Farm Vestn 2014; Special Issue: 1 - 253; UDK 615 CODEN FMVTA, SLO ISSN 0014-8229 September 2014, Vol 65 f FARMACEVTSKI * VESTNIK Special Issue BOOK OF ABSTRACTS 10th Central European Symposium on Pharmaceutical Technology September 18 - 20, 2014 ,L Portoroz, Slovenia «¿¿¿S Se^BL'^S® «a» T -a I?v3r i. I "S ' . ^ > ■■ iP,., iL f i STROKOVNO GLASILO SLOVENSKE FARMACIJE I PHARMACEUTICAL JOURNAL OF SLOVENIA Izdaja / Publisher: SLOVENSKO FARMACEVTSKO DRUŠTVO, Dunajska 184 A, SI - 1000 Ljubljana STROKOVNO GLASILO SLOVENSKE FARMACIJE I PHARMACEUTICAL JOURNAL OF SLOVENIA i FARMACEVTSKI VESTNIK C Special Issue I September 2014 I Vol 65 Guest Editors' Preface With the main theme "Translation of basic concepts into drug delivery for specific populations" the 10th Central European Symposium on Pharmaceutical Technology (CESPT 2014) brings focus to patients' needs as the ever more important aspect of drug formulation and the entire process of providing the medication »from-lab-to-bed«. The needs of specific patient populations can be better met by employing the latest achievements in pharmaceutical technology, biopharmaceutics and related sciences. A step further, truly personalized medicine requires advanced approaches like in silico modelling to predict the needs of every individual patient. As an integral part of drug development, the increasing importance of novel specialized pharmaceutical dosage forms and complex drug release mechanisms call for advanced physiologically relevant in vitro drug testing as many CESPT 2014 participants show by the work presented in this Book of Abstracts. Presentations at this symposium are divided into four sessions: Advanced drug delivery systems for specific populations, Frontiers of bio-pharmaceutical evaluation to promote early drug registration, Tuning physicochemical properties of nanomaterials to efficacy and safety and Recent achievements of modelling approach to personalized medicine. The authors of 5 plenary lectures, 5 keynote lectures, 33 oral and 89 poster presentations have met and exceeded the organizers' expectations with the quality and variety of their contributions. Advanced drug delivery systems such as orodispersible drug formulations, minitablets, nanosized delivery systems (nanoparticles, nanofibers, nanotubes, nanosuspensions, nanoemulsions, nanocrystals, nanohydrogels), mi-crosized delivery systems (microspheres, microemulsions), (magneto) liposomes, thermoresponsive hydrogels, solid self-microemulsifying delivery systems, polymeric micelles, solid dispersions, mucoadhesive delivery systems, chronopharmaceuticals, biopolymers and pellets are investigated, from pysicochemical characterization of drugs and excipients to production technologies, in vitro evaluation and translation "from-lab-to-bed". Moreover, in vitro/ex vivo, in vivo and in silico experimental models for the study of mechanisms and kinetics of LADME processes, as well as recent advances in the areas of biorelevant dissolutions tests, transdermal transport studies and eye-related bioavailability prediction models are presented. Over 165 delegates from 20 countries give this symposium a truly international character and represent a strong basis for creative scientific interactions and wide dissemination of new concepts. The organizers are grateful for all the assistance from the supporting societies and associations in promoting the idea of CESPT in the international community. Prof. A. Mrhar, President of CESPT Assist Prof. S. Zakelj, General Secretary of CESPT Assoc. Prof. I Grabnar, Member of Organizing Committee of CESPT ODGOVORNI UREDNIK / GUEST EDITOR: Borut Štrukelj GOSTUJOČI ODGOVORNI UREDNIKI / GUEST EDITORS: Simon Žakelj Iztok Grabnar Aleš Mrhar UREDNIŠKI ODBOR / EDITORIAL BOARD: Saša Baumgartner Marija Bogataj Jelka Dolinar Rok Dreu Mirjana Gašperlin Julijana Kristl Janez Kerč Igor Legen Odon Planinšek Tomaž Vovk Franc Vrečer Jernej Zadnik NASLOV UREDNIŠTVA / ADRESS OF THE EDITORIAL OFFICE: Slovensko farmacevtsko društvo, Dunajska 184a, 1000 Ljubljana, T.: +386 (01) 569 26 01 Transakcijski račun pri Novi LB d.d. Ljubljana: 02010-0016686585. Izhaja petkrat letno. Letna naročnina je 70 EUR. Za tuje naročnike 100 US$. Tiska: COLLEGIUM GRAPHICUM Naklada: 3.400 izvodov Farmacevtski vestnik (Pharmaceutical Journal of Slovenia) is published 5 times a year by the Slovenian Pharmaceutical Society, Subscription rate in inland 70 EUR other countries US$ 100. Farmacevtski vestnik is regulary abstracted in: BIOLOGICAL ABSTRACTS, CHEMICAL ABSTRACTS, PHARMACEUTICAL ABSTRACTS, MEDICAL & AROMATIC PLANTS ABSTRACTS AND INBASE / Excerpta Medica Farmacevtski vestnik is subsidized by the Slovenian Research Agency. f ) CONTENT 4 PLENARY LECTURES 11 KEYNOTE LECTURES 21 ORAL PRESENTATIONS 68 POSTER PRESENTATIONS 244 INDEX OF AUTHORS PLENARY LECTURES THE ROLE OF DRUG DELIVERY TECHNOLOGY IN DRUG THERAPY A. Goepferich1 1 Department of Chemistry and Pharmacy, University of Regensburg, Universitatsstrasse 31 93040 Regensburg, Germany The development of new medicines depends significantly on the discovery of new disease-specific drug targets and the development of drug molecules with sufficiently high target specificity. 'Classical' small molecular weight drugs are, thereby, either developed via rational design or by testing large compound libraries in cell culture for their interaction with a biological target. Both strategies have led to the discovery of highly affine compounds with outstanding target affinities. In recent years we have concomitantly witnessed the massive development of biolog-ics such as antibodies with superb antigen affinities and high plasma half-lives (1). This tremendous success in developing more 'efficient' drugs seems to have severe implications for drug delivery technology. Many small molecules suffer from adverse physicochemical properties such as low solubility. Biolog-ics have an unfavorably large size and high charge density. Given the fact that the in vivo efficacy of a compound does not only depend on its target affinity but equally important on the sufficient distribution to the target site, these are severe handicaps that may lead to a failure to qualify for the originally intended therapy. This development triggered a change of paradigms making drug delivery science not merely a tool for making good medicines better but an integral part of the therapeutic concept. A prominent example is the distribution of antibodies. While in recent years a number of highly efficient compounds were developed their distribution is size limited. For this reason age related macular degeneration, a severe retinal disease, needs to be treated by intravitreal rather than i.v. injections. While 'classical' drug delivery technology may help to keep the injection frequencies low by releasing antibodies from a reservoir over a period of a few weeks, it could even worsen side effects that stem from the continuous intraocular VEGF knock down. New concepts deem necessary to overcome this limita- tion by either implementing discontinuous intraocular drug release patterns or systemic anti VEGF therapies by targeting the retinal endothelium (2). Another prominent example are nucleic acids such as siRNAs the site of action of which is inside cells and that suffer from a lack of ability to cross cell membranes due to their size and charge density. Drug delivery technology is almost mandatory to overcome these drawbacks. Besides biologics, small molecules profit tremendously from innovative drug delivery concepts. Over recent years we have witnessed the massive development of a number of technologies that increase the water solubility of lipophilic drugs by using nanotechnological approaches such as the development of drug nanoparticles with increased solubility (3). Even though the aforementioned examples suggest that drug delivery could easily rescue drug therapy problems related to drug properties, there are shortcomings and even misconceptions. Nanotechnology, for example, offers tremendous opportunities but does not come along without severe intrinsic handicaps. Targeting drugs with the help of nanoparticles via the blood stream can serve a good example. While we may get rid of a number of unfavorable drug properties by using this strategy we may trade them against a number of handicaps that are intrinsic to colloidal particles such as limited drug loading capacity, increased size, immune system activation and a limited ability to leave the blood stream. The aforementioned examples demonstrate that drug delivery science is a highly active research area with steadily growing significance for the therapeutic success of many drugs. REFERENCES 1. R.P. Junghans and C.L. Anderson, The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor, PNAS, 93 (1996) 5512-5516. 2. Pollinger K, Hennig R, Ohlmann A, Fuchshofer R, Wenzel R, Breunig M, Tessmar J, Tamm ER, Goepferich A, Ligand-function-alized nanoparticles target endothelial cells in retinal capillaries after systemic application. PNAS 110 (2013) 6115-6120. 3. Luschmann C, Herrmann W, Strauss O, Luschmann K, Goepferich A, Ocular delivery systems for poorly soluble drugs: An in-vivo evaluation. Int J Pharm 455 (2013) 331-337. PREDICTION OF ORAL DRUG DELIVERY SYSTEMS' IN VIVO PERFORMANCE: HOW TO SIMULATE PHYSIOLOGICAL VARIABILITY M. Bogataj University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia INTRODUCTION In vitro dissolution testing is a useful tool to predict dosage form performance after its administration. This prediction has an important role in the development of new dosage forms and represents a basis for a choice of a newly developed formulation for testing in bioavailability / bioequivalence study. To predict well in vivo behaviour of the dosage form after administration, we have to know the conditions to which the dosage form is exposed in the human body. Thus, for orally administered dosage forms, the detailed knowledge of the conditions in gastro intestinal (GI) tract is essential. However, the variability of GI tract conditions is huge, so it is frequently very difficult to choose in vitro conditions that will reflect well certain in vivo situation. VARIABILITY OF GI TRACT CONDITIONS The variability of conditions is great already in GI tract of a healthy adult, but the conditions in GI tract of an individual who belongs to one of special populations may vary even more, or deviate from those in healthy adults. Two most important special groups are children and elderly people, but there are other groups like pregnant women, people of different races, and also people with different diseases of GI tract or systemic diseases. GI conditions in individuals from those groups might differ significantly. Thus, some conditions in GI tract of a child differ significantly from the conditions in GI tract of healthy adult, at least in certain life periods. These conditions are very much dependent on the age of the child. Many researchers divide children on the basis of their age in 5 - 7 groups (1, 2), because of biological differences that appear in different periods in child's body, and thus also in child's GI tract. For example, pH in the stomach of a baby changes strongly already in first hours, days and weeks of life (1, 2, 3). And pH in the stomach is very important for many drugs and can influence their ionization, solubility, stability etc. There are many other variable parameters and basing on the knowledge of different conditions in GI tract of children and drugs' properties, paediatric biopharmaceu-tical classification system is in preparation (2). Also, the conditions in GI tract of elderly people deviate from those of healthy adults frequently. There are physiological and anatomical conditions that change with increasing age also in healthy older people. Change in acid secretion in stomach appear frequently in elderly (4), also changes in GI tract motility (5), but increasing age represents also an important risk factor for appearance of different diseases which might additionally and strongly influence the conditions in GI tract (5). However, age is not the only factor that produces changes in human body, including GI tract. There exist also groups of people suffering from different diseases independent of their age, which might have great deviations in their GI tract conditions from healthy adults. Thus, Kokubo et al. (6) showed in their meta-analysis study that oro cecal transit time of people with different diseases like celiac disease, cystic fibrosis, etc. differs significantly from healthy adults. They also found out that people with obesity and even pregnant women have prolonged time of transit from mouth to caecum. IN VITRO SIMULATION OF GI TRACT CONDITIONS VARIABILITY To predict in vivo dosage form performance with high certainty, we have to know well the conditions in GI tract including their variability. In the next phase we have to adapt our dissolution tools in such a way that we will be able to simulate the in vivo situation in wide variability of expected physiological / pathological conditions. We have to use suitable apparatuses; in some cases, conventional apparatuses can be used; for example, to simulate only pH or medium composition variability when this is the only or the most critical parameter influencing dosage form performance. However, frequently non-conventional systems have to be applied as certain / extreme values of some physiological parameters cannot be simulated in pharmacopoeial systems. The influence of variability of GI tract parameters on dissolution profiles has already been shown in many stud- ies; and different dissolution is frequently reflected in different absorption and plasma profiles. Kambayashi et al (7) demonstrated that variability of plasma profiles could be successfully predicted considering variability of physiological conditions with special emphasis on gastric retention time of tablets, while Nagelj-Kovacic et al. (8) additionally showed that variability of both, gastric pH and tablet gastric emptying time in physiological range might produce substantial contribution to overall variability of plasma profiles through the influence on dissolution. Additionally, Kersten et al (9) showed that variable pH values simulating those in the stomach of newborns in combination with low volume of gastric medium might influence significantly the dissolution of drugs with pH dependent low solubility. Also variability of transit times of pellets through fasted or fed stomach might contribute significantly to variability of dissolution profiles. Transit times through stomach are especially important for dosage forms which have pH dependent drug release. In the work of Klein et al (10) it was shown that variability of pellet gastric emptying kinetics can contribute substantially to the variability of dissolution / absorption / plasma profiles. Additionally, different experimental setups were developed to simulate different variable situations in GI tract, different movement patterns (11), exposure to different physical burdens inside different parts of GI tract or in sphincters (12), etc. Also TIM pediatric model was developed mimicking different conditions in GI tract of infants and children to predict bioaccessible amounts of drugs after administration (13). REFERENCES I. Bowles A, Keane J, Ernest T et al. Int. J. Pharm. 2010, 395: 37-43. Abdel-Rahman S, Amidon GL, Kaul A. et al. Clin. Ther. 2012, 34(11): S11-S24. Kaye JL. Int. J. Clin. Pharm. 2011; 33:20-24 Britton E, McLaughlin JT. Proc. Nutr. Soc. 2013; 72: 173-177. O'Mahony D, O'Leary P, Quigley EMM. Drugs Aging 2002, 19 (7): 515-527. Kokubo T, Matsui S, Ishiguro M. Pharm. Res. 2013; 30:402-411. Kambayashi A, Blume H, Dressman J. Eur. J. Pharm. Biopharm. 2013; 85: 1337-1347. Nagelj Kovacic N, Pislar M, Ilic I et al. Int. J. Pharm. 2014, in press. Kersten E, Klein S. 9th World Meeting on Pharmaceutics, Biophar-maceutics and Pharmaceutical Technology, Lisbon, 2014. 10. Klein S, Garbacz G, Pislar M et al. J. Control. Rel. 2013; 166: 286-293. II. Bogataj M, Cof G, Mrhar A. 2010, PCT, WO 2010/014046 A1. 12. Garbacz G, Wedemeyer RS, Nagel S et al. Eur. J. Pharm. Biopharm. 2008, 70: 421-428. 13. Havenaar R, Anneveld B, Hanff LM et al. Int. J. Pharm. 2013; 457: 327-332. 9. CONCLUSION Thus, there is a huge variability of GI conditions in healthy individual and even greater in special groups of people, but their influence on a dosage form performance depends also on the dosage form properties and drug susceptibility to certain parameter. However, we still do not know conditions in GI tract in their specific details well enough; we need more knowledge about the dosage form performance under specific conditions and new tools to simulate and predict it. TAILORING NANOMATERIALS FOR PERSONALIZED DRUG DELIVERY: SAFETY AND EFFICACY E. Fattal1,* 1 Université Paris Sud, Institut Galien Paris-Sud, CNRS-UMR 8612, Faculté de Pharmacie, 92296 Châtenay-Malabry, France INTRODUCTION Poly(lactide-co-glycolide) (PLGA) nanoparticles has been widely applied to the delivery of several drugs among which antiinfective drugs systemically and by pulmonary administration and anticancer drugs mainly by systemic administration. In this latest case, the potentialities of nanoparticles have mainly been based on the observation that the endothelium surrounding solid tumours are permeable to colloidal carriers which could use this pathway to extravasate from blood to tumour tissues. This so-called enhanced penetration and retention (EPR) mechanism is highly discussed today since there are a lot of inter-individual physiological differences in each patient regarding extravasation and diffusion across the extracellular matrix. To circumvent this problem, we are developing a strategy that combine imaging and drug delivery which should, by ultrasound imaging or MRI, allow following the fate of nanoparticles and helping in the decision of pursuing the treatment or not (1,2). Moreover, before getting into clinics, nanoparticles need to be clearly demonstrated as safe. One tissue very sensitive to nanotox-icity is lungs. We have investigated the impact of biodegradable nanoparticles on this specific organ (3). Nanoparticles for theranostics PLGA-PEG nanocapsules containing a liquid core of per-fluorooctyl bromide were synthesized by an emulsion-evaporation process and designed as contrast agents for 19F MRI (4, 5, 6). Physico-chemical properties of plain and PEGylated nanocapsules were compared. The encapsulation efficiency of PFOB, estimated by 19F NMR spectroscopy, is enhanced when using PLGA-PEG in- stead of PLGA. PLGA-PEG nanocapsule diameter, measured by Dynamic Light Scattering is around 120nm, in agreement with Transmission Electron microscopy (TEM) observations. TEM and Scanning Electron Microscopy (SEM) reveal that spherical core-shell morphology is preserved. PEGylation is further confirmed by Zeta potential measurements and X-ray Photoelectron Spectroscopy. In vitro, stealthiness of the PEGylated nanocapsules is evidenced by weak complement activation. Accumulation kinetics in the liver and the spleen was performed by 19F MRI in mice, during the first 90 minutes after intravenous injection. In the liver, plain nanocapsules accumulate faster than their PEGylated counteparts. We observe PEGylated nanocapsule accumulation in CT26 xenograft tumor 7 hours after administration to mice, whereas plain nanocapsules remain undetectable, using 19F MRI. Our results validate the use of diblock copolymers for PE-Gylation to increase the residence time of nanocapsules in the blood stream and to reach tumors by the Enhanced Permeation and Retention (EPR) effect. The same nanocapsules were loaded with paclitaxel. The loading was high and likely to be limited to the shell of the nanocapsules. Pharmacokinetics and biodistribution show that drug loaded nanocapsules follow the same profile observed in imaging experiments. Nanoparticle Safety Modifying nanoparticle surface has large impact on toxic-ity. Whereas PLA nanoparticles induce the secretion of acute phase protein after i.v. administration (7). This is not the case for PLA-PEG nanoparticles. Moreover, we have shown no toxicity on Calu-3 or A549 cells of PLGA nanoparticles (7, 8, 9). To extent the toxicity studies to other organ such as lungs, we have developed a co-culture model of THP-1/A549 cells (Figure 1)) to evaluate the toxicity of the PLGA NPs. A > A549 K THP-1 D DY-700 NPs X f 20 urn 1h- 37°C 20 Mm Fig. 1: Nanoparticle uptake by both A549 and THP-1 cells in cocul-ture. Particles are labelled with DY700 (white dots) This model was shown to be relevant for in vitro pulmonary nanotoxicology studies. It was possible to detect a mild inflammatory response to PLGA nanoparticles stabilized by three different hydrophilic polymers PVA, CS and PF68, but very limited compared to well-known inflammatory compounds (Table 1). In vivo in mice the administration of biodegradable NPs did not induce an inflammation process as opposed to non biodegradable NPs for which all parameters measured clearly evidenced a toxicity after acute administration. CONCLUSIONS Our results validate the use of diblock copolymers for PEGylation to increase the residence time of nanocapsules in the blood stream and to reach tumors by the Enhanced Permeation and Retention (EPR) effect. This added to a low toxicity should support the optimisation of PLGA nanoparticles for drug delivery. Table 1. Neutrophils and macrophage counting and cytokine expression in broncho-alveolar lavage of mice treated with different nanopar-ticles Veh l NP NP NP lcu e PLGA/CS PLGA/PVA PLGA/PF68 NP PS NP TiO2(a) NP TlO2(r) Macrophages — — — — — — + Neutrophils — — — — — + — IL-6 — — — — + + — MCP-1 — — — — + + — TNF-a — — — — — + — P S REFERENCES 1. Diou O., N. Tsapis, E. Fattal Targeted nanotheranostics for personalized cancer therapy. Exp. Op. Drug Deliv., 2012; 9:14751487. 2. Diaz-Lopez R., N. Tsapis, E. Fattal Liquid Perfluorocarbons as Contrast Agents for Ultrasonography and 19F-MRI. Pharm. Res., 2010; 27:1-16. 3. Fattal E., Grabowski N., Mura S., Vergnaud J., Tsapis N., Hillaireau H. Lung toxicity of biodegradable nanoparticles. Journal of Biomedical Nanotechnology (in press 2014). 4. Pisani E., C. Ringard, V. Nicolas, E. Raphaël, V. Rosilio, L. Moine, E. Fattal, N. Tsapis Tuning microcapsules surface morphology using blends of homo and copolymers of PLGA and PLGA-PEG. Soft Mat., 2009; 5, 3054-3060. 5. Pisani E., N. Tsapis, B.Galaz, M.Santin, R.Berti, N.Taulier, E. Kurtisovski, O. Lucidarme, M. Ourevitch, B. Thuy Doan, J-C. Beloeil, B. Gillet, W. Urbach, L. Bridal, E. Fattal - Perfluorooctyl bromide polymeric capsules as dual contrast agents for ultrasonography and magnetic resonance imaging. Adv. Funct. Mat., 2008; 18: 2963-297. 6. O. Diou, N. Tsapis, C. Giraudeau, J. Valette, C. Gueutin, F. Bourasset, S. Zanna, C.Vauthier, E. Fattal. Long-circulating perfluorooctyl bromide nanocapsules for tumor imaging by 19F-MRI. Biomaterials, 2012; 33:5593-602. 7. Fernandez-Urrusuno R., E. Fattal, D. Porquet, J. Feger, P. Couvreur Influence of the surface properties on the inflammatory response to polymeric nanoparticles. Pharm. Res., 1995; 12:1385-1387. 8. Mura S., Hillaireau H., Nicolas J., Kerdine-Romer S., Le Droumaguet B., Deloménie C., Nicolas V., Pallardy M., Tsapis N., Fattal E. Biodegradable nanoparticles meet the bronchial airway barrier: how surface properties affect their interaction with mucus and epithelial cells. Biomacromolecules, 12(11), 4136-4143, (2011). 