 1. INTRODUCTION concentration of the compound of interest and Cytokine storm is a life-threatening systemic activated by 0.5 mM ionomycin and 3.33 ng/ml inflammatory syndrome involving the PMA and incubated for 24 h. The resting, uncontrol ed secretion of cytokines, which in untreated cel s and the ionomycin/PMA severe cases leads to systemic organic activated cel s were used as controls. Cytokine dysregulation, and can end in death (1). production was assessed by BD Cytometric Lymphoblastoid cel lines (LCLs) have proved Bead Array (CBA) Human Inflammatory to be a reliable in vitro model for evaluating the Cytokine kit (Contents: IL-8, IL-1b, IL-6, IL- effect of compounds on cytokine production 10, TNFα and IL-12) using At uneNext flow and secretion (2). cytometer Results are expressed in fold change against untreated activated controls. Many drugs not indicated for the modulation of an over-activated immune response have been 3. RESULTS AND DISCUSSION used in clinical and preclinical trials since the 3.1. Selected compounds exhibit onset of the Covid-19 pandemic (3). Among the immunosuppressive effect more promising, in addition to corticosteroids, We evaluated the impact of 8 compounds on are proteasome inhibitors, Janus kinase cytokine production (Fig. 1). Two known inhibitors (JAKi), Bruton tyrosine kinase immunosuppressive drugs, dexamethasone and inhibitors (BTKi) (4). Serine protease inhibitors cyclosporin A, decreased the production of have also shown potential (5). assessed cytokines. Six tested compounds displayed an immunosuppressive effect trend, 2. MATERIALS AND METHODS evident from the decreased median fold change 2.1. Materials of pooled cytokine concentrations compared to Dexamethasone, cyclosporin A, ibrutinib, untreated activated controls; converesely, acalabrutinib, baricitinib, carfilzomib increase in pooled cytokine production was nafamostat mesylate, camostat mesylate, detected with ibuprofen and EP4 antagonist. ibuprofen (al Sigma–Aldrich), and EP4 3.2. Selected compounds has a specific antagonist (Cayman Chemical) were dissolved cytokine-release signature in DMSO. Next, we evaluated the impact of compounds on Human lymphoblastoid cel lines (LCLs) were specific cytokine production. Dexamethasone obtained from the National Laboratory for the and carfilzomib displayed the highest variation Genetics of Israeli Populations (NLGIP), a in cytokine production among the tested human diversity biobank at Tel-Aviv compounds (Fig. 2). Namely, proinflammatory University, Tel Aviv, Israel. cytokine levels (IL-8, IL-6, IL-12 and TNFα) were reduced, while immunoregulatory 2.2. Cytokine assay cytokine IL-10 was slightly elevated when The production of cytokines was analysed as LCLs were incubated with dexamethasone. described previously (2). Briefly, cel s were This is in line with the previous studies. In pre-treated for 1 h with the non-cytotoxic contrast, al cytokines except IL-6 were 286 P72 decreased when the cel s were incubated with Utilising LCLs, we evaluated several selective proteasome inhibitor carfilzomib. This compounds with the potential for the treatment indicates that immunomodulatory compounds of Covid-19. Serine protease inhibitors, have specific cytokine signature, which is likely proteasome inhibitors, BTKi and JAKi were also relevant for clinical use. shown to display immunosuppressive effects and are as such interesting candidates for drug repurposing. However, special at ention must be given to the specific cytokine-effect a b c 2.0 signature as different immunosuppressive compounds may not decrease the same 1.5 cytokines (and to the same effect). change 1.0 fold 0.5 5. REFERENCES 1. Fajgenbaum, DC., et al., Cytokine Storm. The 0.0 New England Journal of Medicine, 2020. 