重组人源CYP同工酶介导的罗通定O-去甲基代谢

应用重组人源CYP同工酶研究参与罗通定代谢的肝药酶亚型并鉴定相关代谢产物。重组人源CYP同工酶CYP1A2、2C9、2C19、2D6和3A4与1 µmol·L^?1罗通定孵育后, 应用LC-MS法测定孵育液中原形药物的剩余量。结果表明, CYP2C19、3A4和2D6参与罗通定的代谢转化, 用整体归一化法评估各酶对罗通定代谢的贡献, 分别为31.46%、60.37%和8.17%。应用LC-MSD和LC/Q-TOF-MSMS鉴定重组酶温孵样品中的代谢产物, 罗通定在体外重组人源CYP温孵体系中的主要代谢途径为O-去甲基化, 可生成4个O-单去甲基化的同分异构体以及1个双去甲基化产物, 但CYP2C19、3A4和2D6介导罗通定O-去甲基化的位点有所不同。应用LC/Q-TOF-MSMS和LC/iTrap-MSⁿ探讨了罗通定及其代谢产物的ESI质谱裂解规律。

In vitro O-demethylation of rotundine by recombinant human CYP isoenzymes

Rotundine (1 µmol·L⁻¹) was incubated with a panel of rCYP enzymes (1A2, 2C9, 2C19, 2D6 and 3A4) in vitro.  The remained parent drug in incubates was quantitatively analyzed by an Agilent LC-MS.  CYP2C19, 3A4 and 2D6 were identified to be the isoenzymes involved in the metabolism of rotundine.  The individual contributions of CYP2C19, 3A4 and 2D6 to the rotundine metabolism were assessed using the  method of total normalized rate to be 31.46%, 60.37% and 8.17%, respectively.  The metabolites of rotundine in incubates were screened with ESI-MS at selected ion mode, and were further identified using MS² spectra and precise molecular mass obtained from an Agilent LC/Q-TOF-MSMS, as well as MSⁿ spectra of LC-iTrap-MSⁿ.  The predominant metabolic pathway of rotundine in rCYP incubates was O-demethylation.  A total 5 metabolites were identified including 4 isomerides of mono demethylated rotundine and one di-demethylated metabolite.  The results also showed that CYP2C19, 2D6 and 3A4 mediated O-demethylation of methoxyl groups at different positions of rotundine.  Furthermore, the ESI-MS cleavage patterns of rotundine and its metabolites were    explored by using LC/Q-TOF-MSMS and LC/iTrap-MSⁿ techniques.