Cellular Accumulation of Cholyl-Glycylamido-Fluorescein in Sandwich-Cultured Rat Hepatocytes: Kinetic Characterization, Transport Mechanisms, and Effect of Human Immunodeficiency Virus Protease Inhibitors
American Society for Pharmacology and Experimental Therapeutics
Drug metabolism and disposition vol:36 issue:7 pages:1315-1321
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 Km 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.