3;383(23):2255-2273. Non-activated 2. Markovič, T., et al., Characterization of human Dexamethasone Cyclosporine A Acalabrutinib Ibrutinib Nafamostat Camostat Baricitinib Carfilzomib Ibuprofen EP4 antagonist lymphoblastoid cel lines as a novel in vitro test Figure 1. Pooled fold change in cytokine (IL-6, IL-8, system to predict the immunotoxicity of xenobiotics. IL-10, IL-12, and TNFα) concentration compared to Toxicology Letters, 2015. 17;233(1):8-15. ionomycin/PMA activated cel s after 24h. Cytokine 3. Chakraborty, C., et al., The Drug Repurposing for production was assessed in three LCLs. Boxes COVID-19 Clinical Trials Provide Very Effective represent highest, lowest and median value for each Therapeutic Combinations: Lessons Learned From compound. On the left are untreated and non-activated Major Clinical Studies. Frontiers in Pharmacology, LCLs as starting point reference. (a) Known 2021. 18;12:704205. immunosuppressive effect; (b) observed immunosuppressive effect; (c) observed 4. Yang, L., et al., The signal pathways and treatment immunostimulatory effect. of cytokine storm in COVID-19. Signal Transduction and Targeted Therapy, 2021. 1.5 Dexamethasone 7;6(1):255. Carfilzomib 5. Zhu, H., et al., Spontaneous binding of potential 1.0 COVID-19 drugs (Camostat and Nafamostat) to change human serine protease TMPRSS2. Computational and Structural Biotechnology Journal, 2020. fold 0.5 28;19:467-476. 0.0 IL-8 IL-6 IL-10 TNFa IL-12 IL-8 IL-6 IL-10 TNFa IL-12 ACKNOWLEDGMENT cytokine This research was co-funded by the Slovenian Figure 2. Average fold change of cytokine Research Agency (Grant No. P1-0208 and NC- production as determined in three LCLs after 0004) and the European Regional Development 24h. The cel s were pre-treated for 1h with Plan for EATRIS-TRI.SI, and by the European dexamethasone and carfilzomib and activated by Union’s Horizon 2020 Research and Innovation ionomycin/PMA. programme (No. 871096). 4. CONCLUSION 287 P73 HIGHER INCIDENCE OF COMMON POLYMORPHISMS IN THE GENES OF FOLATE AND METHIONINE CYCLES IN CHILDREN WITH OROFACIAL CLEFS Natasa Karas Kuzelicki1, Alenka Smid1, Tina Kek2,3, Eberlinc Andreja4, Irena Mlinaric-Rascan1, Ksenija Gersak2,3 1Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Slovenia 2Department of Obstetrics and Gynaecology, University Medical Centre Ljubljana, Slovenia 3Faculty of Medicine, University of Ljubljana, Slovenia 4Department of Maxillofacial and Oral Surgery, University Medical Center Ljubljana, Slovenia 1. INTRODUCTION (UKCLJ). Of 179 OFC patients fulfil ing the Orofacial clefts (OFC) are one of the most inclusion criteria, 122 had a healthy sibling. common congenital malformations in humans, Thus, 122 sibling pairs were included in the with the global incidence of 1 in 700 births. study. Of these, DNA sample was available for Although the etiopathogenesis of OFC was 45 sib pairs. Al mothers completed extensively researched, the environmental and questionnaires to evaluate demographic risk genetic risk factors are unclear. factors and the exposure to environmental OFC Recently, deficiency of folate intake and genetic risk factors during pregnancy. Al mothers and aberrations of the folate and methionine cycles children older than 18 years signed the informed have been connected to several congenital consent form and the study protocol was malformations, including the OFC. Since then, approved by the National Medical Ethics many studies investigating the genetic variants commit ee (No. 57/02/13). in folate/methionine relevant genes have been 2.2. DNA extraction and genotyping performed, but their results have been ambiguous [1-4]. This might be due to the DNA was extracted from buccal swabs by influence of several environmental and means of QIAamp DNA Mini Kit (Qiagen) endogenous maternal risk factors during according to the manufacturersìnstructions. pregnancy that might mask the genetic Ten common polymorphisms (MAF ≥ 25%) in influence of child`s genome on the OFC nine genes involved in folate and methionine formation. cycles were genotyped by means of TaqMan To overcome this limitation, we