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Application of Sandwich-cultured hepatocytes for analysis of drug-drug interaction on bile canalicular transporters

应用三明治培养肝细胞分析胆小管转运蛋白的药物相互作用

基本信息

批准号：
23659076
负责人：
TAMAI Ikumi
金额：
$ 2.33万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Challenging Exploratory Research
财政年份：
2011
资助国家：
日本
起止时间：
2011 至 2012
项目状态：
已结题

项目摘要

Interplay of transporters and enzymes is essential to understand drug disposition in tissues such as liver and intestine. When we consider drug-drug interaction (DDI) on liver bile canalicular transporters,metabolites formed in the hepatocytes must be considered, since many of conjugated metabolites show higher affinity to those transporters like MRP2. We established a quantitative time-lapse imaging-based analysis (QTLI) to assess Mrp2-mediated DDIs in sandwich-cultured hepatocytes (SCHs), utilizing fluorescent probe substrate of MRP2, (5,6)-carboxy-2’,7’-dichlorofluorescein (CDF). When estradiol (E2) was chosen as affecting compound, its metabolite estradiol-17β-glucuronide (E17G) but not E2 itself was confirmed to inhibit Mrp2-mediated CDF transport. When SCRHs were preincubated with E2, fluorescence accumulated in bile canaliculi formed in SCRH was decreased depending upon both length of preincubation period and concentration of E2 given in extracellular medium. The decrease in accumulated fluorescence agreed with an increase in intracellular concentration of E17G generated in hepatocytes, suggesting that the phase II biotransformation is mirrored in MRP2-mediated transport by QTLI. Since SCHs well maintain hepatic uptake transport activity, intracellular binding and drug metabolizing activity as in vitro system, QTLI in SCHs provides a convenient platform to develop an evaluation system for transporter-based DDIs without identifying metabolites of drug candidates.

转运蛋白和酶的相互作用对于了解药物在肝脏和肠道等组织中的分布至关重要。当我们考虑肝胆小管转运蛋白上的药物相互作用 (DDI) 时，必须考虑肝细胞中形成的代谢物，因为许多缀合代谢物对 MRP2 等转运蛋白表现出更高的亲和力。我们利用 MRP2 的荧光探针底物 (5,6)-羧基-2',7'-二氯荧光素 (CDF) 建立了基于定量延时成像的分析 (QTLI)，以评估夹心培养肝细胞 (SCH) 中 Mrp2 介导的 DDI。当选择雌二醇 (E2) 作为影响化合物时，其代谢物雌二醇-17β-葡萄糖醛酸苷 (E17G) 而不是 E2 本身被证实能够抑制 Mrp2 介导的 CDF 转运。当 SCRH 与 E2 预孵育时，SCRH 中形成的胆小管中积累的荧光减少，具体取决于预孵育时间的长度和细胞外培养基中 E2 的浓度。累积荧光的减少与肝细胞中产生的 E17G 细胞内浓度的增加一致，表明 II 期生物转化反映在 MRP2 介导的 QTLI 转运中。由于SCHs像体外系统一样很好地保持了肝摄取转运活性、细胞内结合和药物代谢活性，SCHs中的QTLI提供了一个方便的平台来开发基于转运蛋白的DDIs的评估系统，而无需识别候选药物的代谢物。