外排转运蛋白影响葡萄糖醛酸化代谢及其作用机制研究

Active transport of glucuronide is a critical step in drug elimination via the glucuronidation pathway. In our preliminary work, we found that activity inhibition of efflux transporters significantly reduced the amount of glucuronide excreted and increased the intracellular accumulation of glucuronide. It also significantly altered the levels of parent drug and glucuronide in the systemic circulation. Hence, glucuronide efflux has great potential to regulate the UGT metabolism and alter the tissue distribution and bioavailability of both parent drug and glucuronide. However, how the efflux transporters regulate drug metabolism as well as the underlying mechanisms thereof remain to be fully elucidated. In this proposal, we will continue to study the interplay of efflux transporters with UGTs, and to unravel the mechanisms underlying the interplay. A full Investigation of regulation of drug disposition by glucuronide efflux and the working mechanisms will be performed from three aspects, namely, the simplified systems (UGT expressed HeLa and MDCKII cells), the sandwich cultured human hepatocytes (expressing multiple transporters and many types of drug-metabolizing enzymes), and gene knock-out rodents. Further, we will establish the relationships between efflux capability and alterations in drug disposition. Our study will provide a scientific foundation for understanding of the determinant effects of transporters on drug pharmacokinetics and pharmacodynamics, and will facilitate an accurate prediction of UGT metabolism in vivo.

代谢物外排是机体内葡萄糖醛酸化代谢（UGT代谢）清除药物的关键步骤。我们在前期工作中，发现外排转运蛋白活性抑制显著降低UGT代谢物外排速率、增加代谢物胞内蓄积，影响血液循环中药物/代谢物浓度。因此，代谢物外排具有调控UGT代谢、从而极大改变药物/代谢物组织分布与生物利用度的潜在性。然而，外排影响UGT代谢的作用规律、其调控代谢的作用机制尚需深入阐明。本项目拟在前期工作基础上，继续围绕外排转运蛋白与UGT酶的相互作用关系开展研究，从三个层次、递进式考察代谢物外排对UGT代谢的调控效应、探讨调控作用机制【较简单体系（只表达UGT酶的HeLa及MDCKII细胞）、三明治构型人肝细胞（表达复杂外排转运蛋白和药物代谢酶）及基因敲除老鼠】，建立外排功能调整与药物体内处置改变的相关性，为研究和明确转运蛋白影响药动学及药效学、准确预测体内UGT代谢提供科学依据。

本项目在前期工作基础上，对外排转运蛋白如何影响葡萄糖醛酸化代谢以及影响作用机制进行了系统研究，并取得了以下研究进展：（1）发现外排蛋白功能抑制显著改变代谢物排放及胞内蓄积。在HeLa1A1细胞中，白藜芦醇或白杨素（模型药物）经由UGT1A1酶代谢，产生葡萄糖醛酸苷（代谢物）。代谢物经由细胞膜上外排蛋白介导，外排出细胞进入孵育媒介。使用MRP家族蛋白特异性抑制剂MK-571或外排转运体敲低后，代谢物外排速率及清除率显著减少，而细胞内代谢物水平显著增加。（2）阐明了外排转运调控二相代谢的机制。一般情况下，抑制外排转运体活性或降低其表达会导致代谢物外排的减少、表观外排清除率降低、总代谢率降低以及胞内代谢物水平上升。水解酶介导的结合逆反应（Deconjugation）在代谢-转运间相互作用中扮演关键作用，该作用可形容为“桥联效应”。水解酶可衔接代谢物产生与代谢物外排两个过程，使其相互作用发生。没有水解酶的“桥联效应”，代谢物产生将独立于它的下游过程——代谢物外排，代谢物外排将无法影响代谢过程。（3）构建了高表达UGT2B10的HEK293细胞，对N-葡萄糖醛酸化代谢与代谢物外排机制进行了系统研究。Mianserin或cyclizine与细胞孵育后，产生一个N-葡萄糖醛酸化代谢物及一个葡萄糖化代谢物。化学抑制（采用特异性抑制剂Ko143）或生物抑制（采用shRNA）BCRP转运体活性导致N-葡萄糖醛酸代谢物外排减少。（4）建立与验证了GCS代谢转运分类系统。采用重组酶测定代谢活性，HeLa-UGT细胞测定外排活性。根据代谢活性及外排活性大小，对8个化合物进行GCS分类。采用野生型和Bcrp敲除小鼠，对8个化合物进行生理药代动力学研究。动力学结果表明，Bcrp敲除导致Daidzein和Genistein代谢物的AUC显著增加，而其他化合物（如Maackiain）原型及代谢物AUC变化很小。通过本项目的研究，已在Biochemical Pharmacology、Molecular Pharmaceutics、Drug Metabolism and Disposition、Xenobiotica国际期刊发表SCI收录论文6篇，依托本项目已培养博士生2人、硕士生2人。