Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
Drug Metab Dispos. 2009 Oct;37(10):2103-11. doi: 10.1124/dmd.109.027193. Epub 2009 Jul 23.
Multidrug resistance-associated protein (MRP) 3/ABCC3 and MRP4/ABCC4 are ATP-binding cassette (ABC) transporters expressed in the sinusoidal membrane of hepatocytes. The purpose of the present study was to establish organic anion-transporting polypeptide (OATP) 1B1/MRP2/MRP3 and OATP1B1/MRP2/MRP4 triple transfectants as in vitro model of the hepatobiliary transport of anionic drugs. To find in vivo relevant Mrp3 probes, wild-type and Mrp3(-/-) mice were given gemfibrozil, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridymethyl)benzothiazole (E3040), troglitazone, bisphenol A, and 4-methylumbelliferone orally. Plasma concentrations of the glucuronide conjugates were significantly lower in Mrp3(-/-) mice than in wild-type mice. The systemic exposure of gemfibrozil, E3040, and troglitazone were similar in wild-type and Mrp3(-/-) mice. 4-Methylumbelliferone and bisphenol A were undetectable in the plasma. In MRP3-expressing membrane vesicles, ATP-dependent uptakes of the glucuronide conjugates of estradiol, gemfibrozil, E3040, and troglitazone were markedly greater than those in controls, whereas MRP4-expressing membrane vesicles exhibited significant ATP-dependent uptake of gemfibrozil glucuronide and estradiol glucuronide. MRP3 or MRP4 was expressed in the OATP1B1/MRP2 double transfectants using adenovirus. The expression levels of OATP1B1 and MRP2 proteins were maintained both in the OATP1B1/MRP2/MRP3 and OATP1B1/MRP2/MRP4 triple transfectants, whereas MRP3 and MRP4 were localized in the basal membrane. Significant reductions in the basal-to-apical flux of the glucuronide conjugates of estradiol, gemfibrozil, E3040, and troglitazone were observed in the OATP1B1/MRP2/MRP3 triple transfectants compared with those in the double transfectants, whereas significant reduction was observed only for gemfibrozil glucuronide and estradiol glucuronide in the OATP1B1/MRP2/MRP4 triple transfectants. These results suggest that MRP3- or MRP4-triple transfectants provide a simple and useful in vitro system for evaluating their importance in the hepatobiliary transport of drugs.
多药耐药相关蛋白（MRP）3/ABCC3 和 MRP4/ABCC4 是表达在肝细胞窦状膜上的 ATP 结合盒（ABC）转运体。本研究的目的是建立有机阴离子转运多肽（OATP）1B1/MRP2/MRP3 和 OATP1B1/MRP2/MRP4 三重转染体作为阴离子药物肝胆转运的体外模型。为了寻找体内相关的 Mrp3 探针，野生型和 Mrp3(-/-) 小鼠分别给予吉非贝齐、6-羟基-5,7-二甲基-2-甲氨基-4-(3-吡啶甲基)苯并噻唑（E3040）、曲格列酮、双酚 A 和 4-甲基伞形酮口服。与野生型小鼠相比，Mrp3(-/-) 小鼠的葡糖苷酸缀合物的血浆浓度明显降低。吉非贝齐、E3040 和曲格列酮在野生型和 Mrp3(-/-) 小鼠中的全身暴露情况相似。4-甲基伞形酮和双酚 A 在血浆中无法检测到。在 MRP3 表达的膜囊泡中，雌二醇、吉非贝齐、E3040 和曲格列酮的葡糖苷酸缀合物的 ATP 依赖性摄取明显大于对照组，而 MRP4 表达的膜囊泡表现出吉非贝齐葡糖苷酸和雌二醇葡糖苷酸的显著 ATP 依赖性摄取。用腺病毒在 OATP1B1/MRP2 双转染体中表达 MRP3 或 MRP4。OATP1B1 和 MRP2 蛋白的表达水平在 OATP1B1/MRP2/MRP3 和 OATP1B1/MRP2/MRP4 三重转染体中均得到维持，而 MRP3 和 MRP4 则定位于基底膜。与双转染体相比，OATP1B1/MRP2/MRP3 三重转染体中雌二醇、吉非贝齐、E3040 和曲格列酮的葡糖苷酸缀合物的基底到顶端通量显著降低，而 OATP1B1/MRP2/MRP4 三重转染体中仅观察到吉非贝齐葡糖苷酸和雌二醇葡糖苷酸的显著降低。这些结果表明，MRP3 或 MRP4 三重转染体提供了一种简单而有用的体外系统，用于评估它们在药物肝胆转运中的重要性。
