ISSX European Regional Meeting edition:10 location:Vienna date:18-21 May 2008
Introduction. Interference of drugs with transporter-mediated uptake and excretion of bile acids in the liver has been recognized as a mechanism underlying drug-induced hepatotoxicity. In order to obtain more insight into species differences of the interactions of antiviral drugs with active hepatic bile acid transport, the interaction potential of eight HIV protease inhibitors (PI) with hepatic uptake of the bile salt analogue cholyl-glycylamido-fluorescein (CGamF) was determined in rat, dog and human hepatocytes.
Methods. The bile acid conjugate CGamF (kindly provided by Alan Hofmann, University of California, San Diego) was used as a fluorescent probe substrate. We had previously observed that CGamF accumulation in rat hepatocytes is mainly mediated by the Oatp family of transporters. Sandwich-cultured (SC) rat hepatocytes (isolated in-house) or SC dog and human hepatocytes (kindly provided by Biopredic International, France) were used as in vitro model systems expressing bile salt uptake transporters. CGamF accumulation kinetics were studied in rat and dog hepatocytes, while inhibition by HIV PI was studied in the three species at 1 µM CGamF. Incubations were carried out in duplicate (human, dog) or triplicate (rat) and in 2-3 hepatocyte batches.
Results. Initial hepatic uptake rates of CGamF in SC rat and dog hepatocytes were concentration-dependent and could be described by a model incorporating both a Michaelis-Menten (saturable) and a first-order (linear) component. The average Km and Vmax were 9.3 ± 2.6 µM and 401 ± 73 pmol/mg protein/min for rat, and 22.1 ± 15.3 µM and 781 ± 521 pmol/mg protein/min for dog. The active component of CGamF (1µM) accumulation represented 76% and 58% in rat and dog, respectively. Significant inhibition of CGamF accumulation by the Oatp/OATP inhibitors rifampicin (RFP) and digoxin (DGX) was observed. RFP caused 70% inhibition in rat hepatocytes compared to 40% and 35% in dog and human. DGX decreased CGamF accumulation in rat by more than 60%, while only 20% reduction was noted in dog and human. Inhibition of CGamF accumulation by eight HIV PI differed largely among species. Amprenavir (APV) and atazanavir (ATV) showed concentration-dependent (1-50 µM) inhibition (10-60%) of CGamF accumulation in rat hepatocytes, whereas the inhibition observed in dog (20%) and human hepatocytes (40%) was concentration-independent. Saquinavir and ritonavir elicited the largest extent of inhibition in the rat (50-70 % inhibition at 50 µM), while for human and dog, max 30% inhibition was observed. Indinavir caused concentration-dependent inhibition (20-40%) in dog and human, while a 20% inhibition in rat was only observed at the highest concentration (50µM).
Conclusions. The overall interaction profile for HIV PIs with CGamF accumulation correlated best between dog and human.