【作者】 张鹏；

【导师】 钟大放； 陈笑艳；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2006， 硕士

【摘要】 目的：建立色谱、质谱、光谱定性和定量分析方法，分析两种CYP2C9酶的底物吲哚美辛和格列本脲在真菌中的代谢产物，探讨采用微生物模型部分替代哺乳动物进行药物代谢研究的可行性；利用微生物模型制备吲哚美辛和格列本脲的代谢产物，建立该模型用于制备药物代谢产物的基本方法。 方法：以液相色谱-电喷雾离子阱质谱联用技术为检测手段，进行吲哚美辛和格列本脲微生物转化条件的优化，建立吲哚美辛和格列本脲的微生物转化模型。按照优化的条件进行制备规模转化，应用色谱分离技术从转化液中分离纯化代谢产物，通过ESI质谱和核磁共振光谱进行结构鉴定，确定它们的化学结构。进行吲哚美辛微生物代谢模型的CYP2C9同工酶抑制研究，从细胞水平阐述微生物模型能够模拟哺乳动物药物代谢的物质基础。另外，将吲哚美辛和格列本脲在微生物模型中的代谢情况与人体的代谢情况进行了比较。 结果：从吲哚美辛的微生物转化液中分离纯化，得到吲哚美辛的3种代谢产物，分别为O-去甲基吲哚美辛(IM1)、N-去氯苯甲酰基吲哚美辛(IM2)和O-去甲基-N-去氯苯甲酰基吲哚美辛(IM3)；得到格列本脲的2种代谢产物，分别为4-反式-环己基羟基化格列本脲(GM1)和3-顺式-环己基羟基化格列本脲(GM2)，可将它们作为对照品用于吲哚美辛和格列本脲的进一步代谢研究，同时总结出了吲哚美辛、格列本脲及各自代谢物的ESI质谱断裂规律。通过CYP2C9同工酶抑制研究，直接证明了吲哚美辛在短刺小克银汉霉AS 3.910微生物模型中的Ⅰ相代谢去甲基化反应是由菌体内的CYP2C9同工酶催化的，从细胞水平阐述了微生物模型能够模拟哺乳动物药物代谢的物质基础。 结论：将微生物模型中吲哚美辛和格列本脲的代谢产物与人体的代谢产物进行比较，发现在微生物代谢模型中吲哚美辛和格列本脲的Ⅰ相代谢产物均与人体的Ⅰ相代谢产物相同，说明微生物模型具备一定的模拟人体药物代谢的能力。但吲哚美辛在微生物模型中未能形成结合型代谢产物，而人体内吲哚美辛的三种Ⅰ相代谢产物均可结合葡萄糖醛酸形成结合型代谢物，提示微生物模型与哺乳动物在药物代谢方面还存在着一定差异。更多还原

【Abstract】 Objective: Qualitative and quantitative chromatographic, mass spectrometric, spectrographic methods were developed to analyze the metabolites of indomethacin and glyburide in fungi. Indomethacin and glyburide were used as structural probe to compare the similarities and differences between microbial model and mammals on drug metabolism, and to investigate the feasibility to partly replace mammals by microbial models in drug metabolism studies. The metabolites of indomethacin and glyburide were prepared using microbial models, which proved the generality of using microbial models to prepare drug metabolites, and a basic procedure to prepare drug metabolites using microbial models was also developed.Method: The experiment conditions of the microbial model for indomethacin and glyburide biotransformation were optimized by liquid chromatography-electrospray ion trap mass spectrometric method. In order to isolate the metabolites of indomethacin and glyburide in sufficient quantities for structural elucidation, the biotransformation was carried out on preparative scale according to the optimization experiments. The main metabolites were isolated by chromatographic method and identified by NMR and ESI-MS~n. The inhibitory experiment of CYP2C9 isoenzymes in microbial model was conducted to explain the material base for the microbial models to mimic mammalian metabolism at cellular level.Results: The metabolites of indomethacin in microbial model were identified as O-desmethylindomethacin (DM, IM1), JV-deschlorobenzoylindomethacin (DBI, IM2) and O-desmethyl-N-deschlorobenzoylindomethacm (DMBI, IM3) by NMR and ESI-MS; the metabolites of glyburide were identified as 4-trans-hydroxycyclohexylglyburide (GM1) and 3-cis-hydroxycyclohexylglyburide (GM2) by NMR and ESI-MS. The isolated metabolites could be used as reference substance in human metabolic studies. Using multi-stage ion trap mass spectrometric analysis, the characteristic fragment ions of indomethacin, glyburide and their更多还原