pH dependence of organic anion-transporting polypeptide 2B1 in Caco-2 cells: potential role in antiretroviral drug oral bioavailability and drug-drug interactions.

Human intestinal epithelium expresses a number of drug efflux and influx transporters that can restrict and/or facilitate intestinal drug uptake during absorption. Organic anion-transporting polypeptide 2B1 (OATP2B1), a multispecific organic anion uptake transporter localized at the brush-border membrane of intestinal epithelial cells, is known to transport many endogenous substrates (e.g., steroid conjugates) and xenobiotics (e.g., statins). At present, limited information is available on the mechanism of HIV protease inhibitor (PIs) intestinal uptake. In this study, we examined the interaction of PIs with the OATP2B1 transport system in Caco-2 cells, an in vitro model of human intestinal epithelium, and Madin-Darby canine kidney II cells stably transfected with OATP2B1. The expression of OATP2B1 transcript and protein was confirmed by reverse transcription-polymerase chain reaction and immunoblot analysis, respectively. Estrone-3-sulfate (E3S) uptake demonstrated biphasic saturation kinetics in Caco-2 cells, with dissociation constants (K(M)) of 6 +/- 2 microM and 1.5 +/- 0.2 mM. Several PIs potently inhibited OATP2B1-mediated transport in Caco-2 cells at clinically relevant IC(50) concentrations for ritonavir (0.93 microM), atazanavir (2.2 microM), lopinavir (1.7 microM), tipranavir (0.77 microM), and nelfinavir (2.2 microM). An inwardly directed proton gradient was identified as the driving force of E3S uptake through NH(4)Cl intracellular acidification studies with a H(+):E3S stoichiometry for OATP2B1 of 1:1. In contrast, although atazanavir and ritonavir uptake by Caco-2 cells was stimulated by low extracellular pH, this process was not mediated by OATP2B1 and was not affected by an outwardly directed H(+) gradient. Because OATP2B1 exhibits an increasing number of drug substrates, including several statins, alterations of its function by PIs could result in clinically significant drug-drug interactions in the intestine.