performed the (Applied Biosytems) or LightSNiP (TIB genetic analysis of selected genes involved in MOLBIOL) probes: rs1544105 (FPGS), folate and methionine metabolism and transport rs1677693 (DHFR), rs1801133 and rs1801131 in sibling pairs of OFC patients and their (MTHFR), rs1801394 (MTRR), rs2236225 (MTHFD1), rs3733890 (BHMT), rs10948059 unaffected siblings. Since the siblings shared (GNMT) and rs2424913 (DNMT3B). the prenatal environment we hypothesize that certain genetic influences might be discovered 2.3. Statistical analysis that might be confounded in classical case- Paired samples t-test was used for Gaussian data control approach. and related-samples Wilcoxon signed rank test for non-Gaussian data. Related-samples 2. MATERIALS AND METHODS McNemar test was used for the bi-categorical data, and related samples marginal 2.1. Study subjects and study design homogeneity test for multi-categorical data. OFC cases consisted of children undergoing Holm-Sidak correction for multiple testing was routine fol ow-up examinations before or after applied. the corrective surgery of their OFC at the Department of Maxil ofacial and Oral Surgery at University clinical center Ljubljana 288 P73 3. RESULTS AND DISCUSSION signed-rank test). Boxplots represent median and interquartile range. 3.1. Demographic and environ mental factors Younger siblings in a sib pair were more often These results are not unexpected, since in most affected by OFC than older sibs, and advanced families OFC is inherited as a complex maternal age increased the risk of OFC. multifactorial trait, where contribution of Furthermore, any medicine consumption or individual genes is relatively low, but additive fever in the 1st trimester of pregnancy was contribution of several genes in a same metabolic associated with increased risk of OFC. pathway can have a profound impact on the However, after adjustment for multiple testing disease occurrence. only the presence of a maternal fever during early pregnancy was associated with OFC in a 4. CONCLUSION child. 16,6% of mothers had fever during early To our knowledge, this is the first study to pregnancy with the affected sibling, compared investigate OFC genetic risk factors of the to 3,3% during a non-affected pregnancy (p = folate/methionine metabolic pathways in OFC 0,035). affected – unaffected sibling pairs. 3.2. Genetic factors The only environmental risk factor that differed Of 10 loci in nine investigated genes, four loci between the affected and unaffected in three genes were associated with the pregnancies was the increased body increased risk of OFC: rs1801133 and temperature of the mother in the 1st trimester of rs1801131 (MTHFR), rs2236225 (MTHFD1) the pregnancy. and rs10948059 (GNMT). However, after the None of the investigated genetic loci in the adjustment for multiple testing, none of this genes of folate/methionine cycles was associations stayed significant. individual y associated with OFC. However, when considering al of the investigated loci, a When taking into consideration al the strong correlation between increased number of investigated loci, we found a strong association common polymorphisms in selected genes of between the total number of mutated al eles in folate/methionine cycles and OFC occurrence nine genes associated with folate/methionine was detected. This is indicative of multifactorial cycles and the OFC risk. Siblings who were inheritance pat ern. affected with OFC had higher number of mutations (median (minimum-maximum): 12 5. REFERENCES (5-17)) in the abovementioned genes compared to their unaffected siblings (median (minimum- 1. Krapels IP, et al. Maternal nutritional status and maximum): 9 (4-15)) (p < 0,001) (Figure 1). the risk for orofacial cleft offspring in humans. J Nutr. 2004 Nov;134(11):3106-13 2. Johnson CY, et al. Folate intake, markers of folate status and oral clefts: is the evidence converging? Int J Epidemiol. 