9. Mura S., Hillaireau H., Nicolas J., Le Droumaguet B., Gueutin C., Zanna S. Tsapis N., Fattal E. Influence of surface charge on the potential toxicity of PLGA nanoparticles towards Calu-3 cells. Int. J. Nanomed., 2011; 6:2591-2605. 10. Grabowski N, Hillaireau H, Vergnaud J, Santiago La, Kerdine-Romer S, Pallardy M, Tsapis N, Fattal E. Toxicity of surface-modified PLGA nanoparticles towards lung alveolar epithelial cells. Int. J. Pharm. 2013; 454:686-694. COMPLEX PATIENTS, COMPLEX MODELS: PBPK MADE EASY? GT Tucker12 1 University of Sheffield UK;2 Simcyp Ltd, Sheffield UK The application of physiologically-based pharmacokinetic (PBPK) modelling is coming of age in drug development and regulation, reflecting significant advances over the past 10 years in the predictability of key pharmacokinetic parameters from human in vitro data and in the availability of dedicated software platforms and associated data bases. With respect to understanding co-variates and variability, focus in applying PBPK has been on anticipating the quantitative impact of drug-drug interactions, age, genetics, racial differences, food effects and pharmaceutical formulation. In principle, it is also possible to incorporate pathological features in PBPK models to predict PK in specific disease states defined by aetiology and/or severity. As a start, some progress has been made in predicting the effects of liver cirrhosis and morbid obesity (including bariatric surgery) based on prior knowledge of physiological and relevant biochemical changes. The consequences of impaired drug metabolism as an accompaniment to progressive deterioration in renal function have also been incorporated into prediction of the impact of renal disease. These extensions of PBPK modelling, along with the incorporation of the PK of biologicals and moves towards linking PBPK to pharmacodynamic (PD) outcome, are clearly of benefit in understanding extremes of risk in different patient populations as part of the process of drug development. Indeed, mechanistic PBPK modelling is the only efficient methodology that can anticipate the combined effects of many patient variables acting simultaneously. Apart from its use in drug development, PBPK also has potential application in the health care arena as an educational tool and for the provision of computerised, 'point of care' advice on personalized drug dosage. Multi-drug treatment of the complex patient is a considerable clinical challenge. One day, when sufficient information is available in the patient, clinicians may be able to link that person to his or her virtual twin within a PBPK-PD model on an iPad to provide safe, effective, individualised dosage, and to avoid undesired drug-drug interactions. If the physician considers this too complex, the friendly clinical pharmacist will be looking over his/her shoulder to provide further guidance. KEYNOTE LECTURES SYSTEMS PHARMACOLOGY IN DRUG DEVELOPMENT -TOWARDS PRECISION TREATMENTS M. Danhof12 1 Leiden University, Leiden Academic Centre for Drug Research, Leiden, the Netherlands 2 President European Federation of Pharmaceutical Sciences INTRODUCTION Systems pharmacology is an emerging discipline, which connects systems biology to quantitative pharmacology. A specific feature of systems pharmacology is the focus on biological networks as the basis for drug action. This is important as the network concept explains the well-known plasticity of biological systems with regard to i) drug action (i.e. the often observed lack of efficacy) and ii) disease (i.e. the resilience of disease progression to degeneration) SYSTEMS BIOLOGY - A NOVEL SCIENTIFIC BASIS Systems biology focuses on the structural and functional integration in biological systems to understand (variation in) function. To this end a combination of "reductionist" and "integrationist" approaches is applied. A unique aspect is the multi-dimensionality of this endeavor. The first dimension is the analysis of biological phenomena as dynamic processes across different time scales, which is often referred to as horizontal integration. Next, systems biology also considers integration at different spatial scales (i.e. at the molecular, the cellular, the organ level), which is referred to as the vertical integration [1,2]. Meanwhile, systems biology approaches (i.e. multivariate statistical analysis of metabolome profiles) are increasingly applied to obtain novel insights in the molecular pathways of disease. So far less attention has been paid to the analysis of the effects of drugs on proteome and metabolome profiles. It is anticipated that the analysis of the effects of "drug challenges" can provide unique insights in the functioning of biological systems [3]. SYSTEMS THERAPEUTICS -TOWARDS PRECISION TREATMENTS Systems biology constitutes a scientific basis for the development of "systems therapeutics" interventions for serious and chronic progressive disorders. Such therapeutic interventions are personalized treatments, both with regard to the selection of the drug(s) (to account for variation in the characteristics of the disease), and the dosing regimen (to account for variation in the pharmacology between patients). Moreover, these interventions are intended to be disease modifying rather than symptomatic. As a result the emphasis is on pre-emptive and preventive treatments, which may be initiated prior to the appearance of the clinical manifestations of the disease. This emphasizes the need for accurate and precise diagnostic tools. Finally, to overcome the plasticity of biological systems, these therapeutic interventions are likely to be complex, including the use of multi-target drugs or rational drug combinations. The successful application of systems therapeutics interventions requires the availability of novel biomarkers not only for diagnostic purposes (the characteristics of the disease, to assess the responsiveness to a given treatment) but, in the absence of clinical signs in the early phases of the disease, in particular also for monitoring of the treatment response. Systems therapeutic interventions are "precision treatments". The development and the implementation of "precision treatments" in clinical practice will have major implications for both the pharmaceutical sciences and pharmacy practice. MECHANISM-BASED PKPD MODELING In recent years important progress has been made in the field of mechanism-based pharmacokinetic-pharmacodynamics (PKPD) modeling. PKPD modeling aims at the characterization and prediction of the time course of drug effects in vivo. To this end it considers the cascade of processes on the causal path between drug administration and response. The pertinent processes considered are: a. the target site distribution kinetics, b. the target binding and activation kinetics, and c. the transduction and ho-meostatic feedback [4,5]. For this purpose, mechanism-based PKPD modeling utilizes concepts from physiology-based pharmacokinetic (PBPK) modeling, receptor theory and dynamical systems analysis. Most recently, to characterize the effect of drug treatment on disease progression, the concept of disease systems analysis has been introduced [6]. Mechanism-based PKPD modeling relies on biomarkers which reflect, in a quantitative manner, the processes on the causal path between drug administration and effect [7]. To date mechanism-based PKPD modeling is widely used in translational research for the prediction of drug effect in man, as the basis of early proof-of-concept studies. Meanwhile mechanism-based PKPD modeling is increasingly used in clinical research, as the basis to understand inter-individual variation in drug response. This is important for optimization of the design of clinical trials in special patient groups and for the individualization of drug treatment in clinical practice [8]. CONCLUSION Through the interfacing of systems biology and mechanism-based PKPD modeling, systems pharmacology arises as a novel discipline. It constitutes the scientific basis for the design, the development and the use of novel systems therapeutics interventions REFERENCES 1. Kohl P Crampin EJ, Quinn TA, Noble D (2010) Systemsbiology -an approach, Clin. Pharmacol. Ther. 88: 25-33. 2. Quantitative and Systems Pharmacology in the Post-genomic Era: New Approaches to Discovering Drugs and Understanding Therapeutic Mechanisms (Sorger PK and Allerheiligen SRB eds.), NIH, 2011. 3. Arrell DK, Terzic A. (2010) Network systems biology for drug discovery. Clin. Pharmacol. Ther. 88, 120-125. 4. Danhof M, de Jongh J, De Lange EC, Della Pasqua O, Ploeger BA, Voskuyl RA (2007): Mechanism based pharmacokinetic-pharmacodynamic modeling: biophase distribution, receptor theory, and dynamical systems analysis. Annu Rev PharmacolToxicol. 47:357-400 5. Westerhout J, Ploeger B, Smeets J, Danhof M, de Lange EC. (2012): Physiologically based pharmacokinetic modeling to investigate regional brain distribution kinetics in rats. AAPS J. 14(3):543-553. 6. Post TM, Schmidt S, Peletier LA, de Greef R, Kerbusch T, Danhof M (2013) Application of a mechanism-based disease systems model for osteoporosis to clinical data. J Pharmacokinet Pharmacodyn. 40(2):143-156. 7. Danhof M, Alvan G, Dahl SG, Kuhlmann J and Paintaud G (2005). Mechanism-based Pharmacokinetic-Pharmacodynamic Modelling - A new classification of biomarkers. Pharm. Res. 22: 1432-1437. 8. Krekels EH, Tibboel D, de Wildt SN, Ceelie I, Dahan A, van Dijk M, Danhof M, Knibbe CA (2014): Evidence-Based Morphine Dosing for Postoperative Neonates and Infants. Clin Pharmacokinet. 2014 Feb 5. [Epub ahead of printI NANOMEDICINES FOR TARGETING THE EPITHELIAL BARRIERS OF GUT, SKIN AND LUNG Claus-Michael Lehr Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) Saarland University, 66123 Saarbrücken, Germany INTRODUCTION The focus of our research over the past few years has been on the epithelial barriers of the gastro-intestinal tract, the skin and the lungs. Whiles these outer biological barriers oft the human body are the major routes for non-invasive drug delivery in general, they may also sometimes represent important therapeutic targets by themselves, in particular in the context of inflammatory and infectious diseases. This presentation will highlight some of our recent work in these three areas, either concerning the development of new in-vitro models or of new drug carriers systems, for which the nano-size often has turned out to be advantageous. PARTICLE TARGETING TO THE INFLAMED INTESTINAL MUCOSA For the treatment of inflammatory bowel diseases (IBD) we could previously demonstrate that anti-inflammatory drugs show a prolonged alleviation of colitis syndromes in animal models and a reduction of central nervous side effects, when delivered by polymeric nanocarriers compared to the same dose of the drug administered as an aqueous solution. [1, 2]. In order to study the mechanisms of this intriguing phenomenon we established 3D co-culture model of the intestinal mucosa, comprising human epithelial as well as dendritic cells and macrophages grown on a collagen support. In this system, an inflammation can be experimentally induced and its level be quantified by measuring interleukin release [3,4] TRANSFOLLICULAR DELIVERY OF ANTIGENS By studying the interaction of nanoparticles with the skin barrier, we found a surprising, size dependent penetration into hair follicles [7]. An excellent correlation was found between follicular penetration in human forearm skin in vivo and pig ear skin in vitro [8]. Hypothesizing that this route would possibly allow the non-invasive delivery of antigens to dendritic cells of the skin hair follicles, ovalbumin was encapsulated in chitosan-coated PLGS nanoparticles and applied to the back of shaved mice, but verifying that the the skin barrier was not impaired, indeed. The results of an adoptive transfer model of TCR transgenic CD4+ cells in mice provided first evidence that such transfollicular immunization through the intact skin is possible, thereby stimulating antigen-specific T cells without the need of using any needles, chemical or physical penetration enhancers [9]. INHALATION NANOPHARMACEUTICALS Due to their large surface area and excellent blood supply, the lungs represent an attractive alternative for drug delivery, both for local as well as for systemic action. However, alveolar surfactant and bronchiotracheal mucus represent significant non-cellular barriers [10,11]. In this context, we are working on new in vitro models as well computational approaches to optimize particle deposition on pulmonary cell culture systems, as well as to modulate their mucociliary clearance, their penetration through mucus, as well as the interaction with lung surfactant proteins [12,13]. In order to engineer safer nanomaterials, there is a need to understand, systematically evaluate, and develop constructs with appropriate cellular uptake and intracellular fates; in this context also NP geometry and surface orientation were found to Influence the mode of cellular uptake [14]. Last but not least, inhalation nanopharmaceuticals bear also some interesting opportunities to improve the efficacy of antiinfective drugs: Ultra-small, mucus-penetrating solid lipid nanoparticles were found to significantly enhance the pulmonary delivery and anti-virulence efficacy of novel quorum sensing inhibitors [15]. 2103-2119 4. Leonard, F, H. Ali, E.-M. Collnot, B. J. Crielaard, T. Lammers, G. Storm, and C.M. Lehr, ALTEX 29 (2012) 275-285 5. Schmidt, C., Lautenschlaeger, C., Collnot, E.-M., Schumann, M., Bojarski, C., Schulzke, J.-D., Lehr, C.-M., and Stallmach, J. Control Rel. 165 (2013) 139-145 6. Ali, H, B Weigmann, MF Neurath, EM Collnot, M Windbergs, and CM Lehr. Budesonide Loaded Nanoparticles with pH-Sensitive Coating for Improved Mucosal Targeting in Mouse Models of Inflammatory Bowel Diseases. J.Control.Rel. 183 (2014) 167-77. 7. Patzelt, A, H Richter, F Knorr, U Schäfer, CM Lehr, L Dähne, W Sterry, and J Lademann. Selective Follicular Targeting by Modification of the Part. Sizes. J.Control.Rel. 150 (2011) 45-48. 8. Raber, AS, A Mittal, J Schäfer, U Bakowsky, J Reichrath, T Vogt, UF Schaefer, S Hansen, and CM Lehr. Quantification of Nanoparticle Uptake Into Hair Follicles in Pig Ear and Human Forearm, J. Control. Rel. 179 (2014): 25-32. 9. Mittal, A, AS Raber, UF Schaefer, S Weissmann, T Ebensen, K Schulze, CA Guzmán, S Hansen, CM Lehr, Vaccine, 31 (2013) 3442-3451 10. Ruge CA, J. Kirch, and CM Lehr. Pulmonary Drug Delivery: From Generating Aerosols to Overcoming Biological Barriers-Therapeutic Possibilities and Technological Challenges. Lancet. Resp. Med. 1 (2013) 402-13. 11. Kirch, J, Schneider, A, Abou, B, Hopf, A, Schaefer, U.F., Schneider, M., Schall, C., Wagner, C., Lehr, CM Optical tweezers reveal relationship between microstructure and nanoparticle penetration of pulmonary mucus, PNAS 109 (2012) 18355-18360 12. Ruge, C.A., Schäfer, U.F., Herrmann, J., Kirch, J., Ca adas, O., Echaide, M., Pérez-Gil, J., Casals, C., Müller, R., Lehr, C.-M., PLoS ONE, 7 (2012) e40775. 13. Schäfer, J., Schulze, C., Marxner, E.E.J., Schäfer, U.F., Wohlleben, W., Bakowsky, U., Lehr, C.M., ACS Nano, 6 (2012) 4603-4614. 14. Herd, H, N Daum, AT Jones, H Huwer, H Ghandehari, and CM Lehr. NP Geometry and Surface Orientation Influence Mode of Cellular Uptake. ACS Nano 7 (2013) 1961-73. 15. Nafee, N, A Husari, CK Maurer, C. Lu, C de Rossi, A Steinbach, RW Hartmann, CM Lehr, and M Schneider. Antibiotic-free nanotherapeutics: Ultra-small, mucus-penetrating solid lipid nanoparticles enhance the pulmonary delivery and anti-virulence efficacy of novel quorum sensing inhibitors, J. Control. Rel. 192 (2014) 131-140 REFERENCES 1. Lamprecht, A, N Ubrich, H Yamamoto, U Schäfer, H Takeuchi, P Maincent, Y Kawashima, and C-MLehr. Biodegradable Nanoparticles for Targeted Drug Delivery in Treatment of Inflammatory Bowel Disease. J.Pharmacol.Expt.Therap. 299 (2001): 775-81. 2. Lamprecht, A, U Schäfer, and CM Lehr, Size-Dependent Bioadhesion of Micro- and Nanoparticulate Carriers to the Inflamed Colonic Mucosa. Pharm. Res. 18 (2001): 788-93. 3. Leonard F., Collnot EM, and Lehr CM, Mol. Pharm 7, (2010) PROTAMINE -OLIGONUCLEOTIDE - NANOPARTICLES: RECENT ADVANCES IN DRUG DELIVERY AND DRUG TARGETING A. Zimmer 1* 1 University of Graz, Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, Universitätsplatz 1, 8010 Graz - member of: BioTechMed-Graz, Austria In the year 2000 for the first time a new method for preparing solid nanoparticles from antisense oligonucle-oides together with the cationic peptide protamine was invented by our group. Before, the aggregation into compact structures with short segments of single-stranded DNA was only reported with the polycation poly(L-lysine). We have named this new drug delivery system "proticles" [1]. Within our next study comparing the polycations protamine, spermine and spermidine in terms of their potential to condense different types of oligonucleotides and antisense drugs, protamine was found to be most efficient to form nanoparticles in the size range of 100 - 200 nm [2]. Protamine a peptide well known as pharmaceutical excipient is derived from the sperm of salmon with a molecular mass of approx. 4000 Da and consists of about 70 mol% arginine. In our first evaluations as colloidal nanosuspension these nanoparticles protected oligonucleotides very efficiently against enzymatic digestion caused by nucleases. Also the very early research in this field demonstrated an improved cellular uptake of oligonucleotides combined with significant antisense effects in-vitro [2]. Antisense drugs in general were evaluated as potential anti-viral substances starting from 1990 and in this field of research we could demonstrate for proticles loaded with an AS-PTO drug directed against human immunodeficiency virus type 1 (HIV-1) tat mRNA a very efficient transfection of HIV-1 target cells. Protamine was used to complex AS-ODN and AS-PTO to form nanoparticles with diameters of about 180 nm and surface charges up to +30 mV. Cellular uptake of these nanoparticles was significantly enhanced compared to naked oligonucleotides and showed the release of the antisense compound leading to a specific inhibition of tat mediated HIV-1 transac-tivation [3]. Further research in this field characterized the physicochemical properties of these new nanoparticles and a comparison between different transfection reagents showed lowest cytotoxicity in-vitro for proticles but highest efficacy for cationic lipids [4,5]. Therefore next steps in research included further optimization of the protamine nanoparticles. Albumin was found to be a potent stabilizer of proticles and in addition to the basic binary systems, ternary systems showed superior properties in terms of cellular uptake and intracellular ODN distribution [6-8]. A combination of proticles with liposomes was reported in 2005. Junghans et al. showed the possibility to coat the protamine ODN particle with a lipid film. Further innovation came from the application of prot-amine sulfate to modify the particle diameter in the lower nanometer range more efficiently [9,10]. One year later the first publication which evaluated the immunogenic properties of proticles showed also the possibility to improve the immune-modulation of CpG oligonucleotides which were loaded into proticles [11]. Most successfully proticles with non-immunogenic CpG control oligonucleotides were found to be not immunogenic at all and all basic proticle formulation including pure protamine were tolerated very well and were also found to be highly biocompatible in-vivo. Therefore the next steps in research addressed the question of activ drug targeting by coating the nanoparticle with targeting sequences or to use the proticles as depot system for peptide drugs [12,13]. Consequently in 2010 it was demonstrated for the first time to target proticles loaded with VIP specifically to tumor cells which overex-pressed the VPAC receptor. This approach could be also demonstrated in human lung tumor tissue ex-vivo [14]. Further, targeting of proticles was established for diagnostic purposes using adiponectin as targeting sequence for enhanced imaging of atherosclerotic plaques [15]. More recently our research included also methods to simulate the self-assembly process which is responsible for the nanoparticle formation and to establish a microreactor technology to scale-up the manufacturing process [16-17]. Up to now, this improved technology again was applied to study the possibility of proticle to act as adjuvant and immune-modulator in-vivo [18] and in combination with an improved IL-10 mediated targeting differences were investigated between proticles and targeted liposomes ex-vivo in mice [19]. All together, this presentation will provide a comprehensive overview about our research in this field during the last 15 years. REFERENCES 1. Junghans M., Kreuter J., and Zimmer A, Antisense delivery using protamine - oligonucleotide - particles, Nucleic Acids Res 28 (2000) E45. 2. Junghans M., Kreuter J., and Zimmer A.,Phosphodiester and phosphorothioate oligonucleotide condensation and preparation of antisense nanoparticles, BBA [Protein Structure and Molecular Enzymology] 1544 (2001) 177-188. 3. Dinauer, N., Lochmann, D., Demirhan, I., Bouazzaoui, A, Zimmer A, Chandra, A, Kreuter, J., and von Briesen, H., Intracellular tracking of protamine/antisense oligonucleotide nanoparticles and their inhibitory effect on HIV-1 transactivation, J Control Release 96 (2004) 497-507. 4. Lochmann, D., Weyermann, J., Kreuter, J., Vogel, V., Schubert, D., Dinauer, N., Von Briesen, H., and Zimmer A, Physicochemical characterization of protamine-phosphorothioate nanoparticles, J Microencapsulation 21 (2004) 625-641. 5. Weyermann, J., Lochmann, D., and Zimmer A, Comparison of antisense oligonucleotide drug delivery systems, J Control Release 100 (2004) 411-423. 6. Vogel, V., Lochmann, D., Weyermann, J., Mayer, G., Tziatzios, C., van den Broek, J. A, Haase, W., Wouters, D., Schubert, U. S., Kreuter, J., Zimmer A, and Schubert, D., Oligonucleotide-protamine-albumin nanoparticles: preparation, physical properties, and intracellular distribution J Control Release 103 (2005) 99-111. 7. Lochmann, D., Weyermann, J., Georgens, C., Prassl, R., and Zimmer A., Albumin-protamine-oligonucleotide nanoparticles as a new antisense delivery system. Part 1: Physicochemical characterization, Eur J Pharm Biopharm 59 (2005) 419-429. 8. Weyermann, J., Lochmann, D., Georgens, C., and Zimmer A, Albumin-protamine-oligonucleotide-nanoparticles as a new antisense delivery system. Part 2: cellular uptake and effect, Eur J Pharm Biopharm 59 (2005) 431-438. 9. Junghans M., Loitsch S.M., Steiniger S., Kreuter J., and Zimmer A, Cationic lipid-protamine-DNA (LPD) complexes for delivery of antisense c-myc oligonucleotides, Eur J Pharm Biopharm (2005) 287-294. 10. Mayer G., Vogel V., Weyermann J., Lochmann D., van den Broek J. A, Tziatzios C., Haase W., Wouters D., Schubert U. S., Zimmer A, Kreuter J., and Schubert D., Oligonucleotide-protamine-albumin nanoparticles: Protamine sulfate causes drastic size reduction, J Control Release 106 (2005) 181-187. 11. Kerkmann M., Lochmann D., Weyermann J., Marschner A, Poeck1 H., Wagner M., Zimmer A, Endres S., Hartmann G. Immunostimulatory properties of CpG-oligonucleotides are enhanced by the use of protamine nanoparticles, Oligonucleotides 16 (4) (2006) 313-322. 