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

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-MRP2 interactions were evaluated by measuring the fluorescence change in bile canaliculi in SCRHs in the presence or absence of MRP2 inhibitors using quantitative time-lapse imaging (QTLI) analysis. Fluorescence was negligible in SCHs from rat (r) Mrp2-deficient Eisai hyperbilirubinemic rat, suggesting that Mrp2 is primarily responsible for CDF accumulation. According to QTLI, rifampicin, cyclosporine, and 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK-571) attenuated CDF accumulation in a concentration-dependent manner, with IC 50 values (IC 50, QTLI ) of 3.02, 1.63, and 2.87 μM, respectively. The ratios of IC 50 values obtained from the biliary excretion index over the IC 50, QTLI were 1.34, 1.94, and 1.94, but ratios over IC 50 values in CDF uptake by Mrp2-expressing membrane vesicles varied more: 6.69, 3.07, and 2.43 for rifampicin, cyclosporine, and MK-571, respectively. When the IC 50, QTLI of rifampicin was corrected for the hepatocyte/medium distribution ratio, the relative ratio of IC 50, VES /IC 50, QTLI was reduced to 2.25 from 6.69 (20.2/3.02) and was close to the ratio for MK-571 (2.43, 6.96/2.87), which is thought to cross the plasma membrane by passive diffusion. Our results indicate that QTLI is a suitable method to evaluate drug-MRP2 interaction at the bile canalicular membrane, when the hepatocyte/medium distribution ratio in SCRHs is taken into account.