利用微生物转化模型研究苯丙哌林的代谢产物

选用短刺小克银汉霉菌AS 3.15 3对苯丙哌林进行微生物转化研究 .根据液相色谱和质谱数据推测 ,转化产物分别为苯丙哌林单羟基化物 (约6 5 % ) ,苯丙哌林双羟基化物和苯丙哌林单羟基化物的硫酸酯结合物 .发现苯丙哌林的微生物转化与其在人体内的药物代谢有很多相似之处 ,转化菌株能产生人体Ⅰ相代谢产物和部分Ⅱ相代谢产物 .考察了影响单羟基转化产物形成的因素 ,发现以pH 6 .5 ,底物浓度 0 .0 5 % ,转化反应持续 72h等条件较为适宜 .通过半制备高效液相色谱法分离出两种主要转化产物 ,经核磁共振光谱分析确证其结构分别为 4 羟基苯丙哌林和 4′ 羟基苯丙哌林 .结果表明 ,微生物转化模型是研究苯丙哌林代谢适宜的体外模型     

短刺小克银汉霉AS 3.153菌株对安非他酮的代谢转化

目的寻找适用体外转化安非他酮的微生物菌株及采用此菌株制备吗啉环羟基化安非他酮纯品的可能性。方法首先采用液相色谱-质谱测定4种小克银汉霉对安非他酮的转化能力,确定菌株,再利用最佳菌株,采用高效液相色谱-多级质谱联用法检测转化液中安非他酮的代谢产物,并针对代谢产物2(M2)的转化进行了培养基初始pH值6水平(分别为6.0,6.5,7.0,7.5,8.0和8.5),底物浓度4水平(分别为0.025,0.05,0.1和0.2g·L-1),转化时间5水平(分别为72,96,120,144和168h)等转化条件的单因素考察,以及采用四因素三水平的正交试验设计对安非他酮进一步优化转化条件。结果根据液相色谱和质谱数据,经短刺小克银汉霉AS3.153转化,安非他酮主要形成单羟基化安非他酮和单羟基化安非他酮的硫酸结合物等3种转化产物,对M2进行分离纯化,根据质谱和核磁共振数据,确证代谢产物M2为吗啉环羟基化安非他酮。M2的最优转化条件为采用短刺小克银汉霉AS3.153培养基、初始pH8.0、底物浓度0.1g·L-1和转化时间168h时转化产率最高。结论短刺小克银汉霉AS3.153对安非他酮的转化与人体代谢结果相同,说明此菌株适宜作为研究人体药物代谢的体外模型,采用此模型及相应的优化转化条件可以制备M2纯品。     

采用微生物转化法合成5—羟基普萘洛尔

采用短刺小克银汉霉菌（Cunninghamella blakesleana AS3.153）对普萘洛尔进行了代谢转化研究。液相色谱-多级质谱和核磁分析结果证明，转化液中主要含有5-羟基普萘洛尔转化产物；考察了单羟化物形成的影响因素，发现在pH6.5、底物浓度0.25%和转化反应持续48h等条件下，5-羟基普萘洛尔的产率可达76%，为活性药物代谢产物的合成开辟了一条新途径。     

短刺小克银汉霉AS 3.153菌株对丁咯地尔的代谢转化

建立能够模拟丁咯地尔在哺乳动物体内代谢的微生物模型,研究丁咯地尔在微生物体内的转化途径,并应用该模型制备代谢产物。菌株筛选实验中考察了4种小克银汉霉对丁咯地尔的转化能力,确定最佳菌株为短刺小克银汉霉AS 3.153;然后针对该菌株进行转化条件(培养基初始pH、底物浓度和转化时间)优化,建立微生物模型。应用LC-MSn法对转化样品进行分析,共检测到3种代谢产物,其中2种为首次发现的丁咯地尔代谢产物,进一步采用半制备液相色谱法制备获得对位-O-去甲基丁咯地尔和12-C-氧化丁咯地尔。哺乳动物对比实验表明,短刺小克银汉霉AS 3.153对丁咯地尔的代谢情况与人和比格犬类似,而与大鼠差异较大。     

