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Cellular Accumulation of Cholyl-Glycylamido-Fluorescein in Sandwich-Cultured Rat Hepatocytes: Kinetic Characterization, Transport Mechanisms, and Effect of Human Immunodeficiency Virus Protease Inhibitors

Laboratory for Pharmacotechnology and Biopharmacy, Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium

The present study was aimed at characterizing the in vitro cellular uptake mechanism and kinetics of the bile salt analog cholylglycylamido-fluorescein (CGamF) in sandwich-cultured rat hepatocytes (SCRHs). Concentration-dependent inhibition of active CGamF accumulation by seven human immunodeficiency virus (HIV) protease inhibitors (PIs) was also determined and compared with inhibition data obtained with taurocholate (TC) as a substrate. A K_m value of 9.3 ± 2.6 µM was obtained for saturable CGamF accumulation in SCRHs. The organic anion-transporting polypeptide (Oatp) inhibitor rifampicin (100 µM) inhibited CGamF (1 µM) accumulation in SCRHs by 72%; sodium depletion did not further reduce CGamF accumulation. In contrast, TC accumulation was reduced by only 25% in the presence of rifampicin, whereas additional sodium depletion resulted in a complete loss of TC accumulation. These data imply that Oatp(s) and sodium taurocholate-cotransporting polypeptide preferentially mediate hepatic uptake of CGamF and TC, respectively. Coincubation of CGamF with HIV PIs (amprenavir, atazanavir, darunavir, indinavir, nelfinavir, ritonavir, saquinavir) revealed that five of them had a concentration-dependent inhibitory effect on CGamF accumulation in SCRHs, with IC₅₀ values between 0.25 ± 0.07 and 43 ± 12 µM. The rank order for inhibition of CGamF accumulation in SCRHs was: ritonavir >> saquinavir > atazanavir > darunavir > amprenavir. Indinavir (up to 100 µM) did not alter CGamF accumulation, whereas nelfinavir solubility was limited to 10 µM. Taken together, these findings illustrate the utility of CGamF as a suitable probe (complementary to TC) for rapid in vitro determination of interaction potential with sodium-independent uptake mechanisms (likely Oatps) in rat liver.