12. Kratzer I., Wernig K., Panzenboeck U., Bernhart E., Reicher H., Wronski R., Windisch M., Hammer A., Malle E.,Zimmer A., Sattler W. Apolipoprotein A-I coating of protamine-oligonucleotide nanoparticles increases particle uptake and transcytosis in an in vitro model of the blood-brain barrier, J Control Release 117 (2007) 301-311. 13. Wernig K., Griesbacher M., Andreae F., Hajos F., Wagner J., Mosgoeller W. and Zimmer A, Depot formulation of Vasoactive Intestinal Peptide by protamine-based biodegradable nanoparticles, J Control Rel 130 (2008) 192-198. 14. Ortner A., Wernig K., Kaisler R., Edetsberger M., Hajos F., Köhler G., Mosgoeller W., and Zimmer A., VPAC receptor mediated tumour cell targeting by protamine based nanoparticles, J Drug Target 18 (6) (2010) 457-467. 15. Almer G., Wernig K., Saba-Lepek M., Haj-Yahya S., Rattenberger J., Wagner J., Gradauer K., Frascione D., Pabst G., Leitinger G., Mangge H., Zimmer A, Prassl R. Adiponectin-coated nanoparticles for enhanced imaging of atherosclerotic plaques, International Journal of Nanomedicine 6 (2011) 1279-1290. 16. Eitzlmayr A., Petschacher Ch., Radl S., Suzzi D., Zimmer A., Khinast J.G., Modeling and Simulation of Polyacrylic acid/Protamine Nanoparticle Precipitation, Soft Matter 7 (2011) 9484-9497. 17. Petschacher C., Eitzlmayr A, Besenhard M., Wagner J., Barthelmes, J., Bernkop-Schnürch A, Khinast J.G. Zimmer A, Thinking continuously: a microreactor for the production and scale-up of biodegradable, selfassembled nanoparticles. Polymer Chemistry (2013) 4, 2342 - 2352 18. Pali-Schöll I., Szöllösi H., Starkl P., Scheicher B., Stremnitzer C., Hofmeister A., Roth-Walter F., Lukschal A, Diesner S.C., Zimmer A., Jensen-Jarolim E., Protamine nanoparticles with CpG-oligodeoxynucleotide prevent an allergen-induced Th2-response in BALB/c mice, European Journal of Pharmaceutics and Biopharmaceutics (2013) 56-64 19. Almer G., Summers K.L., Scheicher B., Kellner J., Stelzer I., Leitinger G., Gries A, Prassl R., Mangge H. and Zimmer A, Interleukin 10-coated nanoparticle systems compared for molecular imaging of atherosclerotic lesions (2014) in press. RECENT DEVELOPMENTS ON NANOTHERAPEUTICS: NANOTUBES, QUANTUM DOTS, LIPID BASED CARRIERS ismail Tuncer Degim Gazi University Faculty of Pharmacy, Department of Pharmaceutical Technology, 06330, Etiler, Ankara, Turkey e-mail: tunc@tr.net Nanotechnology is growing and developing rapidly especially in engineering and medical sciences. In recent years it has been proposed that carbon materials can be used for drug delivery. Latest study results indicate that transdermal route is the safest way for nanosized materials. If carbon nanotubes (CNTs) are able to penetrate through skin layers or if they can be able to provide enough drug molecules on to the skin surface they can be used to deliver active drug substances for therapeutic purposes but exploration of these kind of pharmaceutical delivery systems and their applications are still at a very early stage of development1. It has been shown that single-walled carbon nanotubes (SWNTs) and multiwalled carbon nanotubes (MWNTs) can be internalized by living cells and they can pass across the biological membranes in cell culture studies2. The internalization of carbon nanotubes by corneocytes has been shown3 in the literature but their drug carrying properties through the skin have not been fully evaluated. In our previous study it has been first shown MWNTs can be used to deliver drug molecules through deeper skin layers. The application of iontophoresis using carbon nanotube electrode having adsorb drug molecules on their surface has been shown and molecules transferred successfully to deeper skin layers4. CNTs have been also used to increase dermal penetration of drugs5,6. The penetration enhancement was found to be higher with double walled carbon nano-tubes (DWCNTs) and MWCNTs, however the mechanism was still unknown. Penetration of indomethacin was found to be much higher when indometahcin molecules were introduced to the skin surface with CNTs5. Other materials like quantum dots can also be used for diagnosis and monitoring of living cell trafficking7. Recently it has been proposed that quantum dots can be used for drug delivery8. Similarly, Zhang et al. have synthesized highly uniform quantum dot-doped chitosan nanobeads for traceable siRNA delivery9 and most recently Jia et al. have combined PEI-coated carbon nanotubes with quantum dots for antisense oligodinucletide delivery10. These innovative approaches have opened up exciting opportunities in targeted DNA and RNA delivery. For example, after being treated with quantum dots - oligonucleotides, cells with differential expression levels of the protein of interest, which correlates with quantum dots fluorescence, can be isolated using fluorescence-activated cell sorting; and, if multicolor quantum dots are used, it will allow the screening of siRNA sequences and the monitoring of downstream cell behaviors in a multiplexed manner. Lipid based drug delivery systems including liposomes, their derivatives, solid lipid nanoparticles and recently developed system called "Cochleates" have been shown to deliver actively drug susbstance to targeted tissues. Among them cochleates may be the most interesting ones being recently developed system and they have been recently tested for anticancer drug delivery and enhanced effectiveness on tumor tissues11,12. As a conclusion some literature based information will be discussed with some case studies for carbon materials, quantum dots and lipid based carriers in the talk. Recent developments, important factors for formulations, ingredients will be given and the mechanisms will be evaluated. REFERENCES 1. C.R. Martin, P. Kohli, The emerging field of nanotube biotechnology, Nat. Rev. Drug Discov. 2:29 (2003). 2. Lacerda L, Raffa V, Prato M, Bianco A, Kostarelos K, Cellpenetrating carbon nanotubes in the delivery of therapeutics, Nano Today, 2:38 (2007). 3. Monteiro-Riviere NA, Inman AO, Wang YY, Nemanich RJ, Surfactant effects on carbon nanotube interactions with human epidermal keratinocytes, Nanomedicine: Nanotechnology, Biology and Medicine, 1: 293 (2005). 4. Martínez-Pla JJ, Martín-Biosca Y, Sagrado S, Villanueva-Camañas RM, Medina-Hernández MJ, Evaluation of the pH effect of formulations on the skin permeability of drugs by biopartitioning micellar chromatography, J. Chromatogr. A.,1047: 255 (2004). 5. I.T. Degim, D.J. Burgess, F. Papadimitrakopoulos, Carbon nanotubes for transdermal drug delivery, J Microencapsul. 27: 669 (2010). 6. S. Ilbasmif-Tamer, S. Yilmaz, E. Banoglu, I.T. Degim, Carbon nanotubes to deliver drug molecules, J Biomed Nanotechnol., 6:20 (2010). 7. Sonvico, Fabio,Dubernet, Catherine, Colombo, Paolo,Couvreur, Patrick. Metallic Colloid Nanotechnology, Applications in p c Diagnosis and Therapeutics, Current Pharmaceutical Design, Volume 8,Number 16, June 2005, pp. 2091-2105(15) 8. Tan WB, Jiang S, Zhang Y. Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference. Biomaterials, 28:1565 (2007). 9. Jia N, Lian Q, Shen H, et al. Intracellular delivery of quantum dots tagged antisense oligodeoxynucleotides by functionalized multiwalled carbon nanotubes. Nano Lett, 7:2976 (2007). 10. Degim I.T. and KadiogluD. Cheap, Suitable, Predictable and Manageable Nanoparticles for Drug Delivery: Quantum Dots Current Drug Delivery, 10, 32-38 (2013). 11. Yucel, C, Degim Z, Yilmaz, S, Nanoparticle and liposome formulations of doxycycline: Transport properties through Caco-2 cell line and effects on matrix metalloproteinase secretion, Biomedicine & Pharmacotherapy, 67, 459 (2013). 12. Mutlu Agardan, B, Degim, Z, An investigation on tamoxifen or raloksifen (SERM - Selective estrogen receptor modulators) containing new drug delivery systems, PHD Thesis, Gazi University, Faculty of Pharmacy, Ankara Turkey 2013 p s SELF-ASSEMBLED POLYSACCHARIDE NANOHYDROGELS: A TOOL FOR DRUG SOLUBILIZATION AND DELIVERY P. Matricardi1, c. Di Meo1, G. D'Arrigo1, E. Montanari1, T. coviello1, F. alhaique1* 1 Department of Drug Chemistry and Technologies, "Sapienza" University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy INTRODUCTION Polysaccharide hydrogels can be formulated in the form of macro-, micro- and nano- systems. Macroscopic networks include films, slabs, beads, as well as matrices for tissue engineering. Recently, nanohydrogels (NHs), i.e., nano-sized hydrogel networks, attracted an ever increasing attention because of the relevant variations of their physical (mechanical, electrical, optical, etc.) properties when compared to those of macroscopic systems. Among the different methods suitable for NHs preparation, the self-assembling approach proposed here is based on hydrophobically modified polysaccharides which, by means of an energy supply (ultrasound, heat, etc.), can aggregate in the form of nanosystems. For this purpose, prednisolone (Pred), a poorly water soluble antiinflammatory drug, which represented the hydrophobic moiety responsible for the self assembling process, was chemically conjugated to the carboxylic groups of Gel-lan gum (Ge), whose molecular weight was previously reduced (1). The obtained Ge-Pred NHs have a core-shell structure which allows the entrapment of hydrophobic drugs; thus, considering that an inflammatory microenvironment is an essential component of all tumors and that anti-inflammatory therapy is not cytocidal on its own, paclitaxel (PCT) was physically loaded within the NHs hydrophobic core, leading to an innovative multi-drug delivery system suitable for a combination therapy in cancer. Preparation, characterization, release behaviour and cytotoxicity of the new NHs are reported. Also other polysaccharides can be used for the preparation of NHs according to this approach (2): we report here some recent results obtained with hyaluronic acid (HA). MATERIALS AND METHODS Ge and HA tetrabutylammonium salt were kindly provided by Fidia Advanced Biopolymers, Abano Terme (Italy). Pred, cholesterol (CH) and PCT were Sigma products. Other chemicals were reagent grade and used without further purification. Ge depolymerization was carried out with a probe type sonicator, using a pulsed mode and the obtained polymer was dissolved in N-methyl-2-pyrrol-idone. Due to the steric hindrance of the polysaccharide, a short spacer was needed for the synthesis of Ge-Pred and Ge-CH, thus the Br-butyric derivatives of Pred or CH were previously prepared. The starting polymers as well as their derivatives were characterized by 1H-NMR and FTIR. Self-assembling was obtained by bath sonication and the obtained NHs were analyzed by dynamic light scattering (DLS), Z-potential and transmission electron microscopy (CRYO-TEM). For the preparation of the PCT-loaded NHs a dry film of this drug was hydrated and vortexed with the Ge-Pred or Ge-CH NHs previously prepared; HPLC determination of the not-incorporated PCT, which was removed by centrifugation, allowed to calculate the entrapment efficiency. Cellular cytotoxicity was assessed on several types of cells. For an alternative energy supply leading to NHs, a JunoLiarre autoclave operating at 121 °C and 1.10 bar was employed. RESULTS AND DISCUSSION The sonication treatment reduced the polymer molecular weight without causing any changes in the chemical structure of the macromolecule, as assessed by bidimen-sional NMR analysis. Pred moiety, chemically linked to the carboxylic groups of the sonicated Ge allowed the self-aggregation. The average size of NHs was about 300 nm, with a unimodal size distribution, and their Z-potential values were negative. Biological tests showed that the Ge-Pred NHs are able to promote the pro-apoptotic activity as well as the free Pred, thus suggesting a similar bioavailability of the drug. It is therefore possible to conclude that gellan-based NHs can be considered as efficient innovative Pred carriers. Taking into account that the combination of PCT and steroids is already used in cancer therapy and allows to obtain several advantages, PCT was loaded within the Ge-Pred NHs. Compared to reported PCT solubility in aqueous medium (< 0.1 pg/ ml), a 1000 fold increase was detected for the PCT-Ge-Pred NHs formulation. PCT release from the new formula- tion was evaluated in vitro and PCT-mediated cytotoxicity, which was tested on different cell lines (Fig 1), was always remarkably higher than that of the free drug. In order to confirm the above depicted approach, cell viability tests were carried auto also using reference NHs prepared with CH (i.e. a not anti-inflammatory steroid) instead of Pred. Obtained results indicated that the PCT-loaded Ge-Pred NHs always significantly improved PCT toxicity with respect to the corresponding PCT-loaded Ge-CH NHs, thus confirming, once more, the synergic effect related to the co-administration of the two drugs (3). REFERENCES 1. D'Arrigo G, Di Meo C, Gaucci E, Chichiarelli S, Coviello T, Capitani D, Alhaique F, Matricardi P, Self-assembled gellan gum nanohydrogel as a tool for prednisolone delivery. Soft Matter 2012; 8: 11557-1564. 2. Montanari E, Capece S, Di Meo C, Meringolo M, Coviello T, Agostinelli E, Matricardi P, Hyaluronic acid nanohydrogels as a useful tool for BSAO immobilization in the treatment of melanoma cancer cells. Macromol. Biosci. 2013; 13: 1185-1194 3. D'Arrigo G, Navarro G, Di Meo C, Matricardi P, Torchilin V, Gellan gum nanohydrogel containing anti-inflammatory and anti-cancer drugs: a multi-drug delivery system for a combination therapy in cancer treatment. Eur. J. Pharm. Biopharm. 2014; 87: 208-216 4. Montanari E, De Rugeriis MC, Di Meo C, Coviello T, Alhaique F, Matricardi P, One-step formation and sterilization of gellan and hyaluronan hydrogels using autoclave (submitted). Fig. 1: Scheme of the PCT-Ge-Pred nanohydrogel and of its cellular uptake. As above mentioned, the self-assembling approach was tested also using other polysaccharides and a different energy supply (4). For this purpose HA-CH was obtained according to the same procedure used for Ge-CH preparation. 20 min autoclaving of water dispersions of each polysaccharide derivative yielded sterile NHs in only one step. Furthermore, within the same step, hydrophilic and lipophilic drugs could be loaded, thus leading to a sterile formulation that can be directly used for administration or for vial filling. The features of NHs obtained according to this procedure were perfectly comparable with those obtained by sonication. In order to increase the stability of the self-assembled systems, the obtained water dispersions can be lyophilized in the presence of a cryopro-tectant, leading to the original NHs after re-dispersion in water. CONCLUSIONS Collected data suggest that, using hydrophobically modified polysaccharides, self-assembled NHs can be easily prepared by sonication and by autoclaving (in this last case leading directly to a sterile formulation). The obtained NHs are suitable for single or a multi-drug delivery and their structure is stable also after lyophilization. A synergistic effect of the combination of anti-inflammatory and anti-cancer drugs was detected in vitro on several types of cells. ORAL PRESENTATIONS ORODISPERSIBLE FORMULATIONS WITH PREDNISOLONE TASTE-MASKING MICROPARTICLES E. Maslak1*' W. Brniak1, R. Jachowicz1 1 Department of Pharmaceutical Technology and Biopharmaceutics, Jagiellonian University Medical College, ul. Medyczna 9, 30-688 Krakow, Poland INTRODUCTION Orodispersible tablets (ODT) and orodispersible films (ODF) are solid oral dosage forms disintegrating or dissolving rapidly when placed in the mouth. They are intended mainly to be administered to the patients having difficulty in swallowing, particularly in geriatric or paedi-atric population. The simplest and most popular method of the ODTs manufacturing is direct compression since it requires only conventional tableting equipment and gives the tablets with good mechanical properties. Fast dissolving films (ODF) have the form of a thin strip and are usually produced by solvent casting method. Their main advantage is the low risk of chocking, ease of application, and no need of water to wash down the drug. One of the main issues related to the orodispersible formulation preparation is an efficient taste masking of a bitter drug substance (1). There is a wide spectrum of taste masking technologies (addition of sweeteners and flavors, forming complexes with cyclodextrins, microencapsulation, etc.) (2). The aim of this study was to evaluate the possibility of the formulation of orodispersible dosage forms containing microparticles masking a bitter taste of the prednisolone. MATERIALS AND METHODS Materials Pharmaburst (SPI Polyols, USA), Pruv - sodium stearyl fumarate (JRS Pharma, Germany), prednisolone base (Henan Lihua, China), Eudragit E100 (Degussa, Germany), Aerosil 200 - colloidal silicon dioxide (Evonik, Germany), Pharmacoat 606 (ShinEtsu Chemical Co., Japan), glycerin (Pharma Cosmetics, Poland). Preparation of microparticles Prednisolone was dissolved in the acetone-isopropanol-water solution of Eudragit E100. The solution was spray-dried in temperature 40 °C with Buchi Mini Spray Dryer B-191. The drug to polymer ratio was 1:2. Preparation of tablets Orodispersible tablets containing 15% of microparticles, i. e. 10 mg of prednisolone were directly compressed with single punch tablet press Korsch EK0. They contained Pharmaburst (82,5%), sodium stearyl fumarate (2,0%) and silicon dioxide (0,5%) as the excipients. Their diameter was 9 mm and mass 200 ± 10 mg. Preparation of films The films were prepared by casting method. HPMC (10% Pharmacoat 606) was dissolved in water. The solutions were mixed and glycerol (15% w/w) was added. The microparticles were mixed with the solutions for 30 min. The solutions were poured on the foil using a motorised film applicator (Elcometer 4340 Elcometer, Belgium). The wet films have a thickness of 1 mm. The films were allowed to dry at room temperature for 12 hours and then were cut into rectangular strips (2 x 3 cm). Tablets parameters The hardness, friability and disintegration time of prepared tablets were measured with pharmacopoeial methods. Disintegration time of films Films with dimensions 3,5 x 4,0 cm were placed in a photo slide frames. The 200 pl of distilled water was dropped on the film, and the time until the film disintegrated was measured (3). Mechanical properties of films Mechanical properties were evaluated using the texture analyzer (Shimadzu EZ-SX, Japan). Tensile strength was evaluated according to standard DIN EN ISO 527. Films (2 cm x 15 cm) were placed into the jaws of apparatus and extended with speed 10 mm/min. Percentage of elongation and tensile force was recorded. Puncture resistance test was performed with the specimens 3,7 x 3,7 cm. They were placed in a holder with a circular hole of 14 mm diameter. The 10 mm diameter spherical probe was moved downward to the film with a constant speed of 10 mm/min. The force needed to puncture the film was recorded (4). Dissolution test Dissolution test was performed with a dissolution apparatus type II (SR8 Plus Dissoette II, Hanson Research, USA) in 900 mL of distilled water at 37°C. The rotation speed was 50 rpm. The samples after filtration were assayed with UV-VIS spectrophotometer (V-530, Jasco, Japan). RESULTS AND DISCUSSION Prepared ODTs were of excellent mechanical properties. Their hardness was 63N, friability 0,12% while disintegration time was maintained around 27s. Placebo films prepared with 10% HPMC and 15% glyc-erol were flexible, elastic, smooth, transparent and without entrapped air bubbles. However, after incorporation of microparticles, films became brittle and their surface was rough. Some air bubbles remained in the matrix. Tensile strength and puncture resistance were higher for placebo films compared with films containing active substance. Disintegration time for placebo films was shorter than 1 min but for those containing microparticles exceeded 2 min. Dissolution studies showed good taste masking properties of prepared microparticles. The amount of predniso-lone released during the first 5 min was below 2%. However, after incorporation to the final orodispersible forms, i. e. ODT and ODF, it increased up to 23% after 2 minutes. The increased release of prednisolone in the orodispers-ible form could be the result of the faster wetting of the microparticles while dispersed in hydrophilic matrices. In the case of ODT, also the compression force during tabletting process could destroy the taste masking layer on the microparticles. REFERENCES 1. Ayenew Z. et al.: Trends in Pharmaceutical Taste Masking Technologies: a Patent Review. Recent Pat Drug Deliv Formul. 2009, 3(1), 26-39. 2. Kawano Y. et at.: Preparation of Orally Disintegrating Tablets with masking of Unpleasant Taste: Comparison with Corrective-adding Methods. Yakugaku Zassgi. 2010, 130(1), 75-80. 3. Preis M., Woertz Ch., Schneider K, Kukawka J., Broscheit J., Roewer N., Breitkreutz J.: Design and evaluation of bilayered buccal film preparations for local administration of lidocaine hydrochloride. European Journal of Pharmaceutics and Biopharmaceutics, 2014, 86, 552-556. 4. Preis M., Knop K., Breitkreutz J.: Mechanical strength test for orodispersible and buccal films. International Journal of Pharmaceutics, 2014, 461, 22-29. CONCLUSIONS The research has proved the possibility of effective taste masking of prednisolone by formation of microparticles with Eudragit E100. Nevertheless, the prepared orodispersible formulations need further optimization in order to maintain the desirable release characteristic of the microparticles. DESIGN AND EVALUATION OF SPECIFIC BI-PHASE EXTENDED RELEASE SYSTEM BASED ON DIFFERENTLY COATED I-TABLETS A. Aleksovski1*, R. Dreu1 1 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia INTRODUCTION Mini-tablets - MTs (diameter < 3 mm) can be the basis of the multiple unit systems, which offer possibility of combining different drugs and/or combination of different release kinetics with aim to decrease the dosing frequency, improve the intended plasma concentration profile and/ or improve the compliance in polypharmacy [1-2]. MTs are produced by using ordinary tablets presses equipped with multiple tip tablet compression tooling and are suitable for subsequent coating [1]. Paliperidon is weakly basic compound belonging to the class of atypical antipsychotics. It is present on the market as 24 hours extended release tablet, produced in form of three layer osmotic pump (OROS® push-pull) providing slightly ascending release rate pattern [3]. Pharmaceutical osmotic pumps bring the benefit of the pH independent steady drug release, but also pose disadvantages such as complex and expensive production process of the units and possible bowel blockage in patients with postoperative bowel constriction due to its non-disinte-grative nature and size. The aim of the research was to develop extended release multiple unit oral dosage form of paliperidon based on MTs, containing different matrix-composition and coated with different pH-dependend polymers, a dosage form which would comprise similar in-vitro drug release pattern as the osmotic pump system and would be at the same time more patient friendly and more cost-beneficial. MATERIALS AND METHODS Materials Paliperidone - PAL (MSN Laboratoires); Kollidon® SR -KSR (polyvinyl acetate/ polyvinyl pyrolidone) (BASF); milled (mesh 200) - LM and spray dried lactose - LS (DFE Pharma.); magnesium hydroxide - MGH (Fluka); magnesium stearate - MGST (Faci S.p.A.) HPMC 2910, 6 cps (Harke Group); Eudragit® L30 D-55 and Eudragit® FS 30 D (Evonik). All other reagents were with analytical grade Mini-Tablet preparation and evaluation MTs were produced with instrumented single-punch tablet press (Killian SP300, IMA), equipped with flat multi-tip punches and die of 2.5 mm diameter. Formulation F1 was prepared by direct compression, while F2 by wet granulation of the PAL, MGH and LM, subsequent mixing with the rest of the components and compression. The target core mass was 15 mg. MT's coating was performed in a fluid bed coater (BX-CGD1, Brinox). Obtained MTs were investigated for friability, mass uniformity-MU and units uniformity-UU (HPLC) according to pharmacopoeial tests. Drug release studies on 4 MTs (6 mg PAL) and original drug (Invega®, Jannsen) were performed on a USP III apparatus (BioDis, VanKel) in fasted GIT conditions (transit time and pH) through 24 hours (Table 1). Tab. 1: Dissolution test conditions (pH and cumulative time) Vessel/GIT part pH/medium Cumulative time 1/Stomach 1.2/0.1 M HCl 1h 2/duodenum 5.5/Phosphate buf. 1h 15min 3/jejunum 6.8/ Phosphate buf. 2h 57min 4/ileum 7.4/ Phosphate buf. 4h 15min 5/colon 6.6/ Phosphate buf. until 24 h Combined formulation of F1 (1 MT) and F2 (3 MT) was compared to the original one by calculating the similarity factor f2. RESULTS AND DISCUSSION After careful evaluation of over thirty formulations we chose two basic formulations for creating combined release system (Tab. 2). In both F1 and F2 KSR with dominantly hydrophobic behaviour showed high capability of retarding the drug release and thus its combination with highly soluble diluent such as lactose was required. Due to higher KSR % F1 has shown slower drug release than F2 and with the decay of Eudragite L30 D55 coating at pH 5.5 it was suitable for aprox. 