短刺小克银汉霉AS3.153菌株对雷诺嗪的代谢转化

目的建立能够模拟雷诺嗪在哺乳动物体内代谢的微生物模型,并应用该模型制备代谢产物。方法采用液相色谱-质谱联用法测定雷诺嗪在4种小克银汉霉转化模型中的转化产物,选择其中转化能力最强的菌株,针对雷诺嗪3种主要代谢产物进行了培养基初始pH值、底物质量浓度、转化时间等转化条件的优化。结果短刺小克银汉霉AS 3.153对雷诺嗪的转化能力最强,将其转化为9种代谢产物,采用半制备液相色谱法制备分离得到3种主要代谢产物,确证其结构分别为羟基化雷诺嗪及雷诺嗪硫酸酯结合物。结论短刺小克银汉霉AS 3.153对雷诺嗪的转化与哺乳动物代谢结果类似,可作为模拟哺乳动物体内代谢的体外模型使用。     

短刺小克银汉霉AS 3.910的CYP2C9同工酶抑制作用

目的探索具有药物代谢酶CYP2C9活性的微生物模型对CYP2C9抑制剂的响应。方法选用短刺小克银汉霉AS 3.910为模型菌株， 检测CYP2C9抑制剂对CYP2C9底物特定转化产物产率的影响， 并通过底物间相互影响的程度探索该转化体系中的药物代谢相互作用。 采用液相色谱-多级质谱联用技术检测转化产物。 结果 CYP2C9抑制剂苯溴马隆抑制4′-羟基甲苯磺丁脲的生成，使其产率由100%下降到14.5%；磺胺苯吡唑抑制O-去甲基吲哚美辛的生成，使其产率由75.2%降低到9.9%；丙戊酸抑制4′-羟基双氯芬酸的生成，使其产率由98.6%降低到2.7%。底物药物甲苯磺丁脲、双氯芬酸和吲哚美辛之间存在代谢相互作用，导致生成相应代谢物的产率降低。结论3种CYP2C9抑制剂不同程度地抑制了短刺小克银汉霉的CYP2C9同工酶，而且CYP2C9底物间存在药物代谢相互作用。     

采用微生物转化J法合成5-羟基普萘洛尔

采用短刺小克银汉霉菌(Cunninghamella blakes1eana AS 3.153)对普萘洛尔进行了代谢转化研究.液相色谱-多级质谱和核磁分析结果证明,转化液中主要含有5-羟基普萘洛尔转化产物;考察了单羟化物形成的影响因素,发现在pH 6.5、底物浓度0.25%和转化反应持续48 h等条件下,5-羟基普萘洛尔的产率可达76%,为活性药物代谢产物的合成开辟了一条新途径.     

萘普生的微生物转化

目的研究微生物对萘普生的转化能力。方法以雅致小克银汉霉菌AS 3 1 5 6为转化菌株 ,并对转化条件进行优化 ,利用液相色谱 质谱联用法检测转化液中萘普生的代谢产物。结果根据液相色谱和质谱数据推测 ,主要转化产物为O 去甲基萘普生。对影响O 去甲基萘普生形成因素的考察发现 ,以pH 6 0 ,底物浓度 0 0 2 5 % ,转化反应持续 72h等条件较为适宜 ,其产率大于 95 %。结论雅致小克银汉霉菌AS 3 1 5 6能够高度专一性地对萘普生进行O 去甲基转化反应 ,可以作为药物代谢研究新的体外模型。     

微生物转化法从雷公藤甲素合成雷腾舒

目的:以雷公藤甲素为原料,采用微生物转化法合成新型抗类风湿药物雷腾舒。方法:筛选10株小克银汉霉菌,采用HPLC法检测转化液中目标产物雷腾舒的浓度并计算产率,优化转化系统。结果:模型菌株雅致小克银汉霉ATCC36112在pH 4.5、底物浓度400μg.mL-1和转化10 d等条件下,雷腾舒产率>70%。结论:小克银汉霉ATCC36112转化雷公藤甲素,为活性代谢产物雷腾舒的合成开辟了一条新途径。     

