Cell culture models in the study of drug efflux transporters

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Presentation transcript:

Cell culture models in the study of drug efflux transporters Mgr. Martina Čečková Department of Pharmacology and Toxicology Charles University in Prague Faculty of Pharmacy in Hradec Králové

Cell culture models Primary cultures Cell lines immortalized cells cancer cell lines Genetically modified cell lines Cell cultures as a model of biological barriers Použití buněčných modelů – etické výhody (minimalizace časově, finančně a experimentálně náročných pokusů na zvířatech) - možnost použití lidských buněk (model lidské tkáně) - rychlá metoda (pro studium transepiteliální permeability látek a molekulárních mechanizmů transportu léčiv) Primární kultury – buňky izolované z tkáně živého organizmu (smíšená populace buněčných typů) přeneseny do živného média Buněčné linie – vznik pasážováním primárních kultur, z kmenových bb, buněčné linie imortalizované virovými nebo buněčnými onkogeny, nádorové buněčné linie Kultivace jako bb suspenzní nebo adhezivní Geneticky modifikované buněč. linie – vnesen gen pro expresi studovaného transportéru – vhodné pro studium funkce a substrátové specifity transportérů, a identifikaci (screening) látek, které s ním interagují Pro studium farmakokinetických procesů (Absorpce, distribuce, eliminace léčiv), na kterých se mohou podílet lékové transportéry – použití buněčných modelů biologických bariér

GENETICALLY MODIFIED CELLS MDCKII-parent  MDCKII-MDR1  MDCKII-BCRP  MDCKII-MRP2 MEF 3.8-parent  MEF 3.8-BCRP Transport assays Accumulation / efflux assays ATPase assays MDCK – imortalizovaná buněčná linie odvozená z buněk proximálního tubulu ledvin kokršpaněla, v současnosti nejlépe charakterizovaný epiteliální buněčný model Výhoda – krátká kultivační doba, snadná manipulace, vytvářejí polarizovanou monovrstvu s adhezivními a těsnými spoji – vhodné pro transportní studie - Máme k dispozici geneticky modifikované MDCK linie obsahující gen pro expresi MDR1 (= P-glykoprotein), BCRP a MRP2 transportérů MEF3.8 myší fibroblasty - bez endogenní exprese MDR1 (nízký background pro studium transportérů) Základní metody pro studium funkce a substrátové specifity transportérů Transportní studie – buňky kultivovány na semipermeabilních membránách, sledován transport přes vytvořenou monovrstvu Akumulační studie – vhodné i pro buňky suspenzní ATPasové studie – nepřímo sledovaná funkce aktivních transportérů , nízká citlivost

TRANSPORT ASSAYS Transportní studie – umožňují sledovat kinetiku transportu, lokalizaci transportéru v rámci buňky Polarizované buňky, apikální membrána (strana in vivo obrácená do lumen) – vrchní kompartment, Basolaterální membrána obrácená k membráně (in vivo k pod ní uloženým tkáním) – spodní kompartment Semipermeabilní m Rozdíl v transportu u MDCK buněk a u buněk, které exprimují ve zvýšené míře studovaný transportér Např. MCDK-BCRP – zvýšený eflux topotekanu (svého substrátu) v basolaterálně-apikálním směru

Transepithelial transport of PhiP Pavek P., et al: 2005; J Pharmacol Exp Ther, 312(1): 144-52.

ACCUMULATION EXPERIMENTS

Interactions between BCRP and antiepileptics Accumulation of BODIPY- prazosin in MEF3.8-BCRP cells Cerveny L., et al., 2005

CELL CULTURES AS A MODEL OF BIOLOGICAL BARRIERS Intestine: Caco-2, HCT-8, HT-29, SW-480… Kidney: HK-2 Liver: Cultured hepatocytes HepG2 Blood-brain barrier: BCEC SV-HCEC, HBEC-51, BB19 Blood-testis barrier: isolated Sertoli cells Placenta: BeWo, JEG3, JAR

PLACENTA in vitro model of human trophoblast – cell line BeWo BeWo

Expression of BCRP in BeWo cell line Number of transcripts per microgram of total RNA (Real time RT-PCR) Transcript (104) Human term placenta BeWo Mean ± S.D. Minimum-Maximum BCRP 104.4 ± 16.2 77.6 - 128.1 626.7 MDR1 8.9 ± 2.5 6.3 - 13.3 N.D. Immunodetection of BCRP (Western blotting, BXP-21 antibody) A - MDCKII B - MDCKII – BCRP C – BeWo D – human term placenta Ceckova M., et al., Clin Exp Pharmacol Physiol, 2005

Localization of BCRP in the BeWo cells Indirect immunofluorescence microscopy (BXP-21 antibody) BCRP transporter is localized predominantly in the apical plasma membrane Ceckova M., et al., Clin Exp Pharmacol Physiol, 2005

Functional expression of BCRP in BeWo cells Accumulation assay  FACS analysis Substrate: 20µM mitoxantrone Inhibitors: GF120918, Ko143, PSC833 BCRP actively pumps mitoxantrone out of BeWo cells Ceckova M., et al., Clin Exp Pharmacol Physiol, 2005

BCRP BCRP pumps potentially toxic xenobiotics from trophoblast cells in feto-maternal direction BCRP BeWo

Poděkování Department of Pharmacology and Toxicology Charles University in Prague, Faculty of Pharmacy in Hradec Kralove Doc. PharmDr. František Štaud, Ph.D. PharmDr. Petr Pávek, Ph.D. Mgr. Lukáš Červený Mgr. Antonín Libra Prof. MUDr. Zdeněk Fendrich, CSc. Department of Pathophysiology, Medical University of Vienna Univ. Prof. DI Dr. Renate Fuchs Dr. Marianne Brabec Institute of Clinical Immunology and Alergology, Faculty Hospital, Hradec Kralove PharmDr. Doris Vokurková