23-hours drug release. F2 formulation provided faster drug release compared to F1 and due to that it was coated with the colon-targeting polymer Eudragite FS 30 D (decays in the ileal simulated pH 7.4) and was aimed for PAL release during period of aprox. 21 hours. Incorporation of pH modifier MGH into the F2 formulation was done with the intention to decrease the initial release of the active compound and thus increase the similarity factor f2 betwean the original formulation Invega® and F2. MGH is pointed as suitable pH modifier since it has high basic strength and low water solubility (possibility to remain longer in the matrix). Combination of 1 MT F1 and 3 MTs F2 provided the required bi-phasic release (similar to the original drug) with slower release pattern («10%) in the first 4 hours (due to safety reasons) and faster drug release («100%) up to 24 hours. The combined formulation and the original one showed in-vitro similarity (f2=55.31). Additionally combined MT formulation was compared to a theoretically calculated zero order release kinetics. MT combined formulation showed also high similarity to the linear profile (f2=59.11). The drug release profiles of combined MT formulation and the original drug together with the linear profile are given in Fig. 1. Fig. 1: 24h drug release profiles of dosage forms Besides providing suitable drug release profile, F1 and F2 individaully exhibited acceptable properties regarding pharmacopoeial demands on friability, mass and unit uniformity (Tab. 3) Tab. 3: Friability, mass and unit uniformity of F1 and F2 F1 F2 Friability (%) 0.45 0.39 MU (mg) 15.1 ± 0.3 15.08 ± 0.28 CU (AV) 11.80 12.07 CONCLUSIONS Combining differently coated MTs in one multiple unit formulation proved to be a promising pathway for designing systems with specific extended release patterns (ascending or linear like). Proposed system exhibits low dosage form complexity and low manufacturing costs. REFERENCES 1. Tissen C, Woertz K, Breitkreutz J, Kleindebude P Development of mini-tablets with 1mm and 2mm diameter, Int. J. Pharm. 2011; 416:164-170. 2. Ishida M, Abe K, Hashizume M, Kawamura M. A novel approach to sustained pseudoephedrine release: Differentially coated mini-tablets in HPMC capsules. Int. J. Pharm. 2008; 359:46-52. 3. Scientific Discussion - Invega® tablets. EMEA. 2007: 1-58 SIMPLE, BIOCOMPATIBLE AND COST-EFFECTIVE INULIN BASED siRNA DELIVERY SYSTEMS C. Sardo1, M. Licciardi1, C. Scialabba1, G. Giammona1, G. Cavallaro1, F. Tonon2, R. Farra2, B. Dapas3, M. Grassi2, G. Grassi3. 1 Lab of Biocompatible Polymers, Dipartimento di Scienze e Tecnologie Biologiche, Chimiche, Farmaceutiche (STEBICEF), University of Palermo, Palermo 90123, Italy 2 Department of Engineering and Architecture, University of Trieste, Trieste, Italy 3 Department of Life Sciences, Cattinara University Hospital, Trieste University, 34149, Trieste, Italy INTRODUCTION Short interfering RNAs (siRNAs) (1), represent an emerging paradigm for the treatment of many human diseases (2-3), however, owing to its hydrophilicity and negative charges, siRNA molecules are not readily taken up by cells and are susceptible to nuclease degradation (4). Thus, much effort has been placed in the development of siRNA delivery systems. Polyplexes, colloidal systems originated from the electrostatic interaction between negative nucleic acids and positive charged polymers, emerged as one of the most versatile systems for nucleic acid delivery (5). In this work, diethylenetriamine (DETA) molecules were grafted onto inulin backbone to generate a novel siRNA delivery system (Inu-DETA). RESULT AND DISCUSSION Characterization of Inu-DETA Among synthetic methods, the use of microwave irradiation in organic synthesis has become increasingly popular within the polymer synthesis and modification area (6). Here, an alternative recent method to perform microwave-assisted organic reactions, termed "Enhanced Microwave Synthesis" (EMS), has been employed (7). By cooling the reaction vessel, under simultaneous microwave irradiation, more energy can be applied to the reaction mixture, ensuring a high, constant level of microwave energy into the reaction, while keeping constant the reaction tem- perature. The Inu-DETA polymer so generated was able to completely arrest the run of siRNA in an agarose gel, starting from a weight ratio (R) polymer/siRNA of 7; notably, at lower R an interaction between polymer and nucleic acid is still visible. Additionally, DLS results (Figure 1) revealed that although Inu-DETA formed complexes with siRNA at R lower than 7, polyplexes below R10 exhibited a large size, up to 700 nm, with a high polydispersity, suggesting that aggregation occurs. Starting from R10 poly-plexes exhibited a decrease of sizes that remain in the range between 300 and 400 nm. Figure 1. Characterization of Inu-DETA/siRNA polyplexes: hydrody-namic diameter (red square) and Z-potential measurements (blue circles). The Z-potential of Inu-DETA/siRNA polyplexes (Figure 1) increases by increasing the weight amount of copolymer in the polyplex formation: starting from a value of -30 mV for naked siRNA, Z-potential becomes positive near to R7, and reach the value of ~ 40 mV at R40. These results are in agreements with the data obtained from electrophoresis assay on agarose gel and with size distribution obtained with DLS, in which the higher size and polydispersity values are related to polyplexes with lower Z-potential. Analysis of Inu-DETA/siRNA polyplexes at R20 by TEM confirmed DLS measurements data. Impact of Inu-DETA on cell viability The possible toxic effects were evaluated in 16HBE and JHH6 cell lines. Inu-DETA polymer alone was incubated with cells, in a range of concentrations from 25 to 1000 pg/ml for 4, 24 and 48 hrs. MTT tests on cells treated with Inu-DETA did not show appreciable cytotoxicity, even after 48 hrs of incubation for the entire range of concentrations. Comparable results were obtained incubating the cells with Inu-DETA/siRNA polyplexes. Efficacy of Inu-DETA in siRNA delivery Inu-DETA was complexed with a siRNA directed against the mRNA of E2F1 (siE2F1), a protein involved in the promotion of cell cycle (8). As control, Inu-DETA complexed with siGL2, a non-functional siRNA, was used. At R4 and 3 days after administration in JHH6, Inu-DETA/siE2F1 reduced E2F1 mRNA (60% of control) and protein levels compared to controls (Figure 2). This resulted in a reduction of cell growth as evaluated by cell counting (30% reduction) and cell vitality (MTT test, 30% reduction). MO R40*wcfcr ¡UQ+fiAO Figure 2. Inu-DETA/siE2F1 specifically reduces E2F1 mRNA and protein levels. Uptake mechanisms of Inu-DETA In vitro uptake experiments were performed in 16HBE in the presence of different inhibitors of the cell entry mechanisms; in particular, MpCy, PAO and WORT were used as lipid mediated endocytosis, clatrin dependent endocy-tosis and macropinocytosis inhibitors, respectively. Optimal results were obtained using a weight ratio Inu-DETA/ siRNA of 40 (R40). As MpCy but not PAO or WORT inhibited polyplexes uptake (no fluorescence visible in the R40-MpCy panel compared to R40, Figure 3), it follows that lipid and cholesterol mediated uptake pathways regulate Inu-DETA/siRNA uptake. Figure 3. Inhibition of uptake by MpCy, PAO or WORT. Blue: cell nuclei; red: siRNA CONCLUSIONS In this work, DETA molecules were grafted onto inulin backbone to generate a novel siRNA delivery system. The polymer efficiently complexes siRNA and delivers it to model cell lines preserving siRNA functions. Finally, the lipid and cholesterol mediated uptake pathways regulates polymer/siRNA uptake. REFERENCES 1. Grassi, M., Cavallaro, G., Scire, S., Curr Signal Transd T. 5(2), 92 (2010) Grassi G., Scaggiante B., Dapas B., et al. Current Medicinal Chemistry. 20 (28): 3515-38 2013 Deng, Y, Wang, C.C., Choy, K.W., Du, Q., Chen, J., Wang, Q, Li, L., Kwok Hung Chung, T., Tang, T., Gene, 538, 217 (2014) Li, C.X., Parker A., Menocal, E., Xiang, S., Borodyansky, L. and Fruehauf, J.H., Cell Cycle, 5, 2103 (2006) Ballarin-Gonzalez, B. and Howard, K.A., Adv Drug Deliv Rev., 64, 1717 (2012) Hayes, BL., Aldrichimica Acta, 37(2), 66 (2004) Joubert, J., Sharma, R., Onani, M., Malan, S.F., Tetrahedron Lett., 54, 6923 (2013) B Dapas, R Farra, M Grassi, C Giansante, N Fiotti, Rainaldi, A Mercatanti, A Colombatti, P Spessotto, , G Guarnieri and G Grassi. Molecular Medicine, 2009, 15: 296-306 2. 3. THIOLATED AND S-PROTECTED HYDROPHOBICALLY MODIFIED POLY(ACRYLIC ACID)- A NEW GENERATION OF MULTIFUNCTIONAL POLYMERS s. Bonengel1*, s. Hauptstein1, G. Perera1, A. Bernkop schnürch1 1 Center for Chemistry and Biomedicine, Center for Molecular Biosciences, Department of Pharmaceutical Technology Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria INTRODUCTION Alkyl-modified (C10-C30) carbomers (AC1030) with amphophilic properties are representatives of multifunctional polymers. According to their chemical nature these polymers display additional emulsifying properties compared to non-alkylated carbomers. A further polymer modification, namely thiolated polymers (thiomers), emerged to be valuable multifunctional polymers with noteworthy mu-coadhesive properties. Their ability to form covalent bonds with cysteine-rich subdomains in the mucus gel layer provides excellent mu-coadhesive properties (1, 2). As thiol groups are highly susceptible to oxidation, the concept of S-protection was established. Disulphide bonds between the polymeric thiol group and an aromatic thiol bearing ligand are established providing a higher reactivity and stability towards oxidation (3). In order to combine these promising strategies, thiolated AC1030 as well as the preactivated version thereof were synthesized within this study and evaluated as semisolid emulsifying mucoadhesive delivery systems. MATERIALS AND METHODS Materials Carbomers (Carbopol® Ultrez 20/ AC1030 and Carbo-pol® 981) were kindly supplied by Lubrizol Europe. 2-Mer-captonicotinic acid (2-MNA) was purchased from ABCR GmbH & Co KG, Germany. All other chemicals were purchased from Sigma Aldrich Austria. METHODS Synthesis AC1030-cysteine was synthesized by covalent attachment of L-cysteine via amide bond formation mediated by a water- soluble carbodiimide (EDAC). Purified thiolated AC1030 was obtained after dialysis and lyophilization. Preactivation was achieved by disulphide bond formation between the polymeric thiol groups and 2-MNA according to a previously described method. Dialyses was performed for purification and the preactivated thiomer was lyophilized (Figure 1). Mucoadhesion Adhesion to native small intestinal mucosa of the novel conjugates was evaluated by tensile studies as described by our research group (4). Emulsification capacity Medium chain triglycerides were incorporated into aqueous polymer solutions. Stability of the resulting formulations (30 % oil, 70 % water and 0.5 % polymer) was determined by centrifugation at 273 g (Table 2). RESULTS AND DISCUSSION Synthesis A schematic description of the synthesized polymers is provided in Figure 1. cooh coor. H2 1 H2 -c —c—c h -c- H2 c h r, h H2 -hC- H2 h H2 -hc—c - c -c—c i=o cooh COOH H2 i h2 h H2 t —c —C—c -c—c -C— ■ ■ H H -C COOH H2 I H2 H H2 —HC—C -HC —C -C—C — O OH -HC- CO hN Figure 1: Presumptive structure of thiolated (A) and S-protected (B) AC1030 A B S hn COOH hs cooh n n p / Mucoadhesion As illustrated in Figure 2, S-protection provides full reactivity as premature inter-and intramolecular disulphide bond formation within the polymer is prevented, which allows covalent binding to mucus proteins. REFERENCES 1. Gum JRJ, Hicks JW, Toribara NW, Rothe EM, Lagace RE, Kim YS. The human MUC2 intestinal mucin has cysteine-rich subdomains located both upstream and downstream of its central repetitive region. The Journal of Biological Chemistry. 1992;267:21375-83. 2. Bernkop-Schnürch A, Schwarz V, Steininger S. Polymers with Thiol Groups: A New Generation of Mucoadhesive Polymers? Pharm Res. 1999;16(6):876-81. 3. Iqbal J, Shahnaz G, Dünnhaupt S, Müller C, Hintzen F, Bernkop-Schnürch A. Preactivated thiomers as mucoadhesive polymers for drug delivery. Biomaterials. 2012;33(5):1528-35. 4. Dünnhaupt S, Barthelmes J, Rahmat D, Leithner K, Thurner CC, Friedl H, et al. S-Protected Thiolated Chitosan for Oral Delivery of Hydrophilic Macromolecules: Evaluation of Permeation Enhancing and Efflux Pump Inhibitory Properties. Molecular Pharmaceutics. 2012;9(5):1331-41. 5. Boehmer MR, Koopal LK. Association and Adsorption of Nonionic Flexible Chain Surfactants. Langmuir. 1990;6:1478-84. Figure 2: Mucoadhesive properties of AC1030 polymers determined via tensile studies on porcine native small intestinal mucosa Emulsion stability Due to their lipophilic residues, all polymers were able to form stable emulsions with oils. Reduced stability of emulsions formed with the preactivated thiomer might be a result of the steric hindrance of the substituent 2-MNA (5). Tab. 2: Emulsion stability determined via centrifugation at 273 g Time stable (min) NA - carbomer 0 AC1030 > 30 AC1030-cysteine > 30 Preactivated AC1030 10 CONCLUSIONS The lipophilic C10-C30 alkyl chains provide the potential for using these polymers with emulsifying properties in semisolid delivery systems. The outstanding mucoadhe-sive properties, due to thiolation and S-protection make this novel thiomer a valuable excipient for creams exhibiting a prolonged residence time on mucosal tissues, such as vaginal, ocular or nasal mucosa. FUNCTIONALITY RELATED CHARACTERISTICS OF HYPROMELLOSE TYPE 2208 USED AS MATRIX FORMER IN PROLONGED-RELEASE TABLETS T. Stanic Ljubin1*, M. Horvat1, B. Jankovic1, P. Jurkovic1 1 Lek Pharmaceuticals, d.d., Sandoz Development Center Slovenia, Verovskova 57, 1526 Ljubljana, Slovenia INTRODUCTION Hypromellose is a water-soluble polymer derived from cellulose used as the controlled-release agent in hydro-philic matrix systems. Polymer hydration and swelling play an important role in controlling the rate of drug diffusion from hydrophilic matrices (1,2). Ph. Eur 7.0 recognizes following functionality related characteristic (FRC) of hypromellose: • degree of substitution (hydroxypropyl (HP) and me-thoxy (MC) content), • molecular weight distribution, • viscosity, and • particle size distribution. Hypromellose producers suggest following physico-chemical properties to be critical material attributes (CMA) that influence the dissolution rate: • degree of substitution (hydroxypropyl (HP) content), • viscosity, • particle size - pass through sieve 230 mesh (63 microns) (3,4). The purpose of this work was to evaluate the influence of hypromellose physico-chemical properties (HP content, viscosity, particle size, specific surface area, and moisture content) on dissolution rate. MATERIALS AND METHODS Dissolution results for production scale batches of prolonged-release hydrophilic matrix tablets containing low dose of poorly soluble drug substance and hypromellose 2208 (20% w/w) were used to evaluate the influence of hypromellose physico-chemical properties on dissolution rate. Dissolution in 900 mL phosphate buffer pH 6.8, Apparatus 1, Ph. Eur., 100 rpm, performed within batch release testing, was utilized for evaluation (percent of drug substance dissolved at 6th and 10th hour). Hypromellose properties (HP content, viscosity and pass through sieve 230 mesh) were provided by hypromellose producer and also obtained by in-house analyses. Additional parameters, moisture content (determined by loss on drying measurement (LOD), specific surface area (SSA), and particle size distribution (PSD) measured by laser light scattering method (d01), were obtained by in-house analyses. The influence of hypromellose parameters on dissolution results was assessed by computer software Modde 9.1 and SIMCA-P+ 12.0. RESULTS AND DISCUSSION Average dissolution results for 168 production scale batches and physico-chemical properties of seven batches of hypromellose are presented in tab. 1. Tab. 1: Material attributes of hypromellose batches and dissolution results of tablets batches produced Viscosity (mPas) HP content (%) % thru 230 mesh SSA (m2/g) d01 (^m) LOD (%) Average dissolved at 6th h (%) Average dissolved at 10th h (%) 21362 8.2 63 0.78 23 2.1 32.9 51.8 17596 9.6 63 1.09 21 2.0 36.8 57.1 21091 9.4 67 0.76 23 2.3 35.6 55.6 23035 9.7 62 1.08 23 1.4 36.2 56.7 19836 9.2 65 0.9 23 2.2 35.2 56.0 16683 9.2 64 0.65 25 1.6 38.0 59.3 21632 10.0 57 0.54 25 1.5 39.2 59.9 Data presented in tab. 1 were analysed by MLR (multiple linear regression) method by computer software Modde 9.1. Results are presented by fig. 1 and fig. 2. Scaled & Centered Coefficients fot disso 6 Scaled & Centefed Coefficients for disso 10 if-l€B 3C'£ ME-1,533 H-1ÏB R2-a4324 KSE-2.37S fi?-164 Conf OF-16Î 136 Cant. lav.-0.95 Fig. 1: Coefficient plot - graphical presentation of the influence of viscosity (vis), hydroxypropyl content (HP) and particle size - pass thru sieve 230 mesh (sieve) on dissolution results obtained at 6th and 10th hour Seated & Centefed Coefficients for disso 6 Scaled & Centered Coefficients for dis» 10 Fig. 2: Coefficient plot - graphical presentation of the influence of viscosity (vis), hydroxypropyl content (HP) and loss on drying (LOD) on dissolution results obtained at 6th and 10th hour It was observed that hydroxypropyl (HP) content has the most significant influence on dissolution rate. Also, other two phisico-chemical properties suggested by hyprom-ellose producers - viscosity and pass through sieve 230 mesh were confirmed to affect the dissolution rate (fig 1.). The influence of d01 determined by laser light scattering method and SSA was also assessed. d01 was confirmed to be appropriate parameter for quantifying of PSD, equivalent to pass through sieve 230 mesh, while SSA was demonstrated not to be as good as d01 and sieve analysis. Therefore, the introduction of other methods than sieve analysis for quality control testing of PSD of hypromellose was not identified as beneficial. Besides these parameters, also LOD was identified as potential CMA of hypromellose (fig. 2). However, it was observed that LOD is correlated to sieve analysis results and it was not investigated whether moisture content determined by other methods also shows an impact on dissolution rate. Presented analysis has several limitations. MLR model, obtained from data presented in tab. 1, accounts only for ~30% of the observed variability of dissolution results. Primarily, the selected material attributes and their analyses are correlated and their impacts on dissolution are aliased. Moreover, additional factors, such as drug substance properties and tablets manufacturing process parameters also impact the dissolution results. Therefore, we additionally conducted a hierarchical PLS analysis of all available incoming materials and process data. The improved MVDA model was found to explain ~50% of observed dissolution variability. It confirmed quantitatively that all three material attributes suggested by hypromellose producers (HP content, viscosity, and pass through sieve 230 mesh) significantly impact the dissolution rate. Additionally, the moisture content of hypromellose (measured as LOD) was also indicated to be highly important material attribute in this case. CONCLUSIONS Following physico-chemical properties of hypromellose were identified as critical material attributes that influence the dissolution rate of poorly soluble drug substance from matrix tablets containing 20% w/w of hypromellose: • HP content, • viscosity, • particle size distribution, and • loss on drying. REFERENCES 1. Alderman, D.A., 1984. A review of cellulose ethers in hydrophilic matrices for oral controlled-release dosage forms. Int. J. Pharm. Tech. Prod. Manuf. 5, 1-9. 2. Bowstra, J.A., Junginger, H.E., 1993. Hydrogel. In: Swarbrick, J., Boylan, J.C. (Eds.), Encyclopedia of Pharmaceutical Technology, vol. 7. Dekker, New York, pp. 441-465. 3. Cabelka, T. et all, Application of Quality by Design (QbD) principles to the formulation of a hydrophilic matrix tablet of a high dose/high solubility drug. AAPS annual meeting and exposition, Los Angeles, CA 2009. 