川楝素代谢活化和肝脏毒性机理研究

Toosendanin, a limonoid compound extracted from Melia toosendan Sieb. et Zucc., possesses a wide spectrum of biological activities, among which there have been many researches on its anthelmintic, antitumor and antibotulismic efficacies. However, liver injury has been reported when toosendanin or Melia-family plants, which contain toosendanin are applied. The hepatotoxicity mechanism of toosendanin is still unclear. Our preliminary studies provided evidence for the formation of bioactive metabolites from toosendanin after incubation with NADPH-fortified human liver microsomes. These bioactive metabolites could be trapped with glutathione (GSH), and several GSH conjugates were detected. The formation of the metabolites reactive to GSH was found to require the presence of NADPH, and the coincubation of ketoconazole suppressed the generation of the GSH conjugates. Moreover, pretreatment of rats with ketoconazole reduced ALT and AST concentrations in plasma induced by toosendanin. Further study will be carried out using UPLC/Q-TOF MS technical, multiple in vitro metabolism models (hepatocytes, liver S9, liver microsomes and recombinant enzymes), and different types of nucleophilic reagents, to investigate the bioactivation process of toosendanin. Radiolabeled toosendanin will be synthesized to evaluate the covalent binding of toosendanin to biomacromolecules. Furthermore, animal models and primary human hepatocytes will be used to study the effects of enzyme inhibition, enzyme induction, and other constituents in herbal formulation on the covalent binding and hepatotoxicity of toosendanin. This research will provide scientific basis for the safety application of traditional Chinese medicines containing toosendanin and the further development of toosendanin as a new drug candidate.

川楝素是楝属植物果实和根皮中的主要成分之一，具有驱虫、抗肿瘤、抗肉毒中毒等多种药理活性，是一种重要的柠檬苦素类化合物。临床应用和实验研究都证实该化合物具有肝脏毒性，但其产生毒性的机理尚不明确。我们在以谷胱甘肽（GSH）为捕获剂的人肝微粒体代谢研究中发现了川楝素的多种GSH结合物，这提示有反应性代谢物的生成，并且这些GSH结合物的生成能够被酮康唑完全抑制；大鼠体内毒性研究显示灌胃给药前给予酮康唑能够降低川楝素的肝毒性。本课题拟在前期工作基础上，采用UPLC/Q-TOF MS技术，多种体外代谢模型如肝细胞、肝S9、肝微粒体和重组酶，以及不同类型亲核性捕获剂研究川楝素的代谢活化过程；合成放射性标记药物研究川楝素与生物大分子不可逆结合的强度和靶部位；使用动物模型和人原代肝细胞评价酶抑制、酶诱导、中药复方其它组分对共价结合和对肝毒性的影响，为川楝素深入开发和含川楝素中药的安全合理使用提供科学性依据。

川楝素是重要川楝子中的主要活性成分。本项目系统地研究川楝素的体内吸收、分布、代谢和排泄的变化规律，探讨了川楝素的代谢活化机制和可能的致肝毒性机制。体内代谢研究发现，大鼠灌胃给予川楝素单体后，其在体内发生广泛代谢，主要代谢途径是酯水解和氨基酸结合代谢，此外，大鼠的胆汁中还检测到少量的谷胱甘肽结合物。这些结果提示川楝素可发生代谢活化，生成反应性中间体。人CYP重组酶和化学抑制剂实验均表明川楝素代谢活化过程主要是CYP3A4的参与。通过在肝微粒体中加入不同的捕获试剂并合成结合物的对照品，鉴定了川楝素的反应性中间体和代谢活化途径。川楝素首先在CYP3A4酶的催化下氧化重排生成烯二醛的中间体，该中间体易与氨基酸或者谷胱甘肽中的氨基形成3-吡咯啉-2-酮的结合物；此外，可与巯基形成不稳定的S-结合物，进一步与氨基结合生成吡咯型的结合物。肝细胞和Cyp3a-null小鼠实验证明川楝素导致的肝毒性与原形关系密切，CYP3A介导的代谢活化能减弱川楝素引起的肝毒性，且这一过程与胆汁酸水平的改变相关。当代谢活化被抑制后，原形可通过诱导小鼠肝脏中Cyp7a1、Ostβ和Mrp4 基因mRNA水平使血浆和肝脏中胆汁酸水平显著升高，川楝素诱导的肝毒性也增加。本研究为指导川楝子中药临床合理使用和川楝素新临床适应症的开发提供科学依据。