Preskušanje in izboljšave opredelitve celičnega permeabilnostnega modela s sočasno uporabo velikega števila modelnih učinkovin

Pangeršič, Katarina (author), Žakelj, Simon (mentor)

V magistrskem delu smo preizkušali uporabo kasetne meritve permeabilnosti večjega števila učinkovin pri treh različnih donorskih koncentracijah na tankem črevesu podgane in na celičnem modelu Caco-2. Uporabili smo 66 učinkovin iz vseh štirih razredov biofarmacevtskega klasifikacijskega sistema pri predhodno določeni osnovni donorski koncentraciji ter njenem redčenju na 1/2 in 1/5 osnovne koncentracije. Spojine in njihove osnovne koncentracije so bile predhodno izbrane na osnovi združljivosti v kompleksnih donorskih raztopinah in uporabnosti z vidika končne analize akceptorskih vzorcev. Raziskali smo, kakšne rezultate nam podajata celični model in model tankega črevesa podgane v difuzijskih celicah. Primerjali smo tudi kasetno in individualno določene permeabilnostne koeficiente na podobnem celičnem modelu. Izmerjene permeabilnostne koeficiente in efluksna razmerja smo predstavili v korelaciji z deležem absorbirane učinkovine (fa). Raziskali smo omejitve in prednosti nižanja donorskih koncentracij spojin. Polovična koncentracija se je med raziskavo izkazala za bistveno manj toksično za celično linijo, hkrati pa smo pridobili skoraj enake rezultate kot pri osnovni koncentraciji. Nižanje koncentracije na 1/5 se ni izkazalo za bolj koristno, naleteli pa smo na več težav z detekcijo tako nizkih koncentracij v vzorcih. Kot pričakovano smo s pomočjo ROC krivulje (Receiver operating characteristic) potrdili, da ima individualni pristop merjenja permeabilnosti na celičnem modelu Caco-2 boljšo zmožnost ločevanja in razvrščanja med kategorijami. Prav tako je kasetni pristop na modelu Caco-2 opazno boljši kot na modelu difuzijske celice z uporabo podganjega čreva. Kasetni pristop smo uspešno uporabili za določitev velikega števila permeabilnostnih koeficientov v enem samem poskusu in pokazali, da bo za nadaljnjo uporabo na osnovi naših rezultatov potrebna selekcija do ožjega nabora spojin, ki se ustrezno razvrščajo z različnimi eksperimentalnimi modeli za določanje permeabilnosti.

• Language: Slovenian
• Keywords: permeabilnost, celični model Caco-2, difuzijske celice
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-141191
• Publication date in RUL: 24.09.2022

Secondary language

• Language: English
• Title: Testing and improvements of a cell-based permeability model characterisation by simultaneous use of numerous model compounds

Abstract

This thesis investigated the usage and limitations of a cassette approach for determining the permeability coefficients for a large number of active pharmaceutical ingredients with Caco-2 and Rat intestine permeability models. We conducted our research using 66 active substances from all 4 Biopharmaceutical Classification System classes at pre-determined donor concentration, and their 1/2 and 1/5 dilution. Active substances were previously chosen based on their compatibility in complex donor solutions and usefulness in acceptor sample analysis. We investigated the results of a cell model and the rat intestine model. We also compared cassette and individually determined permeability coefficients on a similar cell model. The measured permeability coefficients and efflux ratios were presented in correlation with the fraction absorbed. Half concentration was shown to be significantly less toxic to the cell line during the experiment, while at the same time the obtained results were almost the same as with the full concentration. Lowering the concentration to 1/5 did not prove to be more beneficial regarding the toxicity, but several more problems with the detection of certain compounds in the acceptor samples were encountered. As expected, the Receiver Operating Characteristic curve has shown that the individual permeability measurement approach on the Caco-2 cell model has a better ability to separate and classify between categories. Besides that, the cassette approach of the Caco-2 model is superior for classification compared to the diffusion cell model using rat intestine. We successfully used the cassette approach to determine a large number of permeability coefficients in a single experiment and showed that further use will require a selection based on our results to a narrower set of compounds that are appropriately classified by different experimental models for determining permeability.

• Keywords: permeability, Caco-2 cell model, diffusion cells