2008 Oct;37(5):1041-58. 3. Butali A, et al. Folic acid supplementation use and the MTHFR C677T polymorphism in orofacial clefts etiology: An individual participant data pooled- analysis. Birth Defects Res A Clin Mol Teratol. 2013 Aug;97(8):509-14. 4. De-Regil LM, et al. Effects and safety of periconceptional folate supplementation for preventing birth defects. Cochrane Database Syst Rev. 2010(10):CD007950. Figure 1. Total number of mutated al eles at 10 loci in a child. A comparison between healthy (median (minimum-maximum): 9 (4-15)) and ACKNOWLEDGMENT OFC siblings (median (minimum-maximum): 12 The work was supported by Slovenian research (5-17)) (p < 0.001, Wilcoxon matched-pair agency grants no. J3-5507 and J3-8207. 289 P74 FACTORS INFLUENCING INTER-INIDIVIDUAL DIFFERENCES IN RESPONSE TO PROSTAGLANDIN EP4 RECEPTOR AGONIST IN CHRONIC LYMPHOCYTIC LEUKEMIA CELLS Tijana Markovič1, Alenka Šmid1, Helena Podgornik1,2, Matevž Škerget2, Irena Mlinarič-Raščan1 1University of Ljubljana, Faculty of Pharmacy, Slovenia 2University Medical Centre Ljubljana, Department of Haematology, Ljubljana, Slovenia 1. INTRODUCTION 2.3. Genotyping and expression of Ptger4 Chronic lymphocytic leukemia (CLL) is a very gene heterogenous disease, which is reflected in the CLL samples were genotyped for the single development of the disease, prognosis and nucleotide polymorphisms (SNPs) rs4495224 response to the therapy. Ptger4 gene, encoding and rs7720838 (TaqMan genotyping assay). the EP4receptor was identified as a potential Ptger4 mRNA expression was determined tumor suppressor and its expression was using TaqMan assay elevated on the malignantly transformed lymphocytes B (1). Our previous studies 3. RESULTS AND DISCUSSION demonstrated that EP4 receptor agonists PgE1- OH and L-902,688 had selective cytotoxic 3.1. Inter-individual variability in the effect on the primary CLL cel s (2, 3). The aim response to EP4 receptor agonist of this study was to evaluate the impact of PgE1-OH induced concentration-dependent Ptger4 expression levels and Ptger4 expression- decrease in cel viability (Fig. 1). The average modulating polymorphisms as wel as other EC50 values were 13.56 µM after 24 h and 7.23 clinical and prognostic factors on inter- after 48 h. The EC50 values ranged from 0.97 individual variability in the response to PgE1- µM to 55.07 µM after 24 h, revealing inter- OH in CLL cel s. individual variability in response to PgE1-OH, 2. MATERIALS AND METHODS which is in agreement with the fact that CLL is very heterogeneous disease. 2.1. Primary CLL cells *** After obtaining informed consent in accordance 6 0 with the ethical approval of the National 5 0 Medical Ethics Commit ee of the Republic of 4 0 Slovenia (Nr. 93/12/10), cel s were col ected )M (m from 151 patients with diagnosis of CLL at the 0 3 0 5C University Medical centre, Ljubljana, Slovenia. E 2 0 Lymphocytes B were isolated using Roset eSep 1 0 Ficol -Paque procedure. 0 2.2. Metabolic activity assay 2 4 h 4 8 h Figure 1. Inter-individual variability in the The viability of cel s treated with PgE1-OH response to EP4 receptor agonist PgE1-OH. (Cayman Chemical) was assessed using the Each dot (24 h) or square (48 h) denotes EC50 resazurin based assay (PrestoBlue® Cel value as determined on CLL cel s from one Viability Reagent). patient. Unpaired t-test, *** p < 0.001 290 P74 3.2. EP4 receptor agonist induces selective The analysis revealed sex-dependent sensitivity cytotoxicity in all subgroups of CLL cells of CLL cel s to PgE1-OH, which was more cytotoxic to the cel s of male compared to PgE1-OH was comparably cytotoxic in al female donors (average EC50 values 14.2 μM subgroups of CLL cel s (Fig. 2), which is of and 17.6 μM, respectively; p=0.02). PgE1-OH prime importance, especial y for the patients with progressed disease with Binet stages B and was shown to be more cytotoxic towards the C and with unfavourable prognostic factors, cel s of