4. Deng H. et all, Application of Quality by Design (QbD) principles to the formulation of extended release, propranolol hydrochloride hydrophilic matrix tablets. AAPS annual meeting and exposition, New Orleans 2010. THE PHARMACOLOGICAL ACTIVITY OF COMPOSITES OF BETULIN ESTERS WITH ARABINOGALACTAN T.P. Shakhtshneider12, M.A. Mikhailenko1, S.A. Kuznetsova34, Yu.N. Malyar3, A.S. Zamai3, V.V. Boldyrev12, E.V. Boldyreva12* 1 Institute of Solid State Chemistry and Mechanochemistry SB RAS, Kutateladze 18, Novosibirsk, 630128 Russia 2 Novosibirsk State University, Pirogov str., 2, Novosibirsk, 630090 Russia 3 Institute of Chemistry and Chemical Technology SB RAS, Krasnoyarsk, Russia 4 Siberian Federal University, Krasnoyarsk, Russia INTRODUCTION Betulin and its esters extracted from the birch bark exhibit antioxidant, hypolipidemic, hepatoprotective, and other forms of pharmacological activity. The main drawback for their internal and external application in pharmacology is the poor water-solubility of the preparations. The mechanical activation of drugs with the polymers is a well-known efficient method for preparing powder systems which are characterized by an increased solubility and dissolution rate. Earlier the mechanocomposites of betulin with water-soluble polymers, polyvinylpyrrolidone and polyethylene glycol were prepared, which have showed a higher solubility of betulin and improved gastroprotective properties [1]. The purpose of this paper was to prepare the composites of betulin esters, diacetate and dipropionate, with water-soluble biopolymer arabinogalactan, in order to see, if an even better form can be suggested. MATERIALS AND METHODS Materials Betulin diacetate (BDA) and betulin dipropionate (BDP) (Fig. 1) as well as arabinogalactan (AG) were obtained by the originally developed methods [2-4]. Methods The mechanocomposites were prepared by mechano-chemical treatment of the mixtures of betulin esters with arabinogalactan in the SPEX 8000 ball mill. The composites of betulin diacetate and betulin dipropionate were also obtained by dissolution of the mechanocomposite or the mixtures of the initial components in water and subsequent solvent evaporation. The physicochemical properties of the composites were studied by gel permeation chromatography, X-ray diffraction, infrared spectroscopy, electron and atomic force microscopy methods. Release of the biologically active substances from the composites was studied using dissolution tester 705 DS (Varian). Antitumor activity of BDA, BDP, AG and their composites against Ehrlich ascites carcinoma cells and lung cancer cells was determined in vitro by estimating the fraction of apoptotic and necrotic cells 24 h after application of the compounds in Hanks solution by fluorescence method. Fig. 1: The molecular structures of betulin (R1=OH, R2=H, R3=CH2OH), betulin diacetate (R1=OAc, R2=H, R3=CH2OAc), and betulin dipropionate (R1=OPr, R2=H R3=CH2OPr). RESULTS AND DISCUSSION Under mechanical treatment of the mixtures of BDA and BDP with AG, the practically amorphous products were obtained. It was found that in the case of the mechanocomposites, the solubility of betulin derivatives increased as compared to the initial substances. It was by IR spectroscopy method detected that there was no hydrogen bonding in the composites of BDA and BDR with AG. Nevertheless, the formation of the complexes of BDA and BDP with AG in the ball-milling mixtures and in the solutions was suggested due to restoration of molecular weight of the mechanically activated polymer and accord- R R Survival of AEC cells, % AG BDP Mechanical composite of BDP - AG (1:9, w/w) Control Necrosis 6.6±0.8 3.9±0.6 5.4±0.7 7.3±0.5 Apoptosis 16.7±1.8 25.0±3.1 41.0±5.4 3.1±2.8 Survival BDA-AG BDA-AG of lung BDA (1:9, w/w) (1:9, w/w) Control cancer physical composite cells, % mixture as a film Necrosis 0.4±0.04 0.5±0.04 0.4±0.04 0.3±0.05 Apoptosis 28.3±0.7 27.7±2.1 82.3±3.9 2.1±0.08 ing to the fact that the esters were not extracted by hex-ane from the aqueous solutions. The obtained composites were non-toxic and exhibited antitumor properties against the Ehrlich ascites carcinoma cells. The data on antitumor activity of BDA-AG mech-anocomposite against EAC cells can be found in [5] and for BDP-AG mechanocomposite, they are presented in the Table 1. Tab. 1: Effect of mechanical composites of BDP with AG and starting components on elimination level of Ehrlich ascites carcinoma cells. After dissolution of the mechanocomposites or the mixtures of betulin esters with AG in water and subsequent solvent evaporation, the composites were obtained as the thin amorphous films. From the films, the complexes of BDA and BDP with AG were totally water-soluble that made the films very attractive for pharmacological applications. As an example, the data on antitumor activity of the BDA-AG films against lung cancer cells are presented in the Table 2. Tab. 2: Effect of the composite of BDA with AG as a film and starting components on elimination level of lung cancer cells. CONCLUSIONS Using mechanochemical methods, the composites of betulin diacetate and betulin dipropionate with water-soluble polysacharide arabinogalactan were obtained. They showed higher dissolution rate and solubility in comparison with the initial substances. The possible reasons of increasing the solubility are disordering of crystal structures of biologically active substances and formation of molecular complexes with the polymer. It has been shown in in vitro studies that mechanocomposites of betulin esters with arabinogalactan as well as the composites prepared as the thin water-soluble films exhibited higher antitumor activity in comparison with the initial substances. ACKNOWLEDGMENTS The work was partly supported by the RFBR grant No. 14-03-31900 and the program of the Russian Academy of Sciences "Fundamental Sciences for Medicine". REFERENCES 1. Mikhailenko MA, Shakhtshneider TP, Brezgunova ME, Kuznetsova SA, Boldyrev VV, The composition of betulin with biocompatible polymers and the method of its preparation, R.F. Patent 2401118, October 10, 2010. 2. Kuznetsova SA, Kuznetsov BN, Red'kina ES, Sokolenko VA, Skvortsova GP, The method of obtaining betulin diacetate, R.F. Patent 2324700, May 20, 2008. 3. Kuznetsova SA, Kuznetsov BN, Mikhailov AG, Skvortsova GP, The method of obtaining arabinogalactan, R.F. Patent 2273646, April 6, 2006. 4. Kuznetsova SA, Skvortsova GP, Malyar YuN, Vasil'eva NYu, Kuznetsov BN, The method of obtaining betulin dipropionate, R.F. Patent 2469043, December 10, 2012. 5. Shakhtshneider TP, Kuznetsova SA, Mikhailenko MA, Zamai AS, Malyar YuN, Zamai TN, Boldyrev VV, Effect of mechanochemical treatment on physicochemical and antitumor properties of betulin diacetate mixtures with arabinogalactan. Chem. Nat. Comp. 2013; 49: 470-474. INVESTIGATION OF DISSOLUTION MECHANISMS BY IMAGING METHODS K. Puncochova 1 3*, J. Beranek 3, A. Dumicic 3, A. Ewing 2, s. G. Kazarian 2, F. Stepanek 1 1 Institute of Chemical Technology Prague, Technicka 3, Prague, Czech Republic 2 Imperial College London, South Kensington Campus, London, Great Britain 3 Zentiva, k.s., U Kabelovny 130, Prague 10, Czech Republic INTRODUCTION The dissolution testing of pharmaceutical formulations is basic methodology for determination of drug release. The design of tablets or capsules plays important role to drug delivery. Basic dissolution test provides information about concentration of active pharmaceutical ingredience (API) as a function of time. The result is a rate of drug release but there is limited information about the mechanism of release.1 Moreover, polymer dissolution significantly influences the release of drug. The understanding of polymer dissolution mechanism and related to drug dissolution process is useful to predict a behavior of poorly soluble drugs during dissolution and choosing of a candidate formulation, polymers or additives.2 Therefore, the purpose of the present work is to investigate the physical and chemical processes of dissolution in a more detailed way, focusing on water penetration, polymer swelling, drug dissolution and precipitation in order to explain the mechanism of drug release from the dosage form, especially the release of poorly soluble drugs from solid dispersions. A several analytical approaches allow investigating the physical and chemical processes of dissolution, including FT-IR imaging, MRI, UV imaging and Raman spectros-copy. MATERIALS AND METHODS The mechanisms of dissolution were observed using imaging methods including ATR-FTIR imaging, Magnetic Resonance Imaging (MRI), and UV imaging in a flow cell using poorly soluble Aprepitant, Valsartan and polymers PVP and Soluplus. The dissolution mechanism of two types of formulations, solid dispersion prepared by spray drying and physical mixture, were studied. RESULTS AND DISCUSSION In spite of the different principles of the imaging methods used, consistent dissolution mechanism of pure API, physical mixtures and solid dispersions was confirmed. PVP had a fast dissolution rate, similar to the rate of water penetration. However, the dissolution of PVP significantly slowed down in combination with a poorly soluble Apre-pitant. Moreover, precipitation of API was recognized by ATR-FTIR imaging and can be explained by fast dissolution of PVP, high concentration of water and local supersaturation of API (see in Figure 1). Presence of Aprepitant also decreases the dissolution rate of Soluplus in solid dispersion but Soluplus creates the wide gel layer which helps to keep Aprepitant dissolved and inhibits the precipitation. MRI showed a good wetability of tablet related to high wetting properties of PVP. Different mechanisms of water penetration were confirmed in the case of physical mixture and solid dispersion, which is illustrated in Figure 2. The physical mixture, as the polymer dissolved and tablet has disintegrated. On the other hand, tablet made from a solid dispersion was wetted and a gel layer as well as a dry core were created. UV-Imaging allowed observing of release the API from the dosage form. a) p. L W (t.* I I rz- - : ------- I 1 1 b) .■•'Hi'*- Aprepitan) Fig. 1: ATR-FTIR Images of dissolution process of solid dispersions with combination a) Aprepitant and PVP with precipitation and b) Aprepitant and Soluplus. 45 min 65 min 83 min 100 min 120 min b) 25 min 35 min 45 min 55 min p s Fig. 2: Penetration of water to the tablet of Aprepitant and Soluplus a) solid dispersion and b) physical mixture. CONCLUSIONS The imaging methods were useful to characterize the physical and chemical processes of dissolution. The interactions API-polymer in solid dispersion decreased the dissolution rate of polymer in contrast with enhancing of dissolution rate of Aprepitant. The concentration gradient of polymer which was determined in gel layer caused by water penetration and dissolution of polymer improved the release of poorly soluble drugs by increasing the supersaturated concentration of drug and by inhibiting precipitation. REFERENCES 1. Dressman, J. F.; Amidon, G. L.; Reppas, Ch.; Shah, V. P. Dissolution testing as a Prognostic Tool for Oral Drug Absorption: Immediate Release Dosage Forms. Pharmaceutical Research 1998, 15 (1), 11-21. 2. Miller-Chou, B. A.; Koenig, J. L. A review of polymer dissolution. Progress in Polymer Science 2003, 28, 1223-1270. IMPORTANCE OF ABSORPTIVE ENVIRONMENT SIMULATION IN DETECTION OF EXCIPIENT-MEDIATED PRECIPITATION INHIBITION OF SIROLIMUS M. Petrusevska1*, M. Homar2, D. Kocjan2, L. Peternel2 1 Institute of pharmacology & toxicology, 50 Divizija 6, Skopje, R. Macedonia 2 Sandoz Development Centre Slovenia, Verovskova 57, Ljubljana, Slovenia INTRODUCTION The rate limiting step of BCS class II drug absorption is either the drug solubility or the dissolution rate in the lumen of the gastrointestinal tract (GIT). A variety of formulation approaches have been developed to tackle this challenge (1). The goal of these approaches is to assure an adequate fraction of dissolved drug in the GI lumen and consequently a satisfactory flux across the intestinal epithelia. However, when the drug is released in the GIT from these formulations drug precipitation can occur, subsequently leading to a decreased fraction of the dissolved drug and a decreased flux across the intestinal epithelia (1, 2). Herein, we present the results of the sirolimus excipient mediated precipitation inhibition in presence of hydroxy-propyl methyl cellulose (HPMC). Additional spectroscopic and permeability studies implied that HPMC inhibits the precipitation of sirolimus out of supersaturated solutions. Finally, in the proof-of-concept human pharmacokinetic study we developed formulation with HPMC that showed a significantly higher AUC and Cmax in comparison to the reference Rapamune®. J?' c MATERIALS AND METHODS Materials Sirolimus was supplied by Biocon (India) and Rapamune® was supplied by Wyeth Medica (Ireland). The tested excipients as precipitation inhibitors are given in Tab. 1. SIF™ Powder (Phares AG, Switzerland) was used for the preparation of fasted/fed state simulated intestinal fluid (Fa/FeSSIF) buffer. Tab. 1. Tested precipitation inhibitors Surfactants: Tween® 80, Tween® 20, Cremophor® RH40, Cremophor® EL, Lutrol® F68, Lutrol® F127 (Poloxamer 407) (BASF, Germany), Gellucire® 44/14, Labrasol® (Gattefosse, Germany), Texapon ®K15, (Cognis, The Netherlands). Polymers: Pharmacoat® 606, Pharmacoat® 603 (HPMC), (Harke, Germany), Klucel EF® (Hercules Inc, Germany), Kollidon® K30, Kollidon® K25, Kollidon ® VA 64, Kollidon® K17 (BASF, Germany). Other: Elvanol ® (DuPont ™), D-Mannitol, Poly(ethylene glycol) Mn 6000, Propylene glycol, Poly(ethylene glycol) Mn 400, (Sigma-Aldrich, Belgium), Kollicoat® IR (BASF, Germany), a - cyclodextrin, (Merck, Germany). RESULTS AND DISCUSSION The screening campaign revealed that some excipients effectively inhibited sirolimus precipitation. Among them, HPMC and Poloxamer 407 were identified as 'hit' precipitation inhibitors and were further evaluated in a physiologically more relevant dissolution media, namely Fa/ FeSSIF (Fig. 1). A. In vitro high-throughput (HT) precipitation inhibitor screening method Solutions containing precipitation inhibitors were prepared by a HT Freedom EVO liquid handler, followed by addition of stock DMSO solution of sirolimus in Mcllvaine buffer pH 6.8, incubated at 37°C followed by sampling at pre-determined time points. Samples were analyzed for the amount of drug remaining in the solution by UPLC method (3). Confirmation of precipitation inhibitors in absorptive environment The identified precipitation inhibitors (HPMC and Poloxamer 407) were tested in FaSSIF and FeSSIF. In addition their impact on increased flux across intestinal epithelia was investigated using PAMPA assay. Investigation of developed interactions between HPMC and sirolimus were characterized using solid state characterization techniques (IR and DSC). A randomized, double blind study under fasting conditions was conducted with 12 healthy adult volunteers. The subjects were given a reference product Rapamune® and the test formulation with a 14 day wash-out period. Samples were taken at predetermined time points and analyzed by a HPLC/MS and automated extraction for the determination of sirolimus in human whole blood (4). Fig. 1: Impact of "hit" excipients HPMC 603 (0.1%) and Lutrol F127 (Poloxamer 407) (0.1%) on sirolimus precipitation in (A). FeSSIF and (B). FeSSIF incubation media. The results are shown as mean ± SEM (n = 4). These results demonstrated that sirolimus supersaturation can be created and maintained to a certain degree in the absence of precipitation inhibitors due to the solubiliz-ing components in Fa/FeSSIF. The solubilizing components of Fa/FeSSIF inhibited the sirolimus precipitation to the extent that the addition of Poloxamer 407 did not cause any additional effects of sirolimus precipitation inhibition. In PAMPA experiment, HPMC significantly increased the sirolimus flux across the lipid bilayer (ANOVA, p<0.01), what is most probably related to the HPMC mediated increase of sirolimus concentrations in the donor compartment (Fig. 2). DSC and IR spectroscopic studies confirmed that certain interactions between sirolimus and HPMC are indeed developed. These interactions most likely prevent sirolimus to precipitate out of solution resulting in increased absorption of sirolimus across intestinal epithelia. Fig.2 The impact HPMC (0.1%) and Poloxamer 407 (0.1%) on the flux of sirolimus in the parallel artificial membrane permeability assay (PAMPA). The results are shown as linear regression lines (R2>0.8243, p<0.05) obtained from the concentration-time profiles (n=4). In vitro sirolimus precipitation data are in line with the outcome of the human PK study (Tab. 2). Test formulation contained HPMC and larger sirolimus particle size in comparison to Rapamune, which in contrast to test formulation did not contain precipitation inhibitor. Tab.2: Sirolimus PK parameters after oral administration of Rapamune® and test formulation to fasted human volunteers. Results are shown as mean (n=11) ± S.D. Cmax (Pg/mU AUC (pg x h/ mL) Rapamune 7655 ± 1896 51089 ± 15754 Test formulation 12616 ± 2867 64793 ± 12878 CONCLUSION Precipitation inhibition can play a significant role in determining the amount of dissolved drug available for absorption and consequently in determining the AUC and Cmax values of investigated drug. REFERENCES 1. Brouwers J, Brewster ME, Augustijns P. Supersaturating drug delivery systems: the answer to solubility-limited oral bioavailability? J. Pharm. Sci. 2009; 98: 2549-2572 2. Bevernage J, Brouwers J, Brewster ME, Augustijns P. Evaluation of gastrointestinal drug supersaturation and precipitation: strategies and issues. Int. J. Pharm. 2013; 453: 25-35 3. Petrusevska M, Urleb U, Peternel L. Evaluation of a high-throughput screening method for the detection of the excipient-mediated precipitation inhibition of poorly soluble drugs. Assay Drug Dev. Technol. 2013; 11: 117-129 4. Petrusevska M Petrusevska M, Homar M, Petek B, Resman A, Kocjan D, Urleb U, Peternel L. Hydroxypropyl methylcellulose mediated precipitation inhibition of sirolimus: from a screening campaign to a proof-of-concept human study. Mol. Pharm. 2013; 10: 2299-2310. DEVELOPMENT OF PHYSIOLOGICALLY RELEVANT DISSOLUTION METHODS FOR EVALUATION OF HYDROPHILIC MATRIX TABLETS USING NEW DISSOLUTION APPARATUS U. Klancar1*, B. Markun1, I. Legen1 1 Lek Pharmaceuticals d.d., Sandoz Development Center Slovenia, Verovskova 57, SI-1526 Ljubljana, Slovenia INTRODUCTION Depending on predominant drug release mechanism, hy-drophilic matrix tablets have different drug release and mechanical susceptibility (1, 2). Consequently, the drug release rate may vary after tablet exposure to variable gastrointestinal conditions in vivo or dissolution apparatus conditions in vitro. Considering in vivo gastrointestinal conditions the in vitro tests should be designed. However, appropriate models simulating stress conditions in vivo are difficult to develop by only using conventional dissolution Apparatus. Some modifications of conventional and novel dissolution methods have been suggested, including the use of the USP dissolution Apparatus 3 (reciprocating cylinder, Bio-dis) (3, 4). In one of our previous studies, USP 3 Apparatus in combination with plastic beads was used to mimic mechanical stress on HPMC matrices. It was concluded that incorporation of beads was crucial in discriminating the tablets in vitro and in establishing a good correlation with in vivo data (2). The aim of the present study was to verify the applicability of newly developed dissolution model on three different additional cases. Obtained results were compared with in vivo results and IVIVC was suggested. MATERIALS AND METHODS Materials Three different matrix tablet formulations for analysing with novel dissolution method were developed in Lek d.d. Basic formulations characteristics are described in Tab. 1. Reference samples were obtained from wholesale supplier. Methods Dissolution testing was performed simultaneously for test and reference product. The dissolution testing model consists of USP 3 Apparatus (Varian Vankel Bio-dis III, US) and 8 mm plastic beads of density about 1.1 g/cm3. Testing was performed in 250 ml of water medium at 37°C ± 0.5°C. Plastic beads to fill approximately % of the vessels were placed into reciprocating cylinders. Then, the tablets were added and cylinders were attached to the Bio-dis. The stainless steel mesh on both sides of the cylinders was 2 mm in size. The DPM rate program was set at 20 DPM with short 10 min interval at 1.5 hours set to 40 DPM. The sampling time points were selected depending on the formulation studied. Withdrawn samples were analysed with HPLC. RESULTS AND DISCUSSION Dissolution results for three different test formulations compared to reference products analysed with beads dissolution method are presented in Fig 2. Fig. 1: Novel dissolution testing device incorporating plastic beads into USP3 dissolution Apparatus (Bio-Diss, Varian, US). Tablet moves reciprocally with plastic beads which exert additional mechanical stress on matrices. Fig. 2: Dissolution results for three case studies with SD values (N=3). Test product T (dotted lines), reference product R (solid lines). Results show that in Case 1, test formulation is slightly more susceptible to mechanical stress compared to the reference formulation. Dissolution rate was accelerated after 10 min stress manipulation at 40 DPM initiated at 1.5 h. This acceleration was not evident when the tablets p s were analysed using conventional methods. Dissolution profiles for Case 2 and Case 3 show good robustness and overall similarity between test and reference formulations. All studied examples were evaluated in bioequiva-lence study and results were correlated with in vitro data. In first two cases we found IVIVC Level C correlation and in Case 3 the correlation was Level A (Tab.1). Tab. 1: Description of formulations and API with corresponding level of IVIVC after comparing dissolution results to in vivo results obtained from bioequivalence studies. Example Matrix type API solubility Correlation Case 1 Eudragit, HPMC pH dependant Level C Case 2 HPC good Level C Case 3 HPMC low Level A Examination of the profiles also indicates that drug release mechanism differs between cases and that is most linear and erosion controlled in Case 3. CONCLUSIONS New, beads dissolution testing method was introduced in the evaluation of three different matrix formulations. Obtained results were in good agreement with in vivo data which indicates the methods bio relevance and usefulness for analysing different hydrophilic matrix tablets. REFERENCES 1. Wang Z, Shmeis RA. Dissolution controlled drug delivery systems. In: Li X, Jasti BR. Design of Controlled Release Drug Delivery Systems. United States: McGraw-Hill; 2006. p. 139-172. 2. Klančar U, Markun B, Legen I, Baumgartner S. A novel beads-based dissolution method for the in vitro evaluation of extended release HPMC matrix tablets and the correlation with the in vivo data. AAPS Journal. 2013; 15(1): 267-277. 3. Kostewicz ES et al. In vitro models for the prediction of in vivo performance of oral dosage forms. Eur. J. Pharm. Sci. 2013; http://dx.doi.org/10.1016/j.ejps.2013.08.024. 4. Fotaki N, Aivaliotis A, Butler J, Dressman JB, Fischbach M, Hempenstall J, Klein S, Reppas S. A comparative study of different release apparatus in generating in Vitro - in Vivo correlations for extended release formulations. Eur. J. Pharm. Biopharm. 