Rapid CommunicationSubstrate- and Species-dependent Inhibition of P-glycoprotein-mediated Transport: Implications for Predicting in vivo Drug Interactions
Section snippets
INTRODUCTION
P‐glycoprotein (P‐gp) is an efflux transporter belonging to the ATP‐binding cassette (ABC) protein superfamily. P‐gp transports a wide range of structurally diverse compounds, and it has been estimated that 50% of marketed drugs are transport substrates or inhibitors of P‐gp.1 In humans, P‐gp is expressed at the blood–brain barrier (BBB)2 and in the hepatocyte bile canalicular membrane, the luminal surface of intestinal epithelia and renal proximal tubule,3 placenta, ovaries, and testes.4 This
Chemicals, Reagents, and Biological Materials
Vincristine sulfate and ketoconazole were purchased from MP Biomedicals (Solon, Ohio). Cyclosporin A (CsA) was acquired from Calbiochem (San Diego, California). Verapamil, quinine, quinidine, itraconazole, and propidium iodide were purchased from Sigma (St. Louis, Missouri). N‐(4‐(2‐(1,2,3,4‐Tetrahydro‐6,7‐dimethoxy‐2‐isoquinolinyl)ethyl)phenyl)‐9,10‐dihydro‐5‐methoxy‐9‐oxo‐4‐acridine carboxamide (elacridar, GF120918) was a generous gift from GlaxoSmithKline (King of Prussia, Pennsylvania).
RESULTS
Prazosin and verapamil are drugs that are also well‐characterized P‐gp substrates,13 and the addition of a fluorescent BODIPY moiety allows for the measurement of their net intracellular accumulation using flow cytometry. As the net accumulation of these compounds is a combination of uptake (e.g., diffusion) and efflux (e.g., active transport) rates, we utilized the parental LLC‐PK1 cell line to provide a measure of net accumulation in the absence of P‐gp, and compared this with cells in which
DISCUSSION
This observation of substrate‐dependent inhibition of P‐gp has significant implications for in vitro to in vivo prediction of P‐gp‐mediated drug–drug interactions. In the case of quinidine, clinical plasma concentrations are in the range of 1.5–3.5 mg/L (4.6–10.8 μM). The free fraction of quinidine in human plasma is around 0.2,14 meaning that unbound plasma concentrations achieved in the clinic will be in the range of 0.9–2.1 μM. This is two‐ to fivefold above the IC50 value we calculated for
ACKNOWLEDGEMENTS
This work was supported by the National Institutes of Health (Grants AG031485 and GM32165), and the University of Washington Drug Metabolism, Transport and Pharmacogenomic Research (DMTPR) program. We would like to thank Dr. Alfred Schinkel for providing LLC‐ABCB1 cells, and also Tasha Ritchie and Peng Hsiao for insightful discussions.
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