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目的研究新型他克林衍生物ST09、ST10的体外代谢稳定性并分别考察了其对人肝微粒CYP450各亚型酶的抑制作用。方法采用人肝微粒体模型分别孵育ST09、ST10,液相色谱-质谱联用法测定不同孵育时间体系中剩余底物浓度,通过底物消除法计算其消除半衰期。利用LC-MS/MS测定6种CYP450主要代谢产物生成量,计算得到IC50,分别评价ST09、ST10对主要人CYP450酶的抑制活性。结果 ST09在0.1 mg·m L~(-1)人肝微粒体中消除半衰期小于1 min;其主要代谢产物ST10为NADPH非依赖性代谢,体外消除半衰期为32 min,体外固有清除率为0.043 m L·min~(-1)·mg(protein)~(-1)。ST09与ST10抑制人肝微粒体CYP3A4(咪达唑仑为底物)、CYP3A4(睾酮为底物)、CYP1A2、CYP2C9、CYP2C19、CYP2D6的IC50分别为0.42/0.25、1.27/0.81、24.92/18.21、36.53/54.34、67.64/144.9、6.43/5.30μmol·L~(-1)。结论 ST09与ST10在人肝微粒体系统代谢消除较快,两者均对人肝微粒体中CYP3A4及CYP2D6有显著抑制作用。
OBJECTIVE To study the in vitro metabolic stability of novel tacrine derivatives ST09 and ST10 and investigate their inhibitory effects on CYP450 subtypes of human liver microsomes respectively. Methods Human liver microsome model was used to incubate ST09 and ST10 respectively. The concentration of remaining substrate in different incubation time was determined by liquid chromatography - mass spectrometry. The elimination half-life was calculated by substrate elimination method. The main metabolites of 6 kinds of CYP450 were measured by LC-MS / MS, and the IC50 was calculated to evaluate the inhibitory activity of ST09 and ST10 on the major human CYP450 enzymes respectively. Results The elimination half-life of ST09 in 0.1 mg · mL -1 human liver microsomes was less than 1 min. ST10, an essential metabolite, was NADPH-independent and had an elimination half-life of 32 min in vitro and an intrinsic in vitro clearance rate of 0.043 m L · min ~ (-1) · mg (protein) -1. The IC50 of ST09 and ST10 on human liver microsomes CYP3A4 (midazolam as substrate), CYP3A4 (testosterone as substrate), CYP1A2, CYP2C9, CYP2C19 and CYP2D6 were 0.42 / 0.25,1.27 / 0.81,24.92 / 18.21, 36.53 / 54.34,67.64 / 144.9,6.43 / 5.30μmol·L -1. Conclusions ST09 and ST10 are rapidly eliminated in human liver microsomes, both of which have significant inhibitory effects on CYP3A4 and CYP2D6 in human liver microsomes.