2737 KB235 KB0 KB2013Brodnjak-Vončina, DarinkaProsen, HelenaVnučec Popov, Tanja114 str., 30 cmCOBISSID:17212694URN:URN:NBN:SI:doc-5ETO0IR1slT. Vnučec Popovvisokošolska deladried blood spotsdried plasma spotsfarmakokinetične študijeliqiud chromatography tandem mass spectrometrymasna spektrometrijapharmacokinetic studysuha lisa krvisuha lisa plazmetekočinska kromatografija s tandemsko masno spektrometrijotopiramattopiramateRazvoj in uporaba tehnike suhe lise plazme za kvantitativno določevanje topiramata v študijah farmakokinetike in bioekvivalence| Development and use of dried plasma spots technique for quantitative determination of topiramate in pharmacokinetics and bioequivalence studies| doctoral dissertation| doktorska disertacija|As a part of my doctoral studies I have developed and evaluated dried plasma spot technique for determining topiramate in pharmacokinetic and bioequivalence studies. Dried plasma spot technique derives from dried blood spot technique. In the last two years the latter was rediscovered by bioanalysts and pharmacokineticians and introduced in pharmacokinetic and bioequivalence studies as an alternative for traditional sampling and determination of substances from liquid human plasma. The collection of whole blood samples on paper, known as the dried blood spot technique (DBS), dates back to the early 1960s, when it was used for detecting metabolic problems in newborns. Today DBS offers a number of advantages over conventional whole blood in pharmacokinetic and bioequivalence studies. These advantages are related to ethical, practical, and cost aspects. DBS provides the ethical benefits of reduced blood volume (less than 100 ŽL compared to more than 1.0 mL blood, which is usually obtained during the conventional blood sample collection) and less invasive sampling (finger or heel prick rather than conventional venous cannula). Furthermore, it provides easier transfer and simpler and cheaper storage (no need for dry ice or freezers). When DBS technique was being reintroduced, the questions from the perspective of its practical advantages, technical improvements and possible analysis problems were considered. It was only until later that the technique got attention fromthe viewpoint of pharmacokinetics and bioanalysis. At that point it was discovered that blood matrix and consequently DBS technique is not useful for determining active substances in all kinds of studies. Therefore, DBS technique hasn't been regulatory acknowledged as an independent method for quantitative determination of active substances when it comes to the registration of human and veterinary drugs. In pharmacokinetic studies, liquidplasma is usually used as biological matrix for the determination of active substances. The aforementioned dried plasma spot technique (from now onDPS - Dried Plasma Spot) offers the same advantages as DBS technique alongside with the advantage of plasma being already acknowledged matrix for the majority of active substances. As a result of that, a question whether DPStechnique can completely replace the method of determining active substances from the basic matrix, liquid plasma, emerged in my mind. The answer to that question was sought in the molecule of topiramate. Topiramate is an anti-epileptic drug that is used for the treatment of various forms of epilepsy in adults and children and for the prevention of frequently recurringmigraines in adults as well as for treatment of Lennox-Gastaut syndrome. Topiramate enhances the action of inhibitory neurotransmitters (GABA), blocks voltage-sensitive sodium channels and mildly inhibits carbonic anhydrase (CA) isoenzymes. Binding to CA, which is highly concentrated in erythrocytes, may affect drug pharmacokinetics. Topiramate's tendency to bind to CA in erythrocytes implies that blood concentration-time profile may be different from plasma profile and the blood-to-plasma ratio might be greater than 1. The purpose of the research was to determine which is the most appropriate dried matrix for the quantitative determination of topiramate in biological samples of pharmacokinetic and bioequivalence studies, on the basisof literature and DBS and DPS samples from the monitoring research on thetherapeutic effect. The doctoral dissertation consists of three parts of experimental work. In the first part, the method for quantitative determination of topiramate from