the carriers of the variant rs4495224 A allele (average EC50 values 14.23 μM in such as del11q and del17p, the lat er leading to patients with rs4495224 AA/AC genotype and aberration of the tumor suppressor gene TP53 18.59 μM in patients with rs4495224 CC and predicting an aggressive disease course and refractoriness to chemoimmunotherapy. PgE1- genotype; p=0.03). The analysis of Ptger4 expression levels revealed that male patients OH may thus show potential also in patients had higher expression compared to female with TP53 aberration. patients (p=0.02) as did the donors with the rs4495224 AA genotype compared to those 5 0 with rs4495224 AC/CC genotype (p=0.046). A 4 0 weak, but significant correlation between an )M 30 increased expression of the PTGER4 gene and (05 lower EC50 values was also shown (p=0.046). C 2 0 E 1 0 4. CONCLUSION 0 In conclusion, we identified EP4 receptor as A B C promising therapeutic target and EP4 receptor agonist PgE1-OH as a promising drug candidate for the treatment of CLL. 5 0 4 0 5. REFERENCES )M 30 1. Markovič T et al. Structural features of subtype- (05 selective EP receptor modulators. Drug Discov C 2 0 E Today. 2017; 22(1):57-71. 1 0 2. Markovič T et al. The Enhanced Cytotoxic Effects 0 in B-Cel Leukemia and Lymphoma Fol owing tri1 2 d e l1 3 q d e l1 1 q d e l1 7 p Activation of Prostaglandin EP4 Receptor and Figure 2. PgE1-OH is cytotoxic against al CLL Targeting of CD20 Antigen by Monoclonal cel s including those from patients in (A) Binet Antibodies. Int J Mol Sci. 2022; Jan 29;23(3):159 stage C and (B) with dysfunctional TP53 (A) 3. Nabergoj S et al. EP4 receptor agonist L-902688 Each dot (Binet stage A), square (Binet stage B) augments cytotoxic activities of ibrutinib, idelalisib, and venetoclax against chronic lymphocytic and triangle (Binet stage C) denotes the EC50 leukemia cel s. Biochem Pharmacol., 2021; value for PgE1-OH determined on cel s obtained 183:114352 from one donor. (B)Each dot (tri12), square (del13q), triangle (del11q) and diamond (del17p) ACKNOWLEDGMENT denotes the EC50 value for PgE1-OH This research was funded by the Slovenian determined on CLL cel s obtained from one Research Agency ARRS (Grant No. P1-0208 donor. and NC-0004) and by the Ministry of Education, Science, and Sport (MIZŠ) and the 3.2. PgE1-OH is more cytotoxic towards the cells of male donors and carriers of variant European Regional Development Fund rs4495224 A allele OP20.05187 RI-SI-EATRIS. 291 P75 DEVELOPMENT OF A METHOD FOR THE THERAPEUTIC DRUG MONITORING OF USTEKINUMAB IN DRIED BLOOD SPOTS Panagiotis-Dimitrios Mingas1, Jurij Zdovc1, Iztok Grabnar1, David Drobne2, Tomaž Vovk1 1Faculty of Pharmacy, University of Ljubljana, Slovenia 2Department of Gastroenterology, University Medical Centre Ljubljana, Slovenia 1. INTRODUCTION 2.2. DBS method development Ustekinumab (UST) is a monoclonal antibody Drug free venous blood was col ected in Li- (mAb) indicated for the treatment of various heparin vacutainers, spiked with UST and autoimmune diseases, including the treatment spot ed on the filter paper cards. The DBS were of adult patients with active Crohn’s disease [1]. air dried for at least 3 h, avoiding direct Several studies associate higher serum sunlight. A punch of approximately 6 mm was concentration of mAbs, with higher chance of taken from the central area of the DBS and it beneficial therapeutic outcomes. Therapeutic was transferred into an Eppendorf tube. 500 mL drug monitoring (TDM) can be a useful tool in of extraction solution, prepared with phosphate- decision-making for possible dose adjustment buffered saline (PBS) containing 0.05% Tween of mAbs and is routinely done, by col ecting 20 and 0.05% NaN3 (sodium azide) were added. patients’ venous blood samples via The samples were then incubated for 4 h at 25 venipuncture [2]. However, a more patient- °C, while gently shaken (200 rpm, HeatMix centric approach could be adopted without