Quantitative Time-Lapse Imaging-Based Analysis of Drug-Drug Interaction Mediated by Hepatobiliary Transporter, Multidrug Resistance-Associated Protein 2, in Sandwich-Cultured Rat Hepatocytes

Abstract

ABSTRACT:There is increasing interest in developing efficient screening platforms to predict drug-induced liver injury. Therefore, we explored a microscope-based analysis to quantitatively evaluate interaction of drugs with multidrug resistance-associated protein 2 (MRP2), essential for hepatic excretion of drugs in sandwich-cultured rat hepatocytes (SCRHs), using 5 (and 6)-carboxy-2,7-dichlorofluorescein (CDF) diacetate, which is intracellularly hydrolyzed to the fluorescent substrate CDF. Drug-

Quantitative intravital microscopy of hepatic transport

“…This result contrasts with previous fluorescence microscopy studies indicating that rifampin reduces canalicular secretion of dichlorofluorescein in cultured hepatocytes 55,56 and studies of transfected cell systems indicating that rifampin blocks human and rat MRP2-mediated transport. 53,55,56,58 The basis of this difference is unclear, but may reflect differences between the in vitro and in vivo context of the different studies.…”

“…The charge renders the molecule membrane impermeant, so that its secretion can be used to assay efflux transporter activity. Previous studies have verified that its efflux is mediated by Mrp2 and Mrp3 57 and 6-CFDA has been used to quantify hepatocyte transport in vitro 56 and in vivo. 37,38 The results of studies using 6-CFDA to assay the effect of rifampin on canalicular secretion are summarized in Figure 4.…”

“…Previous studies of cultured rat hepatocytes indicate that rifampin also inhibits Mrp2-mediated canalicular secretion. 55,56 Assaying the effect of rifampin on hepatocyte secretion using sodium fluorescein as a probe is confounded by the fact that rifampin occurs with chronic administration of rifampin. Based upon previous studies demonstrating that rifampin inhibits transport in cell systems expressing human and rat organic anion transporters, 50,52,53 we interpret our results to indicate that chronic treatment with rifampin results in inhibition of organic anion transporters in the liver of rats.…”

Evaluation of Hepatic Disposition of Paroxetine Using Sandwich-Cultured Rat and Human Hepatocytes

“…These unique features make SCH useful for the prediction of biliary intrinsic clearance, drug-drug interactions, and drug-induced hepatotoxicity (Liu et al, 1999a;Marion et al, 2007;Fukuda et al, 2008;Li et al, 2010;Nakanishi et al, 2011). The use of cultured cells that only express a single ATP-binding cassette (ABC) transporter in a transcellular transport study may lead to results that are not representative of in vivo conditions.…”

MRP2 Inhibition by HIV Protease Inhibitors in Rat and Human Hepatocytes: A Quantitative Confocal Microscopy Study

“…Nakanishi et al were the first to describe the use of fluorescence microscopy to study Mrp2 inhibition by drugs and/or drug metabolites in rat hepatocytes (Nakanishi et al, 2011(Nakanishi et al, , 2012. These investigators looked at a change of fluorescence intensity in the bile canaliculi when potential Mrp2 inhibitors were added.…”