常态及病态条件下药物转运体介导依普罗沙坦的药物相互作用及其分子药代动力学机制

Eprosartan is an angiotensin II receptor antagonist, used in the treatment of hypertension in clinical patients, but the drug-drug-interactions and molecular pharmacokinetics mechanism of eprosartan are still unclear. Our previous studies have demonstrated that eprosartan was transported by Oatp1a1, Oatp1a4/Mrp2 in rat and OATP1B1/MRP2 in human, also it could inhibit the renal uptake of bestatin (an OAT substrate). Base on the results of previous research, the present research was designed to investigate the transporter-mediated molecular pharmacokinetic mechanism of eprosartan in human and rat, and the drug-drug-interactions between eprosartan, rifampicin, cefditoren and JBP485. Also, the molecular mechanism of eprosartan on increasing the expression and the function of transporters in liver and renal were investigated. Furthermore, the relations between the changes of expression of different transporters and ANTI-induced cholestasis (or renal hypertension), and whether the mechanism for the effect of ANTI-induced cholestasis (or renal hypertension) on eprosartan is related to the changing of these transporters expression would be investigated. The research would clarify the drug-drug-interactions and molecular pharmacokinetics mechanism of eprosartan in normal and pathological conditions.

临床常用抗高血压药依普罗沙坦（EPR）经药物转运体介导的药物相互作用及其分子药代动力学机制至今尚不明确。前期工作表明，EPR在肝脏中经OATP1B1及MRP2转运，同时可抑制OAT1/3底物药物的肾排泄。本课题是继前期成果基础上，采用in vivo、in situ在体肝灌流、in vitro游离肝细胞及转运体基因转染细胞摄取等先进技术，拟在整体、器官、细胞、分子以及基因水平上阐明（1）转运体介导的EPR在肝肾的转运机制；（2）EPR诱导下转运体表达改变及其相关核转录因子的调控机制；（3）EPR与利福平、头孢妥仑及JBP485的药物相互作用；（4）高血压及肝损伤病态下转运体表达的改变及此改变对EPR转运的影响。本研究将为阐明EPR药代动力学-药物相互作用-病态下EPR诱导的转运体动态变化对疾病转归的影响，从而探究正常和病态下机体对EPR处置的改变规律，对EPR的临床安全合理用药提供理论依据。

本项目建立了一系列药物的检测方法学，探讨了摄取转运体OATP介导的依普罗沙坦在肝脏的摄取和外排转运体MRP2介导的依普罗沙坦在肝脏的胆汁排泄机制，发现了依普罗沙坦诱导下肝脏外排转运体MRP2表达的改变及其机制，阐明了转运体介导的头孢妥仑在肝脏的摄取机制及其对转运蛋白的调节作用。具体为：建立了三种LC-MS/MS方法用于测定生物样品中依普罗沙坦、地高辛和头孢妥仑的浓度，并将该三种分析方法成功应用于三种药物的药代动力学研究；在体肝灌流实验表明肝脏摄取依普罗沙坦在合用Oatps抑制剂后肝摄取显著降低，提示依普罗沙坦肝脏摄取与Oatps转运体有关；体外肝切片结果表明依普罗沙坦37℃摄取量显著高于4℃，提示依普罗沙坦肝摄取并非被动扩散；而肝切片摄取及大鼠游离肝细胞中摄取抑制实验表明Oatps的抑制剂均可显著抑制依普罗沙坦的肝摄取，提示依普罗沙坦的肝脏摄取与Oatps转运体有关；应用OATP1B3-HEK293单转染细胞进行摄取实验结果提示了依普罗沙坦不经OATP1B3转运；应用OATP1B1-MDCK单转染细胞进行蓄积实验结果提示了依普罗沙坦很可能经OATP1B1转运；应用OATP1B1-, OATP1B1/MRP2-MDCKII转染细胞进行转运实验结果则提示了依普罗沙坦可能经OATP1B1转运；肝灌流实验中胆汁累积排泄率的测定结果说明了Mrp2可能参与依普罗沙坦的胆汁排泄过程，而Bcrp和P-gp不参与其胆汁排泄；应用OATP1B1-, OATP1B1/MRP2-MDCKII转染细胞进行转运实验的结果提示了依普罗沙坦可能经MRP2转运；大鼠连续给与依普罗沙坦一周后， RT-PCR方法进行Mrp2和Bsep的mRNA表达水平分析实验，结果显示Mrp2的mRNA表达上调，而Bsep的mRNA表达不变；Western Blotting 结果表明依普罗沙坦可以增加Mrp2的蛋白表达，而对P-gp、Bsep和Bcrp的蛋白表达无影响；大鼠体内药动学的实验结果显示头孢妥仑大部分经肝脏摄取；大鼠肝灌流实验则进一步确定头孢托仑摄取的靶器官是肝脏；转染细胞摄取实验，结果说明了头孢妥仑是由OATP1B1和OATP2B1摄取进入肝脏的，而与OATP1B3并不参与此过程；PCR实验结果表明，连续给予头孢妥仑能增加Oatp1a1在基因水平的表达，而Oatp1b2和Oatp1a4在基因水平无明显变化。