RT Journal Article
SR Electronic
T1 Quantitative Time-Lapse Imaging-Based Analysis of Drug-Drug Interaction Mediated by Hepatobiliary Transporter, Multidrug Resistance-Associated Protein 2, in Sandwich-Cultured Rat Hepatocytes
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 984
OP 991
DO 10.1124/dmd.111.038059
VO 39
IS 6
A1 Takeo Nakanishi
A1 Yuta Shibue
A1 Yoko Fukuyama
A1 Kenji Yoshida
A1 Hajime Fukuda
A1 Yoshiyuki Shirasaka
A1 Ikumi Tamai
YR 2011
UL http://dmd.aspetjournals.org/content/39/6/984.abstract
AB 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 IC50 values (IC50, QTLI) of 3.02, 1.63, and 2.87 μM, respectively. The ratios of IC50 values obtained from the biliary excretion index over the IC50, QTLI were 1.34, 1.94, and 1.94, but ratios over IC50 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 IC50, QTLI of rifampicin was corrected for the hepatocyte/medium distribution ratio, the relative ratio of IC50, VES/IC50, 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.