2009; 73: 115-120. EVALUATION OF CATIONIC MICROSPHERES WITH MELATONIN USING EYE-RELATED BIOAVAILABILITY PREDICTION MODELS Marieta Duvnjak Romic1*, Marina Juretic2#, Biserka Cetina-Cizmek1, Ivan Pepic2, Anita Hafner2, Jasmina Lovric2, Jelena Filipovic-Grcic2 1 Pliva Croatia Ltd., Prilaz B. Filipovica 25, 10000 Zagreb, Croatia 2 Department of Pharmaceutics, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacica 1, 10000 Zagreb, Croatia * These authors contributed equally to this work INTRODUCTION The conventional dosage forms suffer from poor ocular bioavailability due to fast precorneal elimination owing to lachrymation, tear turnover, nasolachrymal drainage, metabolic degradation and nonproductive adsorption/ absorption, and the relative impermeability of the corneal epithelial membrane. Therefore, current efforts are directed towards the development of innovative drug delivery systems and concomitant development of in vitro models to enable the prediction of eye-related bioavailability (1). The aim of this study was to investigate the potential of chi-tosan/Poloxamer 407 microspheres as carriers for topical ocular delivery of melatonin by evaluating its eye-related bioavailability using cell-based epithelial cornea model. Melatonin, methoxyindole secreted by the pineal gland, has pleiotropic bioactivities among which is its ability to modulate intraocular pressure. Chitosan is a biocompatible and biodegradable polyca-tionic polymer. At pH below 6.5 it is positively charged and electrostatically interacts with negatively charged epithelial surface resulting in mucoadhesion. Moreover, it can act as a permeation enhancer increasing paracel-lular drug permeation. Poloxamer 407, polyoxyethylated nonionic surfactant, has been proposed to increase drug permeability through corneal epithelial cell membranes. J?' c MATERIALS AND METHODS Low-viscosity chitosan (C) and melatonin (M) were purchased from Sigma-Aldrich and Poloxamer 407 (P) from BASF. Microspheres were prepared by spray-drying (Bu-chi 190 mini spray dryer) of formulations prepared by mixing M/P ethanol solution with chitosan acetic acid solution (1 %, w/v). Drying conditions were as follows: compressed air flow rate of 700 Nlh-1, spray flow rate of 2.59 mL/min, inlet air temperature of 145°C. Microspheres were evaluated in terms of size (Olympus BH-2 microscope), zeta-potential (Zetasizer 3000 HS), drug loading and in vitro release (Franz diffusion cell). MTT assay was performed to assess the biocompatibility of microspheres with corneal epithelial cells (HCE-T cell line, Rikken). The cell-based epithelial cornea model was cultivated on Transwell® polycarbonate filter inserts coated with type I rat tail collagen and fibronectin. The HCE-T cells suspended in the culture medium were seeded onto the filter and cultivated submerged for seven days, after which they were exposed to the air-liquid interface during the following three days (2). The permeability experiment was performed directly in the Transwell® using Hank's Balanced Salt Solution buffer (HBSS pH 6.3). For the determination of tight junction opening and cell viability transepithelial electrical resistance (TEER) was measured during the experiment. The quantitative determination of melatonin was performed by HPLC (Agilent 1100). RESULTS AND DISCUSSION Melatonin-loaded C/P microspheres (MCP) are characterized by size and surface charge suitable for ocular delivery (Table 1) offering longer precorneal retention. In vitro melatonin release and permeability from MCP are shown in Fig. 1. Table 1. The composition and the main characteristics of melatonin loaded chitosan/P407 microspheres. Micro-sphere CC (g/l) M/C (w/w) C/P (w/w) Size (mm) Z (mV) MCP 8 1:2 5:1 2.23±0.04 29.6±3.0 800n 1-1.6 700- -1.4 ) ■ 600- -1.2 u 500- -1.0 A T 160 pg/mg lipid) and the lowest by DL (Tab. 1). Regarding the determined degree of liposome membrane elasticity (E), DL and PGL-10 were calculated to have an almost 5-fold higher elasticity than the CL. The assessment of the penetration ability of various liposomes containing DCS was performed using two PVPA barriers: a simple PVPAc consisting of phospholipid vesicles made of egg phosphatidylcholine and cholesterol and more skin-like PVPAs prepared with phospholipid vesicles composed of lipids normally found in stratum corneum. To validate the permeability experiments, the barrier integrity was tested by measuring the electrical resistance across the barriers throughout the study. Fig. 1 shows the slight improvement in the DCS permeation profiles using DL-A and PGL-10-A on PVPAc as compared to the CL-A. However, when experiments were performed on the PVPAs the overall permeation and the difference between formulations were higher. All of the liposomal formulations exhibited significantly higher permeabilities than the DCS aqueous solutions, most probably due to the penetration-enhancing effect of the phospholipids. The highest Papp of DCS was attained with PGL-10-A, followed by DL-A, and the lowest values were obtained by CL-A. These results are consistent with the high elasticity of PGL-10-A and DL-A. Our findings demonstrate the increased permeation of hydro-philic drug by using elastic vesicles. Tab. 1: The physicochemical properties of different types of liposomes containing DCS. Formulation code Mean diameter (nm) Zeta potential (mv) Entrapment efficiency DCS/lipid (Mg/mg) Degree of liposome elasticity (E) CL-A 161±3 - 58,8 ± 0,4 130,7 ± 3,5 1,08 ± 0,11 CL-B 147±4 - 58,6 ± 0,8 88,7 ± 1,6 DL-A 153±1 - 57,3 ± 0,3 61,7 ± 2,5 5,59 ±0,23 DL-B 153±1 - 67,8 ± 0,6 49,0 ± 1,8 PGL-10-A 156±4 - 69,3 ± 1,4 137,9 ± 6,6 5,21 ±0,31 PGL-10-B CD - 70,0 ± 0,8 93,4 ± 3,8 PGL-30-A 166±1 - 67,9 ± 0,6 160,5 ± 7,9 3,69 ±0,17 PGL-30-B 157±4 - 60,7± 0,9 99,3 ± 4,7 Fig. 1: The permeability of DCS from the liposomal formulations on PVPAc and PVPAs barriers. The values denote the mean ± S.D. (n=3). CONCLUSIONS The permeation of the hydrophilic drug from the liposomes was affected by their physicochemical properties, which were influenced by the lipid composition and the presence of the edge activator or penetration enhancer. This study demonstrates the potential of the newly developed skin-PVPA for the screening and optimization of liposomes at the early preformulation stage. REFERENCES 1. El Maghraby GM, Williams AC, Barry BW. (2006). Can drug-bearing liposomes penetrate intact skin? J Pharm Pharmacol 58:415-29. 2. Engesland A, Skar M, Hansen T, et al. (2013). New applications of phospholipid vesicle-based permeation assay: permeation model mimicking skin barrier. J Pharm Sci 102:1588-600. 3. Vanic Z, Hurler J, Ferderber K, et al. (2014). Novel vaginal drug delivery system: deformable propylene glycol liposomes-in-hydrogel. J Liposome Res 24:27-36. A STUDY ON THE APPLICABILITY OF LINE MEASUREMENT IN THE MONITORING OF THE PELLET COATING PROCESS G. Hudovornik1, K. Korasa1*, F. Vrečer1 1 Krka, d.d., Novo mesto, Šmarješka cesta 6, 8501 Novo mesto, Slovenia INTRODUCTION In recent years, regulatory authorities have demanded a better understanding of products and processes by requesting an "enhanced approach" using Quality by Design (QbD) in new registration applications. One of the ways to achieve this is to implement new technologies, such as the process analytical technology (PAT) (1). A part of PAT are new measurement tools, such as in-line process analysers, which enable collection of data in real time (2). In our study we tested the applicability of two in-line measurement probes, a probe for the determination of moisture by near infrared (NIR) diffuse reflectance spectroscopy (3) and a spatial filtering technique (SFT) probe for the evaluation of particle size (4). MATERIALS AND METHODS The probes were evaluated during the process of applying polymer film coating onto drug layered pellets. We manufactured five pilot scale batches in a fluid bed coater (Aeromatic FielderTM MP3/2/4, GEA Pharma Systems). Compositions of coating dispersions for the first three batches were the same (Eudragit® RS 30 D (Evonik Industries, Germany), Eudragit® RL 30 D (Evonik Industries, Germany), talc (Imerys Talc, Italy), triethyl citrate (Vertellus Performance Materials, USA), purified water)), the composition of the coating dispersion of the fourth batch was changed quantitatively and the composition of the fifth batch was changed qualitatively (Eudragit® L 30 D (Evonik Industries, Germany), talc, triethyl citrate, purified water) compared to the first three batches. NIR spectra were collected by Lighthouse ProbeTM (GEA Pharma Systems) and afterwards treated by standard normal variate transformation (SNV) and 2nd Savitzky Golay derivative. The partial least square (PLS) regression was used to predict Karl Fischer (KF) (V30, Mettler Toledo) and loss on drying 85°C/20 min (LOD) (Mettler Toledo, HR73 Halogen Moisture Analyzer) values from pre-treated NIR data. Samples for off-line determination were collected every 10 minutes during the coating of the pellets. A multivariate data analysis was performed with Unscrambler® X 10.2 (CAMO Software).We used the results of the first two batches (19 samples) to calibrate and validate (cross-validation) the PLS model. This model was later used to predict KF and LOD values from NIR data of the last two batches. SFT measurements were made by Parsum IPP70 probe (Malvern Instruments). In-line particle size measurements were compared to the sieve analysis (Air Jet Sieve 200LS-N, Hosokawa Alpine) and static image analysis (Morphologi G3, Malvern Instruments) results. The sieve analysis was made using 250, 500, 710, 1000, and 1250 pm mesh size sieves. Samples for off-line particle size determination were taken every 20-30 min. Samples of the first four batches were used for sieve analysis and static image analysis (26 samples). Samples of the last batch were used to determine the degree of agglomeration (9 samples). RESULTS AND DISCUSSION The NIR data of the first two batches showed high correlation with both off-line methods after PLS regression was made (Tab. 1). The third batch was not used for NIR moisture determination due to attrition of the pellets during the process. els in the range of the measured LOD and KF values of the fifth batch (2.0-5.5%). The predicted values with these models matched well with the measured values. The relative errors were 7.0% and 6.2% for LOD and KF predictions, respectively. The medians of volume distribution (x50) obtained by the SFT probe were lower than x50 values measured by static image analysis. However, a high correlation between the x50 values of both methods was found. The R2 and slope values after linear regression were 0.9933 and 1.044, respectively. When comparing the SFT particle size distribution with the sieve analysis, we could notice that the SFT values were lower and the particle size distribution was wider. However, both methods responded similarly to particle size changes during the process (Fig. 1) Fig. 1: Percentage of particles bigger than 1000 ym for samples of the first, second and fourth batch (n=17). Due to attrition of pellets during the third batch this parameter is not suitable for the particle size evaluation of this batch, thus it is not present on this chart. Tab. 1: R2 values of calibration and validation after PLS regression; we used 2 factors for calculation. Measured off-line values were between 2.5-10.0%. Method R2 calibration R2 validation Karl Fischer 0.9812 0.9640 Loss on drying 0.9855 0.9756 We used above PLS models to predict off-line values (KF, LOD) from NIR data of the fourth and fifth batch. The predicted values of the fourth batch (quantitative composition variation) were not comparable to the measured ones. High deviations were noticed for both off-line methods. At first, the predicted values of the fifth batch (qualitative composition variation) did not correlate well with the measured values. Afterwards, we calibrated both PLS mod- The SFT in-line particle size parameters did not detect agglomeration of particles and were not consistent with the number of the agglomerates determined by off-line counting. CONCLUSIONS The NIR probe correlated well with the traditional methods for off-line moisture determination and it showed a potential to predict moisture content in the pellets with slightly different formulation. The SFT probe proved to be a very useful tool for the inline pellet size measurements. ACKNOWLEDGEMENT Authors would like to thank Krka, d. d., Novo mesto for providing support for the study. REFERENCES 1. Pharmaceutical CGMPs for the 21st Century - A Risk-Based Approach, Final report. Department of Health and Human Services, U.S. Food and Drug Administration, September 2004. 2. Guidance for Industry, PAT - A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance. U.S. Department of Health and Human Services, Food and Drug Administration, September 2004. 3. Bogomolov A, Engler M, Melichar M, Wigmore, A. In-line Analysis of a Fluid Bed Pellet Coating Process Using a Combination of Near Infrared and Raman Spectroscopy. J. Chemometrics 2010; 24 (7-8); 544-557. 4. RoBteuscher-Carl K, Fricke S, Hacker M C, Schulz-Siegmund M. In-line Monitoring of Particle Size in a Fluid Bed Granulator: Investigations Concerning Positioning and Configuration of the Sensor. Int. J. Pharm 2014; 466; 31-37. P S CRITICAL ATTRIBUTES OF NANOFIBERS: PHYSICAL PROPERTIES, DRUG LOADING AND TISSUE REGENERATION J. Kristl1*, P. Kocbek1, S. Baumgartner1, R. Rošic1, J. Pelipenko1, B. Jankovic1, Š. Zupančič1 1 University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia INTRODUCTION In regenerative medicine, therapies generally focus on specific tissue (skin, bone, heart, vessels, ligament, ten-dors) and their cellular behavior due to repair degenerative changes and other damages, occured by ageing of population (1). Skin tissue engineering strategies focus on epidermal, dermal or composite tissue generation or regeneration. In the case of chronic wound is imbalance between synthesis and degradation of natural extracellular matrix (ECM), which offers support for cell growth. In the field of development of modern wound dressings, which would temporary replace natural ECM and offer support for cell growth, nanofibers have gained a lot of attention (2). To construct in vivo like microenviro- nment with nanofabricated structures that mimic biological entities and cell-specific functions, it is necessary to benefit nano-technological approaches. Among available methods is electrospinning a new method in pharmaceutical technology and the most promising tool for generating nanopatterned scaffolds. The objective of this study was to identify skin specific nanostructures, followed by development of electrospin-ning parameters for production of polyvinyl alcohol (PVA) nanofibers, variated in alignment and thickness. Finally, the research was focused on skin cells response grown on nanofibers in order to discover the crucial properties for clinical use. J?-c MATERIALS AND METHODS PVA (Mowiol® 20-98, Mw = 125.000 g/mol) nanofibers empty or loaded with API (antibiotic, resveratrol, blood-derived growth factors for dermal wound healing) were prepared by electrospinning (Fig.1) with static or rotating collector. Morphological (diameter, roughnes) and mechanical properties of nanofibers were determined by SEM and AFM. Cells (keratinocytes and fibroblasts) were seeded on tested support (randomly and aligned nano-fibers or glass coverslip as a control). Based on images the average nanofiber diameter, average interfibrillar pore size and cell size were determined by measuring 40 randomly selected objects using ImageJ 1.44p software (NIH, USA). The speed of cell adhesion was determined by counting of unattached cells, proliferation by MTS assay. The effect of nanofibrillar support on cell morphology was studied as cell shape index, which was calculated on the basis of cell images taken with confocal fluorescent microscope. RESULTS AND DISCUSSION Skin ECM consists collagen reach fibers with diameters 30-130 nm (1), responsible for modulating cell proliferation, migra-tion, differentiation as well as providing mechanical support and a network that enable nutrient transport and diffusion. Scaffolds could emulate native tissue pro-perties, such as ECM architecture, promote cell attachment and clinically assist in the healing and regeneration process. Elecrospinning parameters (Table 1): polymer concentration, relative humidity, applied voltage and collector type and distance to the needle are decisive combination for production of planned nanofibers (more 3-5). Tab. 1: Electrospinning parameters used for production of PVA nanofibers, corresponding diameters and SEM images Sample PVA conc % Tip to collector distance [cm] Applied voltage [kV] Relative humidity [%] Nanofiber diameter [nm] a 8 15 15 60 70 b 10 160 c 33 305 d 4 665 e 15 2 1115 Drug loading depends on physicochemical similarity of nanofiber polymer and API, from dissolution to dry product, and results in molecularly distribution (Fig 1A, B) or crystallization API (Fig. 1C) (more 6, 7). Cell responses on nanofibrillar surface Grown surface influence cell adhesion (Fig. 3A). Random or aligned nanofibers modulated spreading shape and cytoskeletal organization of investigated cells (Fig.2B). The morphology of the same cells differs on various structured scaffolds (Fig.3). Randomly oriented nanofibers limit cell mobility comparing to aligned due interfibrillar penetration of soft part of the cells (more in 8-10). SOLUTION PARAMETERS PROCESS AND AMBIENT PARAMETERS NANOFtBERS Fig. 1: A scheme illustrating the basic principles of the electrospinning process. An electric field is produced between a needle and a collector. The polymer solution is ejected from the needle towards the collector under the influence of the electric field. SEM images of (A) random, (B) aligned, and (C) loaded nanofiers are also shown. I □ Control OfVA-NF "I □ PVA-lilm £ 70 -E 1 ■a ^ » iL B TT 1 o°i « • Control Randomly oriented NF \ ! „ l—_ Ü p s Rccungular oriented NF Aligned NF Fig. 2: Effects of PVA nanofibrillar support on cell growth. A - effect of nanofibrillar substrate on the keratinocytes' adhesion rate, B - effect of nanofiber alignment on keratinocytes' morphology. Fig. 3: Cell shape index on different supports (elongated cells have values close to zero, circular cells closer to 1). ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support provided by the Slovenian Research Agency for Programme P1-0189, Projects J1-4236 and J1-6746, and Grant Numbers 1000 - 09 - 310085, 1000 - 11 -310213. REFERENCES 1. Kim, H.N., et a. Nanotopography-guided tissue engineering and regenerative medicine. Adv Drug Del Rev 65, 2013.536. 2. Engel, E, et al. Nanotechnology in regenerative medicine: the materials side. Trends in Biotechnology, 26, 2008. 39-47. 3. Rosic, R, et al., The role of rheology of polymer solutions in predicting nanofiber formation by electrospinning. Eur Polym J. 48, 2012, 1374-1384. 4. Rosic, R, et al., Physical characteristics of poly (vinyl alcohol) solutions in relation to electrospun nanofiber formation. Eur Polym J 49, 2013, 290-298. 5. Pelipenko, J., et al., The impact of relative humidity during electrospinning on the morphology and mechanical properties of nanofibers. Int. J. Pharm. 456, 2013, 125-134. 6. Rosic, R., et al., Nanofibers and their biomedical use. Acta Pharm. 63, 2013, 295-304, 7. Bertoncelj, V., et al., Development and bioevaluation of nanofibers with blood-derived growth factors for dermal wound healing. Eur. J. Pharm. Biopharm. 2014. doi: 10.1016/j.ejpb.2014.06.001 8. Pelipenko, J. et al., The topography of electrospun nanofibers and its impact on the growth and mobility of keratinocytes. Eur. J. Pharm. Biopharm. 84, 2013, 401-411. 9. Jankovic, B., et al., The design trend in tissue-engineering scaffolds based on nanomechanical properties of individual electrospun nanofibers. Int. J. Pharm. 455, 2013, 338-347. 10. Pelipenko, J., et. al., Nanofiber diameter as a critical parameter affecting skin cell response. Eur. J. Pharm. Sci in Press CONCLUSIONS These results especially highlight a) importance of combination of electrospi-nning parameters for nanofiber production, b) nanofiber alignment are more affected on cells as their size, c) the adhesion and morphology of the skin cells are a consequence of topographical properties of nanofibers, d) cell response is cell line specific. To manage numerous different parameters that affect the production of nanofibres with electrospinning the computer simulation is recommended. IN VITRO STUDY OF PERMEABILITY OF NANONIZED MELOXICAM FROM DIFFERENT NASAL FORMULATIONS BY SIDE-BI-SIDE™ HORIZONTAL CELL MODEL P. Szabö-Revesz1*, T. Horvath1, Cs. Bartos12, R. Ambrus1 1 Department of Pharmaceutical Technology, University of Szeged, Eötvös u. 6, H-6720-Szeged, Hungary 2 Richter Gedeon Ltd., Gyömröi ut 19-21, H-1103 Budapest, Hungary INTRODUCTION Nasal dosage forms of drugs have gained importance in recent years because of (i) the rapid onset of action, (ii) circumvention of the first-pass elimination by the liver and GI tract, (iii) non-invasive and simple daily administration (1). In this work, the Side-Bi-SideTM (Crown Glass, USA) horizontal cell model was applied as a novel approach for an in vitro study of the permeability of nanonized meloxi-cam (MEL) from nasal formulations (2-4). The aim was to optimize the horizontal cell method, and investigate and compare the diffusion of the drug from different nasal formulations (gel, spray and powder). MATERIALS AND METHODS Materials MEL was obtained from EGIS Ltd. (Budapest, Hungary). The grinding additive, polyvinylpyrrolidone (PVP) K25, was purchased from BASF (Ludwigshafen, Germany). Sodium hyaluronate (HA, Mw = 1400 kDa) was obtained from Gedeon Richter Ltd. (Budapest, Hungary). Preparation of MEL nanoparticles and the nasal formulations An aqueous suspension of untreated MEL (rawMEL) (D0.5 = 58.4 pm) was used for the preliminary examinations. During the comparative studies, a co-ground product of MEL and PVP (nanoMEL/PVP) and their physical mixture (MEL/PVP mix) were formulated and investigated. MEL and PVP in a ratio of 1:1 were mixed and ground in a planetary monomill (Fritsch GmbH, Idar-Oberstein, Germany), which resulted in amorphous nanoparticles of MEL (D0.5 = 140±69 nm) (4). To prepare a nasal gel, nanoMEL/PVP and MEL/PVP mix were dispersed in a gel of HA (1:5 mg/ml). Liquid forms containing nanoMEL/PVP and MEL/PVP mix were applied as nasal sprays with HA (1:1 mg/ml). For the dry powders, the co-ground product (nanoMEL/PVP) and the physical mixture (MEL/PVP mix) were used. Preliminary examinations The model comprised compartments, membranes and magnetic fixing (Fig.1). Gel and liquid forms were added by pipette. Dry powder was washed directly into the donor compartment. Samples were removed with a syringe needle. Acceptor compartment Membrane Donor compartment Fig. 1: Side-Bi-SideTM horizontal diffusion system. Comparative permeability studies The investigations were carried out at 37 °C. Each compartment had a volume of 3.0 ml. The pH of the donor compartment was 5.6, and that of the acceptor compartment was 7.4. An artificial membrane impregnated with isopropyl myristate was used between the donor and acceptor compartments (pore size: 0.45 pm). The samples in both compartments were stirred with a magnetic stirrer. Sampling was carried out at 5, 10, 15 and 60 minutes. The quantity of diffused MEL was determined spectro-photometrically at 364 nm. RESULTS AND DISCUSSION Rheological parameters such as the viscosity of the nasal gel and spray forms may influence the rate of diffusion of the MEL. The gel form containing 0.5% HA exhibited a higher viscosity than that of the spray form containing 0.1% HA (Fig. 2). Under shear stress, the structure of the gel form underwent a smaller change than that of the spray form, where the viscosity was 10 times lower than in the starting state. 1.000 » 0,100 c_ & 8 5 0,010 o.oo t H,..,............ M! 1 20 40 60 80 Shear rare (1;f,) 100 120 -MELfPVPlH MEL/PVPih : 1.1 n -1:6 I ix nix gel jaiPUPl i«B.ÏFVP;l 1'1 ïpray - I S gel Since the Side-Bi-Side cell model was not suitable for the investigation of the nasal gel, the Franz vertical cell model (Hanson Research Company, USA) was used in the comparative studies. The results showed that the Franz cell method ensures a contact between the gel and the vertical membrane which results in well- controlled diffusion (Fig. 4). Fig. 2: Viscosity curves of HA-containing nasal gel and nasal spray. For each formulation, the diffusion of the MEL from nan-onized samples was more significant than that from the physical mixture. As concerns the diffusion from the three formulations in the case of the co-ground product, the greatest amount of drug diffused from the nasal powder formulation, followed by the nasal spray and then the gel form (Fig. 3). Fig. 4: Comparative diffusion studies with the Franz cell and Side-Bi-Side methods. CONCLUSIONS It was concluded that the Side-Bi-Side horizontal diffusion system is suitable for permeability studies of formulations for nasal delivery. The small compartment volume (3 ml) well simulates the nasal conditions. Primarily nasal sprays and powders can be investigated with the discussed method. Validation of the Side-Bi-Side method is in progress. The system may be suitable for ex vivo permeability studies, to investigate diffusion through mucous membranes prepared from humans and animals. The presentation is supported by the European Union and co-funded by the European Social Fund. Project number: TÂM0P-4.2.2.A-11/1K0NV-2012-0047 REFERENCES 1. Illum L, Nasal drug delivery: new developments and strategies. Drug Discovery Today 2002; 7: 1184-1189. 