Research Center for Biopharmaceutics and Pharmacokinetics, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou, China; International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education (MOE), College of Pharmacy, Jinan University, Guangzhou, 510632, China.
Research Center for Biopharmaceutics and Pharmacokinetics, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou, China; International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education (MOE), College of Pharmacy, Jinan University, Guangzhou, 510632, China.
Toxicol Lett. 2019 Oct 1;313:188-195. doi: 10.1016/j.toxlet.2019.07.007. Epub 2019 Jul 5.
Brucine is one of the main bioactive and toxic constituents of the herb drug Semen Strychni. Here we aimed to determine dosing time-dependent hepatotoxicity of brucine, and to investigate the role of metabolism in generation of brucine chronotoxicity. Brucine was administered to wild-type or Npas2 (a clock disrupted model) mice at different circadian time points for toxicity and pharmacokinetic characterization. The hepatotoxicity was evaluated by plasma alanine aminotransferase and aspartate aminotransferase measurements and histopathological analysis. The role of Cyp3a11 in brucine metabolism was determined by chemical inhibition assays and Cyp3a11-overexpressing HEK293 cells. Hepatic circadian Cyp3a11 mRNA and protein levels were determined by qPCR and Western blotting, respectively. The toxicity of brucine was more severe in the light phase [Zeitgeber time (ZT) 2 and ZT8] than in the dark phase (ZT14 and ZT20). Chemical inhibition and substrate metabolism assays suggested Cyp3a11 as a significant contributor to brucine metabolism. The Cyp3a11 mRNA, protein and activity in the livers of wild-type mice displayed significant circadian fluctuations. Npas2 ablation markedly down-regulated Cyp3a11 mRNA, protein and activity, and abrogated their circadian rhythms. The circadian time differences in brucine pharmacokinetics and liver distribution were lost in Npas2 mice, so were the time differences in brucine hepatotoxicity. In conclusion, chronotoxicity of brucine was determined by circadian variations in Cyp3a11 metabolism. The findings have implications in improving brucine (and possibly Semen Strychni) efficacy via dosing time optimization.
马钱子碱是士的宁药材中的主要生物活性和毒性成分之一。本研究旨在确定马钱子碱的时间依赖性肝毒性，并探讨代谢在马钱子碱时间毒性产生中的作用。在不同的昼夜时间点，将马钱子碱给予野生型或 Npas2（生物钟破坏模型）小鼠，以进行毒性和药代动力学特征分析。通过测定血浆丙氨酸氨基转移酶和天冬氨酸氨基转移酶水平以及组织病理学分析来评估肝毒性。通过化学抑制实验和 Cyp3a11 过表达 HEK293 细胞来确定 Cyp3a11 在马钱子碱代谢中的作用。通过 qPCR 和 Western blot 分别测定肝组织中 Cyp3a11 的昼夜节律性 mRNA 和蛋白水平。结果显示，马钱子碱的毒性在光照期（ZT2 和 ZT8）比在暗期（ZT14 和 ZT20）更为严重。化学抑制和底物代谢实验表明 Cyp3a11 是马钱子碱代谢的重要贡献者。野生型小鼠肝脏中 Cyp3a11 的 mRNA、蛋白和活性均呈现出明显的昼夜节律性波动。Npas2 敲除显著下调 Cyp3a11 的 mRNA、蛋白和活性，并消除其昼夜节律。Npas2 小鼠中马钱子碱药代动力学和肝分布的昼夜时间差异消失，马钱子碱肝毒性的时间差异也消失。综上所述，马钱子碱的时间毒性取决于 Cyp3a11 代谢的昼夜变化。这些发现对于通过优化给药时间来提高马钱子碱（可能还有士的宁）的疗效具有重要意义。
