Bristol-Myers Squibb, Pharmaceutical Candidate Optimization, Department of Biotransformation, Princeton, NJ 08543, USA.
Chem Biol Interact. 2011 Sep 5;193(2):109-18. doi: 10.1016/j.cbi.2011.05.011. Epub 2011 Jul 1.
We present a comprehensive in vitro approach to assessing metabolism-mediated hepatotoxicity using male Sprague-Dawley rat liver slices incubated with the well characterized hepatotoxicant, precocene I, and inhibitors of cytochrome P450 (CYP) enzymes. This approach combines liquid chromatography mass spectrometry (LC MS) detection methods with multiple toxicity endpoints to enable identification of critical metabolic pathways for hepatotoxicity. The incubations were performed in the absence and presence of the non-specific CYP inhibitor, 1-aminobenzotriazole (ABT) and isoform-specific inhibitors. The metabolite profile of precocene I in rat liver slices shares some features of the in vivo profile, but also had a major difference in that epoxide dihydrodiol hydrolysis products were not observed to a measurable extent. As examples of our liver slice metabolite identification procedure, a minor glutathione adduct and previously unreported 7-O-desmethyl and glucuronidated metabolites of precocene I are reported. Precocene I induced hepatocellular necrosis in a dose- and time-dependent manner. ABT decreased the toxicity of precocene I, increased exposure to parent compound, and decreased metabolite levels in a dose-dependent manner. Of the isoform-specific CYP inhibitors tested for an effect on the precocene I metabolite profile, only tranylcypromine was noticeably effective, indicating a role of CYPs 2A6, 2C9, 2Cl9, and 2E1. With respect to toxicity, the order of CYP inhibitor effectiveness was ABT>diethyldithiocarbamate∼tranylcypromine>ketoconazole. Furafylline and sulfaphenazole had no effect, while quinidine appeared to augment precocene I toxicity. These results suggest that rat liver slices do not reproduce the reported in vivo biotransformation of precocene I and therefore may not be an appropriate model for precocene I metabolism. However, these results provide an example of how small molecule manipulation of CYP activity in an in vitro model can be used to confirm metabolism-mediated toxicity.
我们提出了一种综合的体外方法来评估雄性 Sprague-Dawley 大鼠肝切片中代谢介导的肝毒性，该方法使用了经过充分表征的肝毒物 precocene I 以及细胞色素 P450（CYP）酶抑制剂进行孵育。这种方法结合了液相色谱-质谱（LC-MS）检测方法和多个毒性终点，能够识别出肝毒性的关键代谢途径。孵育在不存在和存在非特异性 CYP 抑制剂 1-氨基苯并三唑（ABT）和同工型特异性抑制剂的情况下进行。在大鼠肝切片中 precocene I 的代谢产物谱与体内谱有一些相似之处，但也有一个主要区别，即环氧化物二氢二醇水解产物没有被观察到可测量的程度。作为我们肝切片代谢产物鉴定程序的示例，报告了 precocene I 的一种次要谷胱甘肽加合物和以前未报道的 7-O-去甲基和葡萄糖醛酸化代谢物。Precocene I 以剂量和时间依赖的方式诱导肝细胞坏死。ABT 降低了 precocene I 的毒性，增加了母体化合物的暴露量，并以剂量依赖的方式降低了代谢产物的水平。在所测试的同工型特异性 CYP 抑制剂中，只有曲安西龙对 precocene I 代谢产物谱有明显的影响，表明 CYP 2A6、2C9、2Cl9 和 2E1 的作用。就毒性而言，CYP 抑制剂的有效性顺序为 ABT>二乙基二硫代氨基甲酸盐∼曲安西龙>酮康唑。呋喃西林和磺胺苯哒唑没有影响，而奎尼丁似乎增强了 precocene I 的毒性。这些结果表明，大鼠肝切片不能再现报道的 precocene I 的体内生物转化，因此可能不是 precocene I 代谢的合适模型。然而，这些结果提供了一个例子，说明如何在体外模型中通过小分子操纵 CYP 活性来确认代谢介导的毒性。
