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Cell culture models in the study of drug efflux transporters

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.

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Cell culture models in the study of drug efflux transporters

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  1. Cell culture models in the study of drug efflux transporters Mgr. Martina Čečková Department of Pharmacology and ToxicologyCharles University in PragueFaculty of Pharmacy in Hradec Králové

  2. Cell culture models • Primary cultures • Cell lines • immortalized cells • cancer cell lines • Genetically modified cell lines • Cell cultures as a model of biological barriers

  3. 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

  4. TRANSPORT ASSAYS

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

  6. ACCUMULATION EXPERIMENTS

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

  8. 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

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

  10. A - MDCKII B - MDCKII – BCRP C – BeWo D – human term placenta Expression of BCRP in BeWo cell line • Number of transcripts per microgram of total RNA (Real time RT-PCR) • Immunodetection of BCRP (Western blotting, BXP-21 antibody) Ceckova M., et al., Clin Exp Pharmacol Physiol, 2005

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

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

  13. BCRP BeWo BeWo BeWo BeWo BeWo BeWo • BCRPpumps potentially toxic xenobiotics from trophoblast cells in feto-maternal direction

  14. 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

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