title: 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: 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 IC(₅₀, 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 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.
authors.affiliation: Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Takeo
authors.initials: T
authors.lastname: Nakanishi
authors.forename: Yuta
authors.initials: Y
authors.lastname: Shibue
authors.forename: Yoko
authors.initials: Y
authors.lastname: Fukuyama
authors.forename: Kenji
authors.initials: K
authors.lastname: Yoshida
authors.forename: Hajime
authors.initials: H
authors.lastname: Fukuda
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Shirasaka
authors.forename: Ikumi
authors.initials: I
authors.lastname: Tamai
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001648:Bile Canaliculi / Q000378:metabolism; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D016572:Cyclosporine / Q000493:pharmacokinetics / Q000494:pharmacology; D004347:Drug Interactions; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D022781:Hepatocytes / Q000378:metabolism*; D006932:Hyperbilirubinemia / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011422:Propionates / Q000493:pharmacokinetics / Q000494:pharmacology; D011804:Quinolines / Q000493:pharmacokinetics / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D012293:Rifampin / Q000493:pharmacokinetics / Q000494:pharmacology
chemical_list: D018528:ATP-Binding Cassette Transporters; C423013:Abcc2 protein, rat; D005452:Fluoresceins; D005456:Fluorescent Dyes; D004364:Pharmaceutical Preparations; D011422:Propionates; D011804:Quinolines; C059141:verlukast; D016572:Cyclosporine; D012293:Rifampin