2. Arora A, Sharma S, Garg S, Permeability issues in nasal drug delivery. Drug Discovery Today 2002; 18: 967-975. 3. Kürti L, Veszelka S, Bocsik A, Khue Dung NT et al. The effect of sucrose esters on a culture model of the nasal barrier. Toxicol. In vitro 2012; 26: 445-454. 4. Kürti L, Gàspàr R, Màrki Â, Kâpolna E et al. In vitro and in vivo characterization of meloxicam nanoparticles design for nasal administration. Eur. J. Pharm. Sci. 2013; 50: 86-92. Fig. 3: Measurements of diffusion of MEL from different nasal formulations. THE DEVELOPMENT AND CHARACTERIZATION OF THIOLATED POLYACRYLIC ACID-POLYALLYL AMINE NANOPARTICLES J. Grießinger1'*, S. Dünnhaupt1, I. Nardin1 and A. Bernkop-Schnürch2 1 ThioMatrix GmbH, Research Center Innsbruck, Trientlgasse 65, 6020 Innsbruck, Austria 2 Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria INTRODUCTION For oral drug administration nanoparticulate delivery systems offer the advantage of providing a prolonged residence time in the small intestine leading to a comparatively higher drug uptake. In order to further improve the intestinal residence time of nanoparticles (NPs) their diffusion into the mucus gel layer followed by their immobilization in the mucus close to the absorption membrane would be advantageous. To render NPs more slippery in the mucus, on the one hand, a high density of positive and negative charges with a neutral net charge on their surface seems beneficial (1), as it is known from viruses exhibiting such surface properties to permeate almost unhindered through mucus (2). On the other hand, a fixation in deeper mucus regions should be feasible by the introduction of thiol groups on the surface of NPs forming comparatively more rapidly disulfide bonds with the mucus close to the absorption membrane where the pH is around 7 then in the more luminal mucus with a pH around 5.5. In order to design such systems, it was the aim of this study to prepare and characterize NPs with a high charge density and thiolation on their surface by combining an-ionic and cationic (thiolated) polymers with each other. MATERIALS AND METHODS Materials Polyacrylic acid (1.8 kDa; PAA), poly(allylamine hydrochloride) (58 kDa, PAH), L-cysteine hydrochloride (Cys), thioglycolic acid (TGA) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimode (EDAC), fluorescein isothiocyanate (FITC), minimal essential medium (MEM), TritonTM X-100 and resazurin were obtained from Sigma-Aldrich, Germany. All other chemicals used in this study were of analytical grade. Methods PAA-cysteine conjugates (PAA-Cys) were synthesized by the covalent attachment of cysteine to PAA in presence of EDAC at pH 4.5. PAH conjugation with thioglycolic acid (TGA) was arranged at pH 5.0 in presence of EDAC. Eight different NPs were prepared and investigated by ionic gelation via complexation of PAA and PAH (Tab. 1). Resazurin assay was performed on Caco-2 cells to determine the in-vitro cytotoxicity of these NPs. For mucus diffusion studies FITC was coupled to PAH and florescence labelled NPs were prepared at pH 7.3. Particle size, polydis-perisity index and zeta potential of NPs were determined using a particle sizer (Nicom PSS 388, USA). Diffusion studies were performed in natural porcine intestinal mucus at pH 5.5 and pH 6.8 across a transwell system over 240 min (3). Tab. 1: Combination of the eight different NPs First polymer solution Added polymer solution PAA PAH PAA PAH-TGA PAA-Cys PAH PAA-Cys PAH-TGA PAH PAA PAH PAA-Cys PAH-TGA PAA PAH-TGA PAA-Cys RESULTS AND DISCUSSION Unmodifed and thiolated nanoparticles were prepared via ionic gelation due to electrostatic interaction between the negatively charged PAA and positively charged PAH. FITC labelled NPs showed a narrow particle size distribution and a slightly negative zeta potential (Tab. 2). Compared with NPs without FITC the particle size increased when more PAH solution was used. FITC was covalently coupled to PAA and PAA-Cys by an oxidative disulfide coupling. Therefore less free amino groups were available for ionic gelation between the two polymers. The zeta potential of the NPs was found between 0 and -1 mV. This indicated a neutral charged surface. Tab. 2: Particle size, polydispersity index and zeta potential of unmodified and modified PAA-PAH-FITC NPs (MD ± SD; n = 3) particle size [nm] polydispersity index [P.I.] Zeta potential [mV] PAA + PAH-FITC 133.2 ± 55.6 0.17 -0.698 ± 0.32 PAA-Cys + PAH-FITC 214.4 ± 74.4 0.12 -0.446 ± 0.30 PAA-Cys + PAH-TGA-FITC 250.5 ± 85.2 0.12 -0.265 ± 0.23 The resazurin-assay after 3 h of incubation showed that the order of the NP preparation between PAA and PAH had an effect on the cell viability. The cytotoxic character of PAH could be eliminated when the PAH solution was added to a propound PAA solution (Fig. 1). Fig. 1: Cytotoxicity potential of the eight NPs (0.5% m/V) in comparison to MEM solution; (MD ± SD; n = 3) The diffusion study through mucus (Fig. 2) showed that thiolated NPs still can permeate the mucus but to a lower extend than unthiolated NPs. NPs prepared of thiolated PAA and thiolated PAH (PAA-Cys+PAH-TGA-FITC) showed a 40% decreased diffusion rate compared to unthiolated NPs. This demonstrates that thiolated NPs have higher mucoadhesive properties due to their capability of forming disulphide bonds with the mucus. pH dependent diffusion behaviour of thiolated NPs, however, could not be demonstrated. Fig. 2: Diffusion rate of PAA-Cys+PAH-FITC NPs and PAA-Cys+PAH-TGA-FITC NPs after 240 min across mucus (pH 5.5 (white bars) and pH 6.8 (black bars) within transwell system; (MD ± SD; n = 3) CONCLUSIONS Within this study a successful preparation of nanoparti-cles with a high positive and negative charge density and thiolation on their surface was demonstrated. The diffusion rates of thiolated NPs and unmodified NPs indicated an improved mucoadhesion of thiolated NPs due to their ability to form disulfide bonds with the mucus. Furthermore, the cytotoxic character of PAH could be eliminated by the preparation order of the NPs. Both the high charge density and the higher mucoadhesive properties suggest thiolated PAA-PAH NPs as a drug delivery system to target various mucosal surfaces. ACKNOWLEDGEMENTS We acknowledge EC for supporting this research through the FP7-2011-NMP-280761 "ALEXANDER" project. REFERENCES 1. Lai SK, Wang YY, Hanes J. Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv Drug Deli Rev. 2009; 61(2): 158-171 2. Olmsted SS, Padgett JL, Yudin AI, Whaley KJ, Moench TR, et al. Diffusion of macromolecules and virus-like particles in human cervical mucus. Biophysical J. 2001; 81: 1930 - 1937 3. Friedl H, Dunnhaut S, Hintzen F. Waldner C. Parikh S et al. Development and evaluation of a novel mucus diffusion test system approved by self-nanoemulsifying drug delivery systems. J. Pharm. Sci 2013; 102: 4406-4413 SURFACE PLASMON RESONANCE AND CONTACT ANGLE MEASUREMENTS FOR DRUG-EXCIPIENT INTERACTION STUDIES L. Peltonen1*, P. Liu1, T. Viitala2, A. Kartal-Hodzic2, H. Liang2, T. Laaksonen2, J. Hirvonen1 1 Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, P.O. Box 56, 00014 University of Helsinki, Finland 2 Division of Pharmaceutical Biosciences, Faculty of Pharmacy, P.O. Box 56, 00014 University of Helsinki, Finland INTRODUCTION Poor solubility is one of the main problems faced in drug delivery and nanocrystallization techniques are widely utilized to improve the solubility properties. Nanocrystals are solid drug particles covered by a stabilizer layer and the size of them is typically from 100 to 500 nm. The most problematic step in production of nanocrystals is the selection of appropriate stabilizer for a certain drug. Some studies are published on that subject but the selection of drug-stabilizer combination is still made on trial and error base (1). The aim of this study was to reach an insight into the interaction mechanisms involved in nanocrystallization between the drug and stabilizer. Accordingly, the affinity of polymers on solid drug particles was measured by surface plasmon resonance (SPR) and contact angle techniques. Indomethacin was used as a model drug and the affinity of five different, but structurally closely related, po-loxamers on indomethacin surface were measured. The corresponding nanocrystals were produced by nanomill-ing technique. MATERIALS AND METHODS Materials Indomethacin was used as a model drug (Hawkins, USA), and Pluronic® F68 and 17R4, Tetronic® 908 and 1107 (Basf, Germany) and Pluronic® L64 (Aldrich, USA) as stabilizers. The water used was ultrapurified Millipore® water (Millipore, France). Ball milling Nanocrystals were produced by wet-ball milling technique (Pulverisette 7 Premium, Fritsch Co., Germany) with 1 mm zirconium oxide pearls. The drug:stabilizer ratio used in this study was 1:0.4. Contact angle The contact angle was measured with the sessile drop method (CAM 200, Attension Biolin Scientific Oy, Finland). Indomethacin powder compact was compressed to a disc with a hydraulic press (Specac, UK) and the contact angle of aqueous stabilizer solutions on compressed powder surface was measured. Surface plasmon resonance, SPR For SPR measurements indomethacin was deposited on the golden SPR sensor surface. The SPR sensograms were obtained by measuring the change in the SPR angle during the injection of a concentration series of aqueous stabilizer solutions over the indomethacin coated sensor surface. Particle size Mean particle size and polydispersity index, PI, of the nanocrystalline suspensions were measured by dynamic light scattering, DLS, technique (Malvern Zetasizer 3000 HS, Malvern Instrument, UK). RESULTS AND DISCUSSION Contact angle When contact angle was measured, the highest contact angle was found with pure water and the contact angle was decreased when any of the stabilizers was added to the solution. Small contact angle between the drug and the stabilizer means higher affinity/wettability. In this study the affinity of the stabilizers was in the following order: L64> 17R4> T1107«F68 «T908. SPR Binding efficiency was calculated from the SPR results. Binding efficiency can be used to describe the interaction strength. The molecular weight of the stabilizer is taken into account when calculating the binding efficiency, stabilizer size/nm PI 17R4 > 3 pm 1 L64 370 0.30 F68 290 0.24 T1107 310 0.26 T908 290 0.22 which enables the comparison of the interaction strength between the drug and different stabilizers independently of the complexity of the interaction mechanisms. Higher binding efficiency means stronger interaction. In this study the binding efficiency was lowered in the order L64 > 17R4 > F68 « T908 « T1107. When binding efficiency was compared to the contact angle results, they agreed very well. Nanocrystallization In nanocrystallization studies good nanocrystals were formed with all the other stabilizers except for 17R4 (Table 1). With 17R4 formed nanocrystals tend to aggregate into bigger clusters. Tab. 1: Size information of nanocrystals produced with different stabilizers. CONCLUSIONS SPR and contact angle measurements were successfully utilized in the interaction studies between drug and stabilizer. For efficient nanocrystal stabilizer high attachment forces and full coverage of the particle surfaces as well as suitable hydrophobic/hydrophilic chain length balance are important requirements, and the interaction measurements alone can't explain the stabilizing efficiency. REFERENCES 1. Peltonen L, Hirvonen J, Pharmaceutical nanocrystals by nanomilling : critical process parameters, particle fracturing and stabilization methods. J. Pharm. Pharmacol. 2010; 62: 15691579. 2. Liu P, Viitala T, kartal-Hodzic A, Liang H, Laaksonen T, Hirvonen J, Peltonen L, Interactions studies between indomethacin nanocrystals and PEO/PPO copolymer stabilizers. Submitted manuscript. Discussion The strongest binding strength was found with L64 and it formed nanosized nanocrystals though the polydispersity was higher. The shorter hydrophilic PEO chains were less efficient in stabilizing the nanocrystals though the binding efficiency was high. 17R4 had second strongest affinity, but its unfavourable telechelic structure with shorter PEO chains lead to larger nanocrystal clusters. F68, T908 and T1107 had lowest binding strengths due to the steric factors caused by the long PEO blocks, which decreased the adsorption of polymers. For efficient stabilization with non-ionic polymers the polymer needs to attach firmly to the nanocrystal surface, fully cover the particles and offer polymer chains long enough for steric stabilization (2). The efficiency can not be explained only by the interaction studies. PREPARATION, CHARACTERIZATION AND EVALUATION OF LIPOSOMAL GELS INCORPORATING TERBINAFINE HYDROCHLORIDE. C. Koutsoulas1*, N. Pippa2, C. Demetzos2, A. Wagner3, E. suleiman3, M. Zabka1 1 Faculty of Pharmacy, Department of Galenic Pharmacy. Comenius University in Bratislava. Odbojärov 10, 832 32 Bratislava, Slovakia 2 Faculty of Pharmacy, Department of Pharmaceutical Technology, University of Athens, Panepistimioupolis, Zografou, Athens 15771, Greece 3 Polymun Scientific, Immunbiologische Forschung GmbH Donaustraße 99 3400 Klosterneuburg, Austria INTRODUCTION Terbinafine hydrochloride (TBH) is an effective, well tolerated broad spectrum antifungal agent, which belongs to the group of allylamines. TBH inhibits specifically squa-lene epoxide activity, thus inhibits ergosterol production, a major component of the fungal cell wall (1). From physi-cochemical point of view, TBH is sparingly soluble in water making it necessary to use solubilization techniques in order to incorporate it in an aqueous dosage form. Liposomes and other vesicles have already been used in order to overcome this water-insolubility problem (2,3). Moreover, liposomes are claimed to achieve improved transdermal permeation compared to conventional dosage forms(4). Their composition is similar to this of the skin, so they are delivering an API in a more physiological way, reducing thus possible hypersensitivity reactions (5). The aim of the present study is to prepare, characterize and evaluate a suitable liposomal dosage form intended for topical application, incorporating TBH. MATERIALS AND METHODS Materials EggPC of >60% purity was purchased from Sigma Al-drich (Germany), EggPC of 99% purity was purchased from Avanti Polar Lipids Inc. (Albaster, AL, USA), POPC of GMP grade was purchased from Lipoid (Germany) and cholesterol was purchased from Dishman (Netherlands). Chloroform and 2-propanol, were purchased from Centralchem s.r.o. (Slovakia). Dialysis membranes used for purification of liposomes and for in vitro drug release study were purchased from Spectrum Laboratories, Inc. (Netherlands). Centrifugal ultrafiltration tubes (centrisart) were purchased from Sartorius (Germany). All reagents used were of analytical grade. Methods Liposomes were prepared by thin film hydration method and down-sized in an Avanti MiniExtruder® (Albaster, AL, USA) through 200 nm polycarbonate membrane. Un-entrapped TBH was removed by dialysis using regenerated cellulose membrane (12000-14000 MWCO) against the same buffer as used for the preparation of liposomes. Size, pdi and physical stability of liposomal populations over time were measured by PCS. TBH retention in liposomes was studied using centrifugal ultrafiltration tubes to separate liposomes from free drug. Hydrogels were prepared by mixing Carbopol 934 or Hydroxyethylcellu-lose with water at different concentrations and liposomal gels were prepared by simple mixing of gels and liposomes to a final TBH concentration of 0.01%. Viscosity of the gels was measured in an oscillatory rheometer at fixed shear rates and in vitro drug release from liposomal gels was studied in vertical Franz diffusion cell system through dialysis membrane (12000-14000 MWCO). RESULTS AND DISCUSSION Fig. 1: Release profiles of terbinafine hydrochloride from HEC 3% liposomal gels. Fig. 2: Release profiles of terbinafine hydrochloride from Carbopol 934 liposomal gels. Tab. 1: Composition and incorporation efficiency of liposome samples prepared. Batch name Lipid composition [mol : mol]* Incorporation efficiency ± sd [%] E1 EggPC (99%) 88.64 ± 4.41 E2 Egg PC (99°%): Chol (92.5 : 7.5) 85.81 ± 0.95 E3 Egg PC (99°%): Chol (85 : 15) 87.55 ± 3.44 E4 EggPC (>60°%) 61.78±3.65 P1 POPC 82.42 ± 0.09 P2 POPC:Chol (92.5 : 7.5) 80.85 ± 2.74 P3 POPC:Chol (85 : 15) 79.77 ± 0.22 P S origin of the phospholipid used to formulate a liposomal gel (natural/synthetic) seems to have minor importance with regard to drug release, physical stability and integrity of a liposomal formulation. Similarly, storage conditions during the studied period (21 days) do not seem to have a significant effect on the stability of liposomal formulations. Moreover, choice and concentration of the gelling agent used to formulate a liposomal gel seem to affect markedly the release behavior and the release kinetics of the final liposomal formulation. REFERENCES 1. N. S. Ryder, Br. J. Dermatol. 126, 2 (1992). 2. Koutsoulas C, Pippa N, Demetzos C, Zabka M, J Nanosci Nanotechnol. 2014 Jun;14(6):4529-33. 3. Koutsoulas C, Suleiman E, Wagner A, Zabka M. J Liposome Res. 2014 Apr 28. [Epub ahead of printI 4. Tanrverdi S.T., Ozer O. (2013). Novel topical formulations of Terbinafine-HCl for treatment of onychomycosis. Eur J Pharm Sci, 48:628-636. 5. J.-P. Zhang, Y.-H. Wei, Y. Zho, Y.-Q. Li, and X.-A. Wu, Arch. Pharm. Res. 35, 109 (2012). CONCLUSIONS EggPC of two different purity grades (>60% and 99%) and POPC of synthetic origin (GMP grade) were used to prepare liposomes with or without cholesterol incorporating TBH. Moreover, effect of pH, drug to lipid ratio, lipid concentration, choice of gelling agent and gel viscosity were examined in order to optimize the final formulation. Physical stability and drug retention in liposomes were investigated over time at two different storage temperatures (2-8°C and RT) and drug release from liposomal gels was studied in vertical Franz diffusion cell system. From the obtained results it is concluded that purity of the phos-pholipid used to formulate a liposomal gel incorporating TBH has a major impact so on in vitro drug release profile from liposomal gels as on the physical stability of the liposomal formulation over time. On the other hand, the NANOSCALE ANALYSIS OF SUBERIZED CELLULOSIC FILMS INTENDED FOR PHARMACEUTICAL MOISTURE BARRIER APPLICATIONS J. Heinämäki1*, a. Halenius2, M. Paavo1, U. Paaver1, s. alakurtti3, P. Pitkänen3, M. Pirttimaa3, K. Kogermann1, P. Veski1, J. Yliruusi2 1 University of Tartu, Faculty of Medicine, Department of Pharmacy, Nooruse 1, 50411 Tartu, Estonia 2 University of Helsinki, Division of Pharmaceutical Chemistry and Technology, P.O. Box 56, FI-00014 University of Helsinki, Helsinki, Finland 3 VTT Technical Research Centre Finland, P.O. Box 1000, FI-02044 VTT, Espoo, Finland INTRODUCTION Suberin is a biopolyester found in the cell walls of external tissues of plants where it plays a fundamental role as a protective barrier between the organism and its environment (1). Suberin polymer can be hydrolyzed by base treatment and fractioned to fatty acids (FA), which are potential raw materials in many industries (1,2). It is evident that the application of suberin FA as such, or combined with polymers could lead to new manufacturing opportunities for a wide range of pharmaceutical and biomedical systems intended for human and/or veterinary medicine applications. The objective of the present study was twofold: (a) to investigate film formation and surface morphology of suberin FA loaded aqueous cellulosic film and (b) to evaluate the water vapor barrier properties of the present suberized films. MATERIALS AND METHODS Suberin FA (Batch Pilot 1/14.01.2013 VTT, Espoo, Finland) was used as such in free films. Chemical composition of suberin FA mixture was similar as reported in literature (3). Hydroxypropyl methyl-cellulose, HPMC (Methocel E5, Dow Chemicals, USA) was used as a cellulosic film forming polymer. Polyethylene glycol (PEG 400) was used as a plasticizer. For preparing polymer solution, HPMC was first dispersed in ethanol at a ratio of 5-8 parts of solvent to 1 part of HPMC. Cold water was then added to produce the final weight (0-8% of HPMC w/w). The solution was mixed until HPMC completely dissolved. This solution was gently heated on a water bath up to 40°C. Suberin FA was added by gently mixing into a warm solution. For plasticized films, PEG 400 was added to the polymer solution(s) (20% w/w of the solid polymer mixture weight). Free films were prepared in polytetrafluoroethylene (Teflon®) molds by a pouring method. The thickness of each film sample was measured at least at five different points by a micrometer. The final concentration levels of suberin FA in the HPMC free films were selected as 2.5%, 5%, 10% and 15% (w/w) from the HPMC weight. Surface topography and morphology of free films were investigated with a high-resolution scanning electron microscope, SEM (Zeiss EVO® 15 MA, Germany) and atomic force microscope, AFM (Autoprobe CP, Thermomicroscopes, USA). The water vapor permeability (WVP) of the films was determined as described by Jia et al. (4) with some modification. The films were cut into a suitable size, fixed onto the mouth of anhydrous calcium chloride containing glass vials and immediately tightly sealed with a thin elastic band and Parafilm® M barrier film. The exposed surface area was 113.1 mm2. The glass vials were held at 23 ± 2°C and 85% RH, and the increase in weight was measured at regular intervals after 1,2,3,4,5 and 7 days of storage. RESULTS AND DISCUSSION The dry thickness of the films ranged from 140 mm to 260 mm. Film formation was dependent on the presence or absence of an external plasticizer and the concentration of suberin FA (Figure 1). The inclusion of suberin FA in the HPMC films resulted in a clear improvement in the moisture barrier of films compared to that obtained with the reference non-suber-ized HPMC films. The WVP of the films decreased with increasing suberin FA consentration in the films (Table 1). Tab. 1: Water vapor permeability (WVP) of the plasticized and non-plas-ticized suberized hydroxypropyl methylcellulose (HPMC) films (n = 3). Fig. 1: SEM micrographs of the suberized HPMC free films. Key: Plasticized (A) and non-plasticized (B) reference HPMC film without added suberin FA; Plasticized (C) and non-plasticized (D) HPMC film with suberin FA (2.5% of the polymer weight); Plasticized (E) and non-plasticized (F) HPMC film with suberin FA (5.0% of the polymer weight). Magnification 1000x. Surface topography and morphology analysis (AFM) showed differences in film structure between the non-suberized and suberized cellulosic films (Figure 2). HPMC film composition* WstoT vapor permeability (WVP) [■ !