“UGT2B10酶-外排转运体”通路调节生物碱代谢与处置的作用机制研究

Alkaloids contain a large number of drugs/natural products with various structures and different bioactivities, many of which exhibit obvious toxicities to humans. Understanding the metabolism and disposition pathway of alkaloid is helpful in guiding their safety and rational use. Our previous studies have discovered that the UGT2B10-mediated N-glucuronidation is a common metabolic pathway for many alkaloids. We unravel that N-glucuronidation show significant species differences, providing a good explanation for the ignorance of such pathway in pre-clinical studies. In addition, we reveal that the N-glucuronidation of alkaloid is regulated by efflux transporters in UGT2B10-overexpressing HEK293 cells. However, the metabolic patterns of UGT2B10-mediated N-glucuronidation are not clear, and the roles of efflux transporters in regulating intracellular N-glucuronidation remain to be determined. In this proposal, we will systematacially evaluate the N-glucuronidation of alkaloids by using several methods, including reaction phenotyping, kinetic analysis, correlation analysis, inhibition experiment, and species difference analysis. The contribution and significance of UGT2B10 in N-glucuronidation will be clarified. Further, we will establish HEK293/MDCKII cell lines overexpressing UGT2B10 and efflux transporters (BCRP and MRPs) to study the metabolism and disposition of alkaloids. Vesicular transport assay and sandwich cultured human hepatocytes will also be used to elucidate the underlining mechanism of efflux transporter in regulating cellular N-glucuronidation reaction. This study will help to deepen our understanding of the metabolism and disposition of alkaloids in human, and provide guidance for pharmacokinetic and toxicological studies of alkaloids.

生物碱包含了多种结构及不同活性的药物和天然产物，其中多数物质还具有明显的毒性。明确生物碱的代谢和处置行为有助于指导该类物质的安全与合理使用。我们发现由UGT2B10酶介导的N-葡萄糖醛酸化代谢（NG代谢）是许多生物碱的共有代谢途径，但其在临床前研究中往往被忽略。并且，我们发现外排转运体可调控细胞内UGT2B10介导的NG代谢。然而，UGT2B10对生物碱的NG代谢行为和规律尚不明确，外排转运体对细胞内NG代谢的调控作用及机制仍需进一步阐明。本项目拟对生物碱的NG代谢进行全面表征，明确UGT2B10的重要性及其底物选择性。此外，我们将构建高表达/敲低UGT2B10和外排转运体的细胞模型，并结合囊泡转运模型和“三明治”肝细胞模型，阐明外排转运体对UGT2B10介导的NG代谢的调控作用及机制。本研究将有利于阐明生物碱类药物/毒物在人体内的代谢和处置过程，为生物碱的药代动力学和毒理学研究提供指导。

本研究在前期工作基础上，对UGT酶及外排转运体介导的生物碱代谢及处置过程进行了研究，并取得了以下研究进展：1）筛选发现多种生物碱能被UGT1A4和UGT2B10代谢，且该代谢途径具有明显的种属差异性；2）测定了一系列生物碱（士的宁、马钱子碱、米氮平、米安色林、苯甲嗪、氯苯甲嗪、洛沙平、多塞平、度硫平、环苯扎林、阿塞那平、氯氮平、伊马替尼、异丙嗪和氯丙嗪）在HLM、UGT1A4和UGT2B10中的代谢动力学，发现UGT2B10的代谢亲和力更高（Km更小），表明UGT2B10对该类生物碱的葡萄糖醛酸化代谢具有重要贡献；3）构建并表征了高表达UGT1A4和UGT2B10代谢酶的细胞系，发现阿米替林、米安色林和苯甲嗪在UGT2B10细胞中可同时产生葡萄糖醛酸苷和葡萄糖苷代谢物，并采用人微粒体对该反应进行了表征；4）进一步研究发现BCRP和MRP4是介导葡萄糖醛酸苷代谢物外排的主要转运体，而BCRP是介导葡萄糖苷代谢物外排的主要转运体，抑制外排转运体活性会改变细胞内代谢酶活性，且葡萄糖苷化代谢可作为葡萄糖醛酸化代谢的补偿途径；5）发现士的宁和马钱子碱的细胞毒性在发生UGT代谢的细胞中显著降低，表明UGT代谢是士的宁和马钱子碱的解毒途径；6）发现中药马钱子和雷公藤的毒性具有时辰依赖性，且主要毒性成分（士的宁、马钱子碱、雷公藤甲素）的时辰药代动力学是引起中药时辰毒性的原因之一。通过本项目的研究，已在Drug Metabolism and Disposition、Biochemical Pharmacology、Current Drug Metabolism和Journal of Pharmacy and Pharmacology期刊上发表SCI论文5篇，依托本项目已培养硕士研究生2名，出版学术专著2部，获广东省药理学会2020年科技成果奖二等奖。