芦西丁在不同种属肝微粒体和肝S9中Ⅰ相代谢及Ⅱ相磺酸化代谢的稳定性考察

Investigation of Metabolic Stability of Phase Ⅰ and Ⅱ Sulfation of Lucidin in Liver Microsomes and Liver S9 from Different Species

目的:采用4个种属体外肝微粒体和肝S9孵育体系评价芦西丁Ⅰ相代谢、Ⅱ相磺酸化代谢稳定性,比较其体外代谢的种属差异。方法:建立并验证了基于超高效液相色谱-高分辨质谱法(UHPLC-HRMS)的芦西丁定性、定量检测方法,将芦西丁与大鼠、小鼠、比格犬、人肝微粒体和肝S9分别进行孵育,考察其代谢稳定性参数、代谢产物和代谢途径。结果:在Ⅰ相和Ⅱ相磺酸化孵育体系中,芦西丁代谢的肝清除率大小排序为小鼠>大鼠>比格犬>人,在大鼠、比格犬、人肝微粒体和肝S9中,其代谢稳定性均良好,而在小鼠肝微粒体和肝S9中,分别表现为代谢不稳定和代谢稳定性中等;在4个种属中快速识别了5个代谢产物,其中Ⅰ相氧化产物3个,Ⅱ相磺酸化产物2个,代谢产物的生成速率与代谢稳定性结果较为一致。结论:建立的UHPLC-HRMS简便、专属性强,可用于芦西丁的代谢稳定性和代谢产物研究。芦西丁Ⅰ相和Ⅱ相磺酸化的体外代谢稳定性、代谢产物生成速率存在明显的种属差异,其在人、大鼠、比格犬中的代谢特征较为相近,可为芦西丁体内代谢研究、安全性评价及动物模型选择提供参考依据。

Objective:To evaluate the metabolic stability of lucidin by incubating liver microsomes and liver S9 from 4 species,and to compare the species differences in metabolism of lucidin in vitro.Method:A qualitative and quantitative method of lucidin based on ultra-high performance liquid chromatography-high resolution mass spectrometry(UHPLC-HRMS)was established and verified.Lucidin was incubated with rat,mouse,beagle dog,human liver microsomes and liver S9 to investigate the metabolic stability parameters,metabolites,metabolic pathways.Result:Hepatic clearance(CLh)of lucidin was in order of mouse>rat>beagle dog>human in both phase Ⅰ and phase Ⅱ incubation system.Its metabolic stability was good in rat,beagle dog and human,while it showed metabolic instability and moderate metabolic stability in mouse microsomes and liver S9,respectively.A total of 5 metabolites were rapidly identified,including 3 oxidation metabolites of phaseⅠ and 2 sulfation metabolites of phase Ⅱ.The production rate of metabolites was consistent with the results of metabolic stability.Conclusion:The established UHPLC-HRMS is simple and specific,which can be used for the study on the metabolic stability and metabolites of lucidin.Its metabolic stability and metabolite production rate in vitro are significantly different among species,the metabolic characteristics of rat and beagle dog are similar to human,which provides an important reference for subsequent research in vivo,safety evaluation and animal model selection of lucidin.