Development of the system for prediction of drug-drug interactions in hepatobiliary transport process

1. 我们成功构建了表达人有机阴离子转运多肽（OATP） 2和多药耐药相关蛋白2 （MRP2）以及大鼠Oatp4和MRP2的人、大鼠双转染。我们证实了OATP2（Oatp4）和MRP2分别在基侧和根尖侧表达，这与肝脏的生理极化表达模式是一致的。在该系统中，与其他方向相比，雌二醇-17β-葡糖苷和普伐他汀等OATP2(Oatp4)/MRP2双底物可以有效地从基室转运到根尖室。此外，通过乘以一个比例因子，大鼠体内肝脏清除率与大鼠双转染物的跨细胞清除率具有良好的相关性，这表明该系统可以用于预测体内肝脏清除率。为了评估环孢素A（CyA）和cerivastatin（CER）在临床情况下的副作用和药物相互作用，我们假设转运体参与其中，我们利用OATP2表达系统和人冷冻保存肝细胞分析了环孢素A（CyA）和cerivastatin（CER）的药物相互作用机制。结果表明，CyA能有效抑制oatp2介导的CER摄取，其抑制常数与人肝细胞和临床未结合CyA浓度几乎相当。另一方面，CyA轻微抑制CYP酶对CER的代谢。因此，我们认为其相互作用的机制之一是OATP2介导的肝脏摄取过程的抑制。为了探讨双胍类药物（降糖药）严重副作用（乳酸性酸中毒）的表达机制，我们利用人有机阳离子转运蛋白（OCT）1和2表达系统进行了转运实验，考察其在肝脏和肾脏中的转运机制。双胍类化合物可通过OCT1和OCT2转运，这表明OCT1参与肝摄取，而OCT2参与肾摄取，OCT1介导的肝摄取可能是乳酸性酸中毒的触发因素之一。少

1. We succeeded in the construction of human and rat double transfectants which express human organic anion transporting polypeptide(OATP) 2 and multidrug resistance asssociated protein 2 (MRP2), and rat Oatp4 and Mrp2, respectively. We confirmed that OATP2(Oatp4) and MRP2 expressed on the basal and apical side, which is consistent with the physiological polarized expression pattern in liver. In this system, OATP2(Oatp4)/MRP2 bisubstrates such as estradiol-17β-glucuronide and pravastatin can be transported from the basal to apical compartment efficiently, compared with other direction. Moreover, rat in vivo hepatic clearance was well correlated with transcellular clearance of rat double transfectant by multiplying a scaling factor, which suggested that this system may be able to utilize the prediction of in vivo hepatic clearance.2. To evaluate the side effects and drug-drug interactions in the clinical situations, which we hypothesized the involvement of transporters, we analyzed the … More mechanism of drug-drug interaction between cyclosporin A(CyA) and cerivastatin(CER) using OATP2 expression system and human cryopreserved hepatocytes. In result, CyA potently inhibited the OATP2-mediated CER uptake and its inhibition constant was almost comparable with that of human hepatocytes and clinical unbound concentration of CyA. On the other hand, CyA slightly inhibited CER metabolism by CYP enzymes. So we suggested that one of the interaction mechanism is inhibition of hepatic uptake process mediated by OATP2. To investigate the mechanism for the expression of severe side effects (lactic acidosis) of biguanides(antidiabetes drugs), we performed the transport assay using human organic cation transporter(OCT)1 and 2 expression system to check their transport mechanism in liver and kidney. Biguanides can be transported by OCT1 and OCT2, which suggested that OCT1 is involved in hepatic uptake, whereas OCT2 in renal uptake and that OCT1-mediated hepatic uptake may be one of the trigger for lactic acidosis. Less