the Tehtnica, Domel). After the incubation, the need of regularly visiting healthcare ELISA assay was utilized to determine the institutions. Such is the method of dried blood concentration of UST. spots (DBS), which can be performed by the 2.3. DBS method validation patients at home. The aim of the study is to The analytical validation fol owed the latest develop a method for the determination of UST guidelines of the International Association for concentrations in DBS. Therapeutic Drug Monitoring and Clinical Toxicology [3]. The method was validated for 2. MATERIALS AND METHODS selectivity, recovery, dilution integrity, 2.1. Materials linearity, accuracy, precision, lower and upper The DBS samples were col ected on filter paper limit of quantification, and stability. cards (Whatman™ 903 Protein Saver Cards, GE Healthcare, Dassel, Germany). Drug free 3. RESULTS AND DISCUSSION venous blood was used for the preparation of 3.1. Selectivity, recovery, and dilution DBS calibrators and quality control (QC) integrity samples; it was obtained from healthy For the selectivity, the drug free venous blood, volunteers using lithium heparin (Li-heparin) obtained from five donors, was used to prepare vacutainer tubes (Becton and Dickinson, US). blank DBS samples with specified Hct values The enzyme-linked immunosorbent assay (0.2, 0.3, 0.4, 0.5, 0.6). After the analysis, no (ELISA) kits, as well as the UST stock solution significant differences were detected between used to spike blood, were purchased from blank DBS samples prepared from different ImmunoGuide® (AybayTech Biotechnology, donors. The mean recovery for the low (QCL) Ankara, Turkey). and high (QCH) quality control concentrations at two Hct levels (0.25 and 0.55), was 90 % and 82 %, respectively. The dilution integrity was 292 P75 evaluated at two concentrations, 30 and 120 mg/L. After the additional dilution steps, the accuracy was 95 % and 96 %, respectively. 3.2. Calibration model, accuracy, precision, and limits of quantification The developed method is linear from 3 to 12 mg/L, and it was evaluated by back-calculating the concentrations of the calibrators. The concentrations were within the limits described by the guidelines, ≤ 15 % of nominal value and ≤ 20 % for the lower limit of quantification Figure 1. Stability of UST DBS at 3 different (LLOQ). The slope and intercept values of the conditions (40°C, RT and -20°C) and two calibration curve are given in Table 1. The concentrations (QCL and QCH). accuracy of LLOQ and QC were within the recommended limits, ≤ 20 % and ≤ 15%, 4. CONCLUSION respectively. The intra and inter-day precision TDM is an important tool in dosage of UST QC samples was below 19.9% and optimization of UST. We developed an 11.2%, respectively. The intra- and inter-day alternative method, for the determination of accuracy of UST QC samples was between 84.2 concentration of UST in DBS. This method was – 101.1% and 90.1 – 106.4%, respectively. The validated in the range from 3 to 12 mg/L for LLOQ was 3 mg/L. UST. The long-term stability of DBS samples at room temperature could be advantageous, for Linear response the application of the method in clinical range (mg/L) Intercept Slope r2 practice. With the ongoing clinical validation of Day 1 3-12 0.2011 0.0955 0.9858 the method we aim to demonstrate that DBS can Day 2 (mg/L) 0.3188 0.0969 0.9984 serve as an alternative to venipuncture for the Day 3 0.1527 0.0892 0.9912 Table 1. Linear response range, intercept values, TDM of UST. slope, and coefficient of determination (r2) for 5. REFERENCES calibration curves of UST. 1. Benson, JM., et al., Discovery and mechanism of 3.3. Stability ustekinumab: A human monoclonal The stability of the UST DBS samples was antibody targeting interleukin-12 and determined at two concentration levels (QCL interleukin-23 for treatment of immune- and QC mediated disorders. Vol. 3, mAbs. 2011. p. H) and the cards were stored