title: Cellular accumulation of cholyl-glycylamido-fluorescein in sandwich-cultured rat hepatocytes: kinetic characterization, transport mechanisms, and effect of human immunodeficiency virus protease inhibitors.
abstract: 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 microM was obtained for saturable CGamF accumulation in SCRHs. The organic anion-transporting polypeptide (Oatp) inhibitor rifampicin (100 microM) inhibited CGamF (1 microM) 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 IC(50) values between 0.25 +/- 0.07 and 43 +/- 12 microM. The rank order for inhibition of CGamF accumulation in SCRHs was: ritonavir >> saquinavir > atazanavir > darunavir > amprenavir. Indinavir (up to 100 microM) did not alter CGamF accumulation, whereas nelfinavir solubility was limited to 10 microM. 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.
authors.affiliation: Laboratory for Pharmacotechnology and Biopharmacy, Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven, O&N2, Herestraat 49-bus 921, 3000 Leuven, Belgium.
authors.forename: Zhi-Wei
authors.initials: ZW
authors.lastname: Ye
authors.forename: Patrick
authors.initials: P
authors.lastname: Augustijns
authors.forename: Pieter
authors.initials: P
authors.lastname: Annaert
mesh_terms: D000818:Animals; D001692:Biological Transport; D005452:Fluoresceins / Q000493:pharmacokinetics*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D022781:Hepatocytes / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature
chemical_list: D005452:Fluoresceins; D017320:HIV Protease Inhibitors; C090312:cholylglycylamidofluorescein