FIGON Medicines Days 2008 location:Lunteren (NL) date:6-8 October 2008
Introduction. In addition to drug metabolizing enzymes, hepatic transporters are nowadays recognized as major factors determining hepatic drug disposition and elimination. Moreover, drug transporters have been implicated in severe drug-drug interactions and hepatotoxicity. Interference of drugs with transporter-mediated uptake and excretion of bile acids in the liver is one such mechanism underlying potential hepatotoxicity. Primary cultured hepatocytes remain the gold standard as a model for this kind of in vitro interaction studies, in order to obtain more information on the interactions of antiviral drugs with efflux transport we deemed it useful to examine the differential interaction potential of eight HIV protease inhibitors with efflux of 5 (and 6)-carboxy-2',7'-dichlorofluorescein diacetate (CDFDA) in sandwich-cultured human hepatocytes.
Methods. Non fluorescent CDFDA passively diffuses into hepatocytes, where it is rapidly hydrolyzed by intracellular esterases to fluorescent CDF, which is actively excreted by MRP2 across the canalicular membrane into the bile canalicular networks of sandwich-cultured human hepatocytes. The efflux of CDF was evaluated by comparison of efflux in the presence and absence of Calcium in the medium after loading of 10 µM CDFDA for 10 min. inhibition tests were conduct in the presence of 8 HIV protease inhibitors during the loading process. All data are the result of three independent experiments (3 livers) with triplicate incubations.
Results. The difference of efflux of CDF between standard buffer and calcium free buffer were significant, more than 40% higher in calcium free buffer were found due to the open of tight junction. Selective MRP2 inhibitor MK571 completely blocked the efflux of CDF from canalicular membrane; the efflux of CDF in standard buffer is comparable with these in calcium free buffer after treatment of MK571. almost all the HIV PIs show inhibition of efflux of CDF in sandwich-cultured human hepatocytes. Atazanavir, lopinavir, saquinavir and darunavir were the most potent inhibitor in present study, while amprenavir and nelfinavir were less potent.
Conclusions. SCRH is a useful model for in vitro evaluation of inhibition of efflux transporters by drugs. In vitro data on inhibition of efflux transporters may help to understand mechanisms underlying side-effects of certain PIs.