in airtight bags at RT and in a freezer at -20°C, for a period 3–18. up to 5 months. Additional y, the DBS were also 2. Papamichael, K., et al., Therapeutic drug monitoring with biologic agents in immune stored at 40°C, for 2 days. The above- mediated inflammatory diseases. Expert Rev mentioned storing conditions did not cause a Clin Immunol.2019;15(8):837–48. significant change to the UST concentration, 3. Capiau, S., et al., Official International except QC Association for Therapeutic Drug L samples stored at -20°C for over 28 days, and QC Monitoring and Clinical Toxicology H samples stored at -20°C for 152 days. The results of the stability studies can be Guideline: Development and Validation of Dried Blood Spot-Based Methods for seen in Figure 1. Therapeutic Drug Monitoring. Ther Drug Monit. 2019;41(4):409–30. 293 P76 ASSOCIATION OF OXIDATIVE STRESS-RELATED BIOMARKERS WITH DISEASE ACTIVITY IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE Armando Tratenšek1, Jurij Zdovc1, Igor Locatelli1, Iztok Grabnar1, David Drobne2, Tomaž Vovk1 1Faculty of Pharmacy, University of Ljubljana, Slovenia 2Department of Gastroenterology, University Medical Centre Ljubljana, Slovenia 1. INTRODUCTION active phase of IBD, and healthy controls. There is emerging evidence that an imbalance Additional y, the most prominent markers were between oxidants and antioxidant defence, then further evaluated in plasma samples of commonly referred to as oxidative stress, plays patients with active and inactive CD. a pivotal role in the pathogenesis of 2. MATERIALS AND METHODS inflammatory bowel disease (IBD), namely Crohn’s disease (CD) and ulcerative colitis 2.1. Systematic review and meta-analysis (UC) [1,2]. Activated immune cel s present in The literature search was conducted in the inflamed intestinal mucosa excessively accordance with the Cochrane Handbook and produce various reactive oxygen and nitrogen PRISMA guidelines from various databases species which cause damage to (PubMed, Embase, Web of Science). We biomacromolecules. To counteract the harmful included studies focusing on enzymatic (e.g. processes, reactive species are neutralized by SOD, GPx, CAT) and non-enzymatic superoxide dismutase (SOD), catalase (CAT), antioxidants (bilirubin, albumin, vitamins, free glutathione peroxidase (GPx) and non- thiols) and on markers of oxidative damage to enzymatic antioxidants [1,2]. Oxidative stress- proteins (AOPP), DNA, and lipids (MDA) in related biomarkers in patients with IBD have both active and inactive IBD patients and been detected in saliva, urine and blood. healthy controls. Two reviewers independently However, the relationship between these extracted the markers of oxidative stress biomarkers and disease activity remains elusive (marker name, mean and standard deviation) in and previously published studies yield each group. contradictory results [3]. The statistical analyses were performed in Patients in remission normal y do not Review Manager 5.4. A mean difference in the experience any clinical signs of the disease, levels of biomarkers between the investigated hence we expect differences in the levels of groups, divided by their respective standard oxidative stress-related biomarkers between deviation was used as a measure of effect size, patients with the active and inactive disease. i.e. standardized mean difference (SMD). Given the possibility that the clinical y 2.2. Patients quiescent yet endoscopical y active IBD Frozen plasma samples from thirty-seven patients are not recognized in time, these patients (eighteen females and nineteen males) biomarkers could potential y serve as surrogate diagnosed with CD and treated with markers of underlying inflammation processes ustekinumab were included in our study. and help to bet er monitor the IBD itself without Seventeen were defined as having active CD, subjecting the patients to unpleasant endoscopic and twenty patients were