<)" g/(mm:li) xmnVPnj HPMC 2.00 HPMC + PEG 40ü 2.13 HPMC +■ Su her in (15%) 2.08 HPMC t S«I)trill (2 5%) + PEG 4011 1 82 fipmc + siftenn 1 71 HPMC + Suberin (5%) + PEO-WO ! 70 HPMC • Suberin (LOH) 1.13 HPMC + Suberin (10%) - PEG >1110 1,25 HPMC 1 Suberin (15%) 069 HPMC H Suberin (15%) + PEG 400 0,77 Fig. 2: Surface topography and morphology analysis of the suberized HPMC films with AFM. Key: Plasticized (A) and non-plasticized (B) reference HPMC film without added suberin FA; Plasticized (C) and non-plasticized (D) HPMC film with suberin FA (5.0% of the polymer weight). * A : r "! !i ! I'T nib cr. :i enltulntod II'I the HP M C polviYUT weight CONCLUSIONS Suberin FA is a readily available bio-material and potential new excipient for pharmaceutical coating applications. Clear relationships can be found between the film surface morphology, WVP and the amount of suberin FA added in the HPMC films. Inclusion of suberin FA improves the moisture barrier properties of HPMC films. ACKNOWLEDGEMENTS This work is part of the targeted financing project no SF0180042s09 and ETF grant project no ETF7980. The research was also supported by the European Social Fund's Doctoral Studies and Internationalization Program DoRa, and GREASE Woodwisdom-ERANET (Tekes decision 40375/11). REFERENCES 1. Gandini A, Neto CP, Silvestre AJD. Suberin: A promising renewable resource for novel macromolecular materials. Prog. Polym. Sci. 2006; 31: 878-892. 2. Alakurtti S. Synthesis of betulin derivatives against intracellular pathogens, Doctoral dissertation (article-based), University of Helsinki, Finland, 2013. 3. Ekman R. The suberin monomers and triterpenoids from the outer bark of Betula verrucosa Ehrh. Holzforschung 1983; 37: 205-211. 4. Jia D, Fang Y, Yao K. Water vapor barrier and mechanical properties of konjac glucomannan-chitosan-soy-protein isolate edible films. Food Bioprod. Process. 2009; 87: 7-10. THE ROLE OF NEURO-FUZZY MODELS FOR PREDICTION OF BIOEQUIVALENCE STUDIES OUTCOME J. opara1*, I. Legen1, 1 Lek Pharmaceuticals d.d., Verovskova 57, 1526, Ljubljana, Slovenia. INTRODUCTION Bioequivalence (BE) studies play an important part in generic drug development. They are unavoidable while requested by regulatory agencies, but on the other hand expensive and time consuming for sponsors. For this reason it is important to take the right decision before the start of a study while expensive or failed studies can limit the development of a generic product. We were looking for tools that are able to reduce the number of bioequiva-lence studies and have significant effect on development costs and timeliness. Modeling can be used to facilitate BE outcome predictions and with such enhanced knowledge of IVIVR incorporated in a model, the number of developed formulations and failed BE studies are reduced. Modeling in vivo data meet many challenges like limited databases, high variability of data and high complexity. For these reasons it is often difficult or impossible to build mathematical models for In Vitro / In Vivo correlations (IVIVC) that are preferred by regulatory agencies and described in Guidance (1,2). In vitro dissolution data are not always sufficient to describe in vivo behavior and for drugs with complicated pharmacokinetics or for immediate release drugs, we need to develop more complex models than level A, B, C or multiple level C correlation. Neuro-fuzzy (NF) systems were recognized as a reasonable method in comparison to the published approaches for development of IVIVR. The usefulness of NF models as an alternative in vitro-in vivo relationship (IVIVR) tool is presented in the continuation. MATERIALS AND METHODS NF systems belong to a group of artificial intelligence models and combine features of neural nets and fuzzy systems to provide a hybrid between the two techniques. Although both fuzzy and neural approaches possess remarkable properties when employed individually, there are great advantages to using them synergistically (4,5). The applicability of neuro-fuzzy models is increased with the transparency of included knowledge into the system. NF models were built to predict 144 pharmacokinetic (PK) parameter ratios required for demonstration of bioequivalence (BE) for 88 pivotal BE studies. The modeling process involved building hybrid artificial intelligence systems—NF models with the use of commercial software Neuframe version 4.0 (Neusciences 2000). Input parameters of models included dissolution data and their combinations in different media, presence of food, formulation strength, technology type, particle size, and spray pattern for nasal sprays. Ratios of PK parameters Cmaxor AUC were used as output variables. Database used for modeling consisted of PK parameters obtained in bioequiva-lence studies intended for regulatory submissions. Internal and external predictability of models were calculated and evaluated based on the currently valid guidelines for IVIVC (1,2). RESULTS AND DISCUSSION Statistical evaluation of prediction performance of models was performed. The prediction performance of models resulted in the following values: 79% of models have acceptable external prediction error (PE) below 10%, 13% of models have inconclusive PE between 10 and 20%, and remaining 8% of models show inadequate PE above 20%. Average internal predictability (LE) is 0.3%, and average external predictability of all models results in 7.7%. Basic statistics of external predictability of all models is presented in Table 1 (3). Tab. 1: Basic statistics of external predictability of all models PE% All models c models max AUc models Mean 7.7% 8.9%% 5.6%% Median 5.5% 6.6%% 4.3%% Min 0.01%% 0.20%% 0.01%% Max 34.4% 34.4%% 28.0%% N of models 144 88 56 With kind permission from Springer Science+Business Media: The AAPS Journal, Neuro-fuzzy Models as an IVIVR Tool and Their Applicability in Generic Drug Development, Mar;16(2), 2014, 324-34, J.Opara, I.Legen, Table III. p s Based on results in Table 1 we can conclude that all mean and median values of external prediction errors for Cmax and AUC models are below requested 10%. Drugs we used for modeling were also members of different BCS classes. Mean external prediction errors for individual BCS class were the following: 6.9, 8.4, 2.9 and 7.6, for BCS 1, 2, 3 and 4, respectively. All BCS 1 class drugs we used for modeling were incorporated in modified release formulations. One-way ANOVA was performed with BCS class as a factor to define statistical differences of PE% among BCS classes. We were not able to reject the H0 about equality and we can conclude that there are no statistical differences in PE% between different BCS class drugs (3). CONCLUSIONS In average, models have acceptable internal and external predictabilities with PE lower than 10% and are therefore useful for IVIVR needs during formulation development. NF models facilitate the determination of impact factors for rate and extent of drug absorption and can be used as a support to QbD and for the prediction of BE studies outcome. With their properties, NF models were recognized as an alternative and reasonable method in comparison to the published approaches for in vitro in vivo correlation and preferential for solving of complex IVIVR problems. Prediction of BE studies outcome with NF models can support decisions when they need to be taken based on incomplete data. ACKNOWLEDGMENTS Many thanks to our colleagues at Lek Pharmaceuticals d.d. for their hard work in achievement of in vivo relevant dissolutions, BE data and other analytical measurements needed as inputs into the models. REFERENCES 1. Guidance for Industry, Extended Release Oral Dosage Forms: Development, Evaluation, and Application of In Vitro/In Vivo Correlations. CDER, September 1997. 2. Note for Guidance on quality of modified release products: A: oral dosage forms B: transdermal dosage forms. EMA, CPMP July 1999. 3. J. Opara, I. Legen. Neuro-fuzzy Models as an IVIVR Tool and Their Applicability in Generic Drug Development. AAPS J. 2014; Mar;16(2): 324-34. (DOI) 10.1208/s12248-014-9569-8. 4. Tsoukalas LH, Uhrig RE. Fuzzy and Neural Approaches in Engineering. John Wiley & Sons, Inc., New York 1997. 5. Manual Neuframe version 4.0, Neusciences 2000. LONGITUDINAL STUDY OF BISOPROLOL PHARMACOKINETICS IN ELDERLY PATIENTS WITH CHRONIC HEART FAILURE K. Cvan Trobec1, I. Grabnar2, M. Kerec Kos2, T. Vovk2*, J. Trontelj2, M. Lainscak1 1 University Clinic of Respiratory and Allergic Diseases, Pharmacy Department, Golnik 36, 4204 Golnik, Slovenia 2 Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia INTRODUCTION Bisoprolol is one of the most commonly used beta-blockers in the treatment of chronic heart failure (HF). It has a balanced clearance, with half of the dose being excreted via the kidney and another half via liver. It is equally hydro-philic and lipophilic drug, only 30% bound to plasma proteins, and its volume of distribution of more than 200 litres suggests binding to tissue proteins (1). It was also showed that bisoprolol pharmacokinetics is altered in obese subjects and thus suggests the dependence of bisoprolol pharmacokinetics (PKs) on body composition (2). For patients with HF, little is known about bisoprolol PKs. In the extremes of renal or liver function and body composition, the beta-blockers associated side effects or even toxicity may develop. The aim of this study was to investigate the PKs of bisoprolol in chronic HF patients by population pharmacokinetic modelling. We specifically assess changes over time, also in relation with body composition, renal and liver function. Finally, potential differences in pharmacokinetics between cachectic and non-cachectic subjects were evaluated. MATERIALS AND METHODS Patients and study design Patients with chronic HF (class I-III) were screened for inclusion. Patients on regular therapy with bisoprolol were seen at baseline visit and at follow-up visit at least 6 months after inclusion. Patients attended the visit in the morning, fasting and before taking their morning dose of bisoprolol. Blood samples were drawn prior to morning bisoprolol dose (trough sample), followed by 2, 3 and 4 hours post dose sampling. Assays On visits, body composition was determined by dual-energy X-ray absorptiometry (DEXA) to assess fat mass, lean mass and body mineral content. Sum of lean mass in both arms and legs was calculated to obtain appendicular skeletal muscle mass (ASM) and skeletal muscle index (SMI) was calculated by dividing ASM with the square of patient height. Patients were screened for cachexia according to Evans et al (3). Renal function was measured with iohexol clearance and with four variable Modification of Diet in Renal Disease (MDRD4) equation. Liver function was assessed with direct (BLRD) and total bilirubin (BLRT), aspartate transaminase (AST), alanine transaminase (ALT), gamma glutamyl transpeptidase (yGT), and alkaline phosphatase (AF) concentration. Bisoprolol plasma concentrations were determined by solid phase extraction of plasma samples and analysis using high performance liquid chromatography with tandem mass spectrometry. Pharmacokinetic modelling PK analysis was performed by a population PK modelling approach using NONMEM software. The structural model used was a one-compartment PK model with first-order absorption and elimination. The first-order conditional estimation method with interaction was used for estimation of apparent clearance (CL/F), apparent volume of distribution (V/F) and absorption rate constant (Ka). Effects of continuous and categorical covariates were tested against the base model. Alternative models were compared by the likelihood ratio test (a = 0.05). Significant covariates were rank-ordered and introduced into the full model. The final model was determined by backward elimination of covariates one by one from the full model to see if they should remain in the model using the likelihood ratio test. The models were evaluated by standard diagnostic plots, absence of substantial n- and e - shrinkage, convergence of minimization, successful covariance step, and gradients in the final iteration (range 10-3-102). RESULTS AND DISCUSSION Mean clearance of bisoprolol was 10.2 L/h, which is about one third lower than in healthy population (1). This result is comparable to the clearance determined in study of Nikolic et al (11.4 L/h) who also studied bisoprolol pharmacokinetics in chronic HF patients (4). With the final model it was demonstrated that CL/F is influenced only by MDRD4 (power model with the exponent of 0.620), while V/F is linearly related with body weight (WT) and SMI (power model). The final models are presented by the following equations: ,0.620 CL / F [L / h]= 10.4 • (MDRD4[mL/min]/60) V/F[L] = 218 • (1 + 0.00568(WT[kg] - 70)) • (SMI/7.32)0715 The difference in volume of distribution between the patient with the lowest SMI and the highest SMI in our study was almost 100%. Patients with lower body weight and SMI have lower volumes of distribution, which reflects in wider concentration fluctuations and higher maximal plasma concentrations of bisoprolol. Bisoprolol clearance did not change significantly between the two visits, although measured renal function declined. Since bisoprolol is eliminated both via kidney and liver, decreasing renal elimination can be partly compensated by increasing hepatic elimination. On the other hand, the absorption rate constant declined during follow-up, which could suggests slower absorption process (Fig. 1). Moreover, apparent volume of distribution significantly increased, but this was not related to changes in the body composition. Fig. 1: Changes in pharmacokinetic parameters during follow-up (N=39; paired sample t test, p< 0.05). p s Cachexia was not found to significantly influence bisopro-lol pharmacokinetics. However, study sample was small and only four patients were cachectic at baseline and only three at follow-up. CONCLUSIONS Bisoprolol clearance is decreased in patients with chronic HF and is dependent on estimated renal function. Patients with lower body weight and skeletal muscle index have reduced volume of distribution, which results in greater fluctuations and higher peak plasma concentrations of bisoprolol. Bisoprolol pharmacokinetics changes during the course of the disease, but these changes are not related to the changes in body composition. REFERENCES 1. Leopold G, Pabst J, Ungethum W, Buhring KU. Basic pharmacokinetics of bisoprolol, a new highly beta 1-selective adrenoceptor antagonist. J. Clin. Pharmacol. 1986; 26: 616-21. 2. Le Jeunne C, Poirier JM, Cheymol G, Ertzbischoff O, Engel F et al. Pharmacokinetics of intravenous bisoprolol in obese and non-obese volunteers. Eur. J. Clin. Pharmacol. 1991; 41: 171-4. 3. Evans WJ, Morley JE, Argilés J, Bales C, Baracos V et al. Cachexia: a new definition. Clin. Nutr. 2008; 27: 793-9. 4. Nikolic VN, Jevtovic-Stoimenov T, Velickovic-Radovanovic R, Ilic S, Deljanin-Ilic M et al. Population pharmacokinetics of bisoprolol in patients with chronic heart failure. Eur. J. Clin. Pharmacol. 2013; 69: 859-65. 2D DYNAMICAL SIMULATION OF A VIBRATIONAL MILL M. Abrami1, D. Hasa2, B. Perissutti2, D. Voinovich2, G. Grassi3, R. Farra1, M. Grassi1* 1 Department of Engineering and Architecture, Trieste University, via A. Valerio 6, I-34127, Trieste, Italy. 2 Department of Chemical and Pharmaceutical Sciences, Trieste University, Piazzale Europa 1, Trieste, I-34127, Italy. 3 Department of Life Sciences, Cattinara University Hospital, Strada di Fiume 447, I-34149, Trieste, Italy. INTRODUCTION Many delivery systems, especially oral administration ones, consist essentially of a crosslinked polymeric carrier hosting the active agent (drug) inside the three-dimensional network (1). The advantage of using such delivery systems is double. On the one hand, the polymeric network in the swollen state is able to control the drug release kinetics, due to the contact with the external physiological fluid. On the other hand, in the dry, shrunken state, the polymeric network is able to stabilise drug nano-crystals and/or amorphous drug. The interesting aspect of the amorphous drug and drug nanocrystals lies in their improved solubility (2, 3) that reflects in an increased bio-availabity, aside from permeability problems (Amidon class II drugs (4)). Among the available techniques (solvent swelling and supercritical fluids, among others) used for drug loading into crosslinked polymers in form of nano-crystals or amorphous state, co-grinding has the considerable advantage of not requiring the use of solvents. Indeed, eliminating solvents from the final formulation is both very difficult and expensive. Albeit not exceptionally energetic (5), the vibrational mill seems a promising tool for drug-polymer co-grinding. Indeed, energy transfer from the grinding media to the drug-polymer system is a continuous (6) rather than an abrupt process that may induce undesired effects such as the occurrence of chemical reactions and/or the formation of polymorphic structures. In order to optimise the milling process, it is desirable, if not mandatory, to grasp the mill dynamics which are strictly connected to the energy transfer among grinding media and the drug-polymer ensemble. Accordingly, the aim of this work was to perform a two dimensional simulation of mill dynamics in order to theoretically select the best mill setup. This was achieved J?-c by means of a home-made FORTRAN software solving the cardinal equations describing mill dynamics. 2D SIMULATION The vibrational mill under study is a Sweco M18/5 (Florence, USA), equipped with 8 springs, 5 plastic vials (each with an internal volume of 250 cm3) and adopting alumina cylinders (0.5 cm diameter, 0.5 cm height) as grinding media. Co-grinding process optimization was performed focusing on the amount of grinding media (Mm) and on the different angular position occupied by the two mill eccentrics (this angular displacement was indicated as y) connected to the rotating mill engine shaft and inducing the complex mill dynamics. Accordingly, a two dimensional simulation (in the X, Y plane) of mill dynamics was performed keeping in mind that the 2D model behaves like the real 3D when both share the same total mass (Mt), centre of mass (abscissa XC, ordinate YC) and inertia momentum (/). In order to greatly simplify the dynamic analysis, we chose to neglect the real movement of grinding media inside each vial. Grinding media were thus considered to be a unique body partially filling vials. The cardinal equations describing mill dynamics are: RESULTS AND DISCUSSION Figure 1, showing the dependence of grinding chamber bottom velocity (VCB) on grinding media mass (Mmm) and eccentrics angular displacement (y), tells us that whatever MMM, the best choice for y is always 180°. In addition, although VCB increases with MMM, the choice of MMM = 2.5 kg (all 5 vials are completely filled by grinding media) cannot be considered as it would make grinding media movements impossible inside vials. where t is time, n is the number of forces acting on the mill, FXi and Fyi are, respectively, the horizontal and vertical components of the ith force acting on mill body, a is the angular rotation around mill mass centre and M, indicates the ith momentum acting on mill body. In order to turn the 3D problem into a simpler 2D problem, we considered only the XY component of the forces acting on the upper and lower eccentrics due their rotation induced by the rotating mill engine shaft. Eqs.(1) and (2) were numerically solved by a 5th order adaptive step Runge-Kutta method. Mill geometrical and mechanical properties (such as spring elastic properties and materials density) have been experimentally determined or assumed according to mill manufacturer indications. As it is very difficult to establish a unique parameter indicating the grinding process optimal conditions (from the energy transfer point of view), we decided to select vials bottom velocity (VCB) as control parameter. Indeed, grinding media impact energy can be assumed to be proportional to vials kinetic energy. Fig. 1: Dependence of vials bottom velocity (VCB) on grinding media mass (Mmm) and eccentrics angular displacement (y). CONCLUSIONS This analysis led to the conclusion that the best mill set up (from the energy input point of view) corresponds to an angular displacement between eccentrics of 180°. In addition, the increase of grinding media mass improves the kinetic energy. Of course, these theoretical results need to be confirmed by experimental tests correlating the different mill set-up with the co-ground drug-polymer characteristics expressed by amorphous drug content and the nano-crystals dimensions. REFERENCES 1. Lee PI, Diffusion-controlled matrix systems, in Treatise on Controlled Drug Delivery; Marcel Dekker: New York, 1992. 2. Hasa D, Perissutti B, Voinovich D, Abrami M, Farra R, S. M. Fiorentino SM, Grassi G, Grassi M. Drug Nanocrystals: theoretical background of solubility increase and dissolution rate enhancement. Chem. Biochem. Eng. Quart. 2014, in press. 3. Hasa D, Voinovich D, Perissutti B, Grassi G, Fiorentino SM, Farra R, Abrami M, Colombo /, Grassi M. Eur. J. Pharm. Sci. 2013; 50: 17-28. 4. Amidon GL, Lennernas H, Shah VP, Crison JR. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 1995; 12: 413-420. Colombo I, Grassi G, Grassi M. Drug mecahnochemical activation. J. Pharm. Sci. 2009; 98:3961-3986. Castillo J, Coceani N, Grassi M. Theoretical and experimental investigation on the dynamics of a vibrational mill, GRICU 2004 Symposium, "Nuove Frontiere di Applicazione delle Metodologie dell'Ingegneria Chimica", Porto d'Ischia (Na), 12-15 September 2004, Volume I, 165-168 APPLICATION OF MIXTURE EXPERIMENTAL DESIGN AND ARTIFICIAL NEURAL NETWORKS IN THE DEVELOPMENT OF TERNARY CARBAMAZEPINE-SOLUPLUS®-POLOXAMER 188 SOLID DISPERSIONS B. Medarevic1*, P. Kleinebudde2, K. Bekcic1, Z. Buric1, S. Ibric1 1 Department of Pharmaceutical Technology and Cosmetology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia, e-mail: djordje.medarevic@pharmacy. bg.ac.rs 2 Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University Duesseldorf, Universitaetsstr.1, 40225 Duesseldorf, Germany, kleinebudde@uni-duesseldorf.de INTRODUCTION Since number of currently available solid dispersion (SD) carriers is limited, it is often necessary to combine them to tailor their properties with regards to processability, solubilizing capacity and stabilization of the drug within the dispersion (1). Finding an optimal composition of SD can be difficult, since both proportions of each component of the carrier mixture and proportion of drug can significantly influence on dispersion properties. This study investigates potential of using mixture experimental design (MED) and artificial neural networks (ANNs) for evaluation the influence of the composition of ternary car-bamazepine (CBZ)-Soluplus-poloxamer 188 (P188) SDs on CBZ release rate. MATERIALS AND METHODS Materials CBZ, donated by Galenika AD (Belgrade, Serbia), Solu-plus®, micronized poloxamer 188 (P188-Kolliphor® P188 micro), both kindly donated by BASF (Ludwigshafen, Germany) and absolute ethanol (Merck, Darmstadt, Germany) were used for SDs preparation. SDs preparation, in vitro drug release testing and data modeling SD formulations were prepared according to D-optimal mixture experimental design (Table 1.), with the following constraints: 20%