Pomen lastnosti dveh modelov s celično linijo Calu-3 za razvrščanje učinkovin glede na njihovo permeabilnost

Hribar, Uroš (author), Kristl, Albin (mentor), Kristan, Katja (co-mentor)

Med razvojem novih učinkovin je ključnega pomena napovedovanje njihove absorpcije v človeškem telesu, kar lahko dosežemo z izvedbo permeabilnostnih testov. Priprava celičnih modelov z različnimi celičnimi linijami je zaradi številnih prednosti vse bolj uporabljena metoda za določevanje permeabilnosti substanc, vključena pa je tudi v mednarodne smernice. Celična linija Calu-3, ki izvira iz človeškega pljučnega adenokarcinoma, se je zaradi svojih ugodnih lastnosti, kot so tvorjenje čvrstega monosloja na membranah, ekspresija številnih membranskih prenašalcev, hitra rast in možnost uporabe velikega števila celičnih pasaž, uveljavila kot ena izmed možnosti za pripravo omenjenih celičnih modelov. Naš namen v tej magistrski nalogi je bil oceniti sposobnost dveh celičnih modelov (model tekočina-tekočina in model zrak-tekočina) za razvrščanje učinkovin glede na njihovo permeabilnost ob upoštevanju njunih lastnosti. Integriteto celičnih slojev na modelih smo ocenili z merjenjem transepitelijske električne upornosti (TEER) ter s paracelularno permeabilnostjo spojine Lucifer rumeno (Lucifer yellow, LY), med samim gojenjem celic pa smo spremljali okuženost celic z mikoplazmami. Rezultati permeabilnostnih testov z uporabo nizko, zmerno in visoko permeabilnih učinkovin na podlagi smernic ICH M9 so pokazali, da sta oba celična modela primerna za razvrščanje učinkovin med nizko in visoko permeabilne glede na BCS klasifikacijo učinkovin. Na podlagi določevanja permeabilnostnih koeficientov v apikalno-bazelateralni in bazelateralno-apikalni smeri smo določili, katere učinkovine so podvržene efluksnemu mehanizmu, in sicer nadolol (substrat P-glikoproteina) na obeh pripravljenih celičnih modelih, furosemid (substrat P-glikoproteina in proteina BCRP) na modelu tekočina-tekočina, hidroklorotiazid (substrat proteina BCRP) na obeh pripravljenih celičnih modelih in klorotiazid (substrat proteina BCRP) na modelu tekočina-tekočina. Z izvedbo testov z inhibitorji P-glikoproteina (PSC833 in verapamil) smo potrdili ekspresijo P-glikoproteina v obeh pripravljenih celičnih modelih, saj so se izračunane vrednosti efluksnega razmerja (ER) za spojino rodamin 123 ob njihovi uporabi signifikantno znižale.

• Language: Slovenian
• Keywords: permeabilnost, Calu-3, celični modeli, P-glikoprotein, efluksni mehanizem
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-118108
• Publication date in RUL: 20.08.2020

Secondary language

• Language: English
• Title: Relevance of two Calu-3 cell line models' properties for drug permeability classification

Abstract

In the development of new active substances, predicting their absorption in human body is crucial and can be achieved by performing permeability assays. Cell models based on different cell types have become commonly used tools for permeability determination of active substances due to many advantages and are also recognized by international guidelines. Cell line Calu-3, which was derived from human lung adenocarcinoma, has been used as one of the options for cell model’s preparation because of its ability to form monolayers with tight junctions, different membrane transporter expression, fast growth and source of extensive cell proliferation which can be used for permeability assays. Our aim in this work was to evaluate the ability of two prepared cell models (model liquid-liquid and model air-liquid) for permeability classification of the active substances according to their properties. Tests for mycoplasma contamination were performed during the cell culturing. Integrity of the cell layers was evaluated by transepithelial electrical resistance (TEER) measurements and paracellular permeability determination of zero permeability marker Lucifer yellow (LY). Model substances with low, moderate and high permeability according to ICH M9 guidelines were used in permeability assays. Results indicate suitability of both prepared cell models for classification of active substances to high and low permeability classes based on BCS (Biopharmaceutical Classification System) classification. By performing permeability assays both in apical-to-basolateral and basolateral-to-apical directions we determined which tested substances are involved in efflux mechanism. Those substances are nadolol (P-glycoprotein substrate) on both cell models, furosemide (P-glycoprotein and BCRP substrate) on cell model liquid-liquid, hydrochlorothiazide (BCRP substrate) on both models and chlorothiazide (BCRP substrate) on cell model liquid-liquid. By performing permeability assays with P-glycoprotein inhibitors (PSC 833 and verapamil) we confirmed the expression of that protein in Calu-3 cells. Efflux ratio (ER) for rhodamine 123 decreased significantly on both cell models after using both P-glycoprotein inhibitors.

• Keywords: permeability, Calu-3, cell models, P-glycoprotein, efflux