dried plasma spot was developed and validated. In the second and third part of experimental work, the method for quantitative determination of topiramate from liquid human plasma and the method for quantitative determination of topiramate from dried blood spot weredeveloped and validatedV okviru doktorskega dela sem razvila in ovrednotila tehniko suhe lise plazme za kvantitativno določevanje topiramata v študijah farmakokinetike in bioekvivalence. Tehnika suhe lise plazme je izpeljana iz tehnike suhe lise krvi. Slednjo so v zadnjih dveh letih bioanalitiki, farmakokinetiki in klinični raziskovalci na novo odkrili in jo uvedli v študije farmakokinetike in bioekvivalence kot alternativo za tradicionalno vzorčenje in določevanje učinkovin iz tekoče humane plazme. Tehnika odvzema vzorcev krvi na papir, znana kot suha lisa krvi (v nadaljevanju DBS - Dried Blood Spots), se je začela uporabljati v zgodnjih šestdesetih letih prejšnjega stoletja v presejalnih testih za novorojenčke. Danes pri izvajanju farmakokinetičnih in bioekvivalenčnih študijah DBS nudi v primerjavi z običajno plazmo veliko prednosti, ki se nanašajo na praktične, etične in stroškovne aspekte. DBS omogoča manj invazivno vzorčenje (vbod v prst ali peto namesto uporabe običajne venske kanile) in etične prednosti odvzema manjšega volumna krvi (manj kot 100 ŽL v primerjavi z več kot 1,0 mL krvi pri običajnem vzorčenju krvi). Poleg tega omogoča lažji transport in enostavnejše ter cenejše shranjevanje (ni potrebe po suhem ledu ali zamrzovalnikih). V obdobju obujanjaDBS tehnike so se obravnavala predvsem vprašanja iz vidika njenih praktičnih prednosti, tehničnih izboljšav ter morebitnih analiznih težav in soji šele pozneje namenili pozornost s stališča farmakokinetike in farmakodinamike. Tedaj so ugotovili, da matrica krvi in s tem DBS tehnika ni primerna za določevanje zdravilnih učinkovin v vseh vrstah študij. DBS tehnikatako še ni regulatorno priznana kot samostojna metoda za kvantitativno določevanje zdravilnih učinkovin pri registraciji humanih in veterinarskih zdravil. Pri farmakokinetičnih študijah običajno kot biološko matrico za določevanje zdravilnih učinkovin uporabljamo tekočo plazmo. Zgoraj omenjena tehnika suha lisa plazme (v nadaljevanju DPS - Dried Plasma Spots) nudi enake prednosti kot DBS tehnika, poleg tega pa je plazma že uveljavljena biološka matrica za analizo večino zdravilnih učinkovin. Pri tem se mi je porajalo vprašanje, ali lahko tehnika suhe lise plazme popolnoma zamenja klasično metodo določevanja zdravilnih učinkovin iz temeljne matrice, tekoče plazme. Odgovor sem iskala v molekuli topiramat. Topiramat (TPM) je antiepileptično zdravilo, ki ga uporabljamo za zdravljenje različnih vrst epilepsije pri odraslih in otrocih, za preprečevanje pogosto ponavljajočih se napadov migrenepri odraslih ter kot dodatno zdravilo za zdravljenje Lennox-Gastautovega sindroma. Topiramat spodbuja delovanje inhibitornih nevrotransmiterjev (GABA), blokira Na-kanale in šibko inhibira karbonsko anhidrazo. Vezava na karboanhidrazo, ki je v veliki meri prisotna v eritrocitih, pa lahko vpliva na farmakokinetiko zdravilne učinkovine. Topiramat ima tendenco vezave na karboanhidrazo v eritrocitih, kar pomeni, da se koncentracije v krvi v odvisnosti od časa lahko razlikujejo od plazemskega profila, ter da je razmerje koncentracij kri - plazma večje od 1. Namen raziskave je bil na podlagi literature, DBS ter DPS vzorcev študije spremljanja terapevtskega učinka, oceniti, katera suha biološka matrica je najprimernejša za njegovo kvantitativno določevanje v bioloških vzorcih študijfarmakokinetike in bioekvivalence. Doktorska naloga je sestavljena iz treh raziskav. Razvila in preizkusila sem tri bioanalizne metode za določevanje topiramata v bioloških matricah. V prvi fazi sem razvila in preizkusila metodo za kvantitativno določevanje topiramata iz suhe lise plazme. V drugem in tretjem delu raziskave, sem razvila in preizkusila metodo iz tekoče humane plazme ter metodo določevanja topiramata iz suhe lise krvi. Za kvantitativno določitev koncentracij topiramata sem uporabila metodo devteriranega internega standarda (topiramat-d12) in tekočinsko kromatografijo s tandemskim masnim spektrometromTEXTvisokošolska delatheses and dissertationsSlovenian National E-content AggregatorNational and University Library of SloveniaUniverza v Mariboru, Fakulteta za kemijo in kemijsko tehnologijo