in remission. procedures [3]. The aim of our study was to conduct a meta- 2.3. Plasma malondialdehyde, free thiols analysis to evaluate oxidative stress-related advanced oxidation protein products biomarkers in patients in remission, those in the The concentration of malondialdehyde (MDA) was measured using high-performance liquid 294 P76 chromatography with an ultraviolet detector Table 1. Measurements of oxidative stress- after prior derivatization with related markers in plasma samples from active dinitrophenylhydrazine. and inactive CD patients. Free thiols (R-SH) were measured spectrophotometrical y at 412 nm using a Active disease Remission (n = 17) (n = 20) modified El man’s method applicable for a MDA 4.89 ± 0.91 4.39 ± 1.23 microplate reader. [µmol/l] p=0.240* Advanced oxidation protein products (AOPP) R-SH 436.5 ± 138.7 433.6 ± 95.2 were determined spectrophotometrical y by [µmol/l] p=0.856* measuring the absorbance at 340 nm. AOPP 229.2 ± 101.3 215.5 ± 94.8 [µmol/l] p=0.833* All values are given as the mean ± SD; *independent 3. RESULTS AND DISCUSSION samples t-test. 3.1. Systematic review and meta-analysis 4. CONCLUSION Concentrations or activities were increased Based on meta-analysis results, measurements (p<0.05) in IBD patients compared to healthy of concentrations or activities of bilirubin, controls for the fol owing markers (SMD): CAT, transferrin and AOPP could serve as AOPP (1.81), MDA (0.87) and GPx (0.67). surrogate markers of underlying inflammation Concentrations or activities were decreased in processes and help to bet er monitor the IBD. IBD patients compared to healthy controls for MDA and AOPP measurements in plasma GSH (-1.56), total antioxidant capacity and total samples were in agreement with meta-analysis antioxidant status (-1.32), albumin (-1.29), results. The next step is to conduct a prospective bilirubin (-1.09), vitamin A (-0.86), transferrin study with homogenous, comparable and wel - (-0.75), R-SH (-0.69), vitamin C (-0.66) and defined groups of patients. vitamin E (-0.41). Additional y, differences between the active and inactive group of 5. REFERENCES patients were observed for bilirubin (active 1. Tian T, Wang Z, Zhang J. phase -3.10, inactive phase -0.78), catalase Pathomechanisms of Oxidative Stress in (active phase -0.68; inactive phase -0.10), Inflammatory Bowel Disease and Potential transferrin (active phase -1.17, inactive phase - Antioxidant Therapies. Oxid Med Cel Longev. 2017;2017:4535194. 0.33) and AOPP (active (3.59) inactive IBD (0.89)). 2. Maor I, Rainis T, Lanir A, Lavy A. Oxidative stress, inflammation and neutrophil 3.2. Plasma malondialdehyde, free thiols and superoxide release in patients with Crohn’s disease: Distinction between active and non-active disease. advanced oxidation protein products Digestive diseases and sciences. 2008 measurements Aug;53(8):2208–14. Results are depicted in Table 1. In accordance 3. Krzystek-Korpacka M, Kempiński R, with meta-analysis results, MDA and AOPP Bromke MA, Neubauer K. Oxidative Stress Markers concentrations were increased in patients with in Inflammatory Bowel Diseases: Systematic active CD, whereas R-SH levels did not differ Review. Diagnostics. 2020 Aug 17;10(8):601. between the two groups. Results were, however, not statistical y significant, possibly due to a smal sample size and poorly defined patient characteristics. 295 SPONSORS PLATINUM SPONSORS KRKA At Krka, our dedication lies in producing high quality products, and keeping people healthy is what motivates us. From start to finish, everything we do revolves around our patients and our attempts to help them preserve and improve their health. Each day, more than 50 million patients in 70 markets are treated with our medicines, which are marketed under our own brand names. This is possible because of our innovative products, investments, and market expansion. 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