短刺小克银汉霉菌对马兜铃酸A的代谢转化

选取短刺小克银汉霉菌（Cunninghamella blakesleana AS3．910）用微生物转化的方法对马兜铃酸A（Aristolochic acid）进行了代谢转化研究并比较，微生物转化产物和哺乳动物体内的代谢产物的异同。考察了影响微生物转化产物形成的各种因素，并对该转化体系进行了优化，采用高效液相色谱、质谱对代谢产物进行了检测和分析。多种因素条件会对微生物的转化产生影响，发酵液中主要的转化产物是马兜铃酸A的O去甲基化物，该产物和报道的NSL动物在摄入马兜铃酸A后尿中发现的一种代谢产物结构相同。利用小克银汉霉菌的转化可以方便地制备马兜铃酸A在哺乳动物体内的某些代谢产物，它可以作为研究马兜铃酸A代谢物的一个体外模型进行探索。     

Biotransformation of metoprolol by the fungus Cunninghamella blakesleeana

Aim： To investigate the biotransformation of metoprolol, a β-cardioselective adrenoceptor antagonist, by filamentous fungus, and to compare the parallels between microbial transformation and mammalian metabolism. Methods： Five strains of Cunninghamella （ C elegans AS 3.156, C elegans AS 3.2028, C echinulata AS 3.2004, C blakesleeana AS 3.153 and AS 3.910） were screened for the ability to transform metoprolol. The metabolites of metoprolol produced by C blakesleeana AS 3.153 were separated and assayed by liquid chromatography-tandem mass spectrometry （LC/MSn）. The major metabolites were isolated by semipreparative HPLC and the structures were identified by a combination of LC/MSn and nuclear magnetic resonance analysis. Results： Metoprolol was transformed to 7 metabolites; 2 were identified as new metabolites and 5 were known metabolites in mammals. Conclusion： The microbial transformation of metoprolol was similar to the metabolism in mammals. The fungi belonging to Cunninghamella species could be used as complementary models for predicting in vivo metabolism and producing quantities of metabolite references for drugs like metoprolol.     

小克银汉霉菌对萘普生的微生物转化

目的：通过分离和鉴定萘普生由真菌转化生成的代谢产物,研究微生物转化和哺乳动物体内药物代谢之间的相似性．方法：选用3种小克银汉霉菌对萘普生进行微生物转化研究．采用液相色谱—质谱联用法检测代谢产物,并通过半制备高效液相色谱法分离出主要代谢物,经质谱和核磁共振光谱确证其结构．结果：萘普生被转化成去甲萘普生和去甲萘普生硫酸结合物,这两种代谢产物都是已知的哺乳动物代谢产物．其中去甲萘普生硫酸结合物是首次在微生物转化样品中发现．结论：小克银汉霉菌对萘普生的转化结果与哺乳动物的体内代谢有某些相似性,有可能成为一种新的体外模型进行药物代谢研究,用于预测和制备可能的药物代谢产物．     

Biotransformation of pantoprazole by the fungus Cunninghamella blakesleeana

To investigate the biotransformation of pantoprazole, a proton-pump inhibitor, by filamentous fungus and further to compare the similarities between microbial transformation and mammalian metabolism of pantoprazole, four strains of Cunninghamella (C. blakesleeana AS 3.153, C. echinulata AS 3.2004, C. elegans AS 3.156, and AS 3.2028) were screened for the ability to catalyze the biotransformation of pantoprazole. Pantoprazole was partially metabolized by four strains of Cunninghamella, and C. blakesleeana AS 3.153 was selected for further investigation. Three metabolites produced by C. blakesleeana AS 3.153 were isolated using semi-preparative HPLC, and their structures were identified by a combination analysis of LC/MS(n) and NMR spectra. Two further metabolites were confirmed with the aid of synthetic reference compounds. The structure of a glucoside was tentatively assigned by its chromatographic behavior and mass spectroscopic data. These six metabolites were separated and quantitatively assayed by liquid chromatography-ion trap mass spectrometry. After 96h of incubation with C. blakesleeana AS 3.153, approximately 92.5% of pantoprazole was metabolized to six metabolites: pantoprazole sulfone (M1, 1.7%), pantoprazole thioether (M2, 12.4%), 6-hydroxy-pantoprazole thioether (M3, 1.3%), 4'-O-demethyl-pantoprazole thioether (M4, 48.1%), pantoprazole thioether-1-N-beta-glucoside (M5, 20.6%), and a glucoside conjugate of pantoprazole thioether (M6, 8.4%). Among them, M5 and M6 are novel metabolites. Four phase I metabolites of pantoprazole produced by C. blakesleeana were essentially similar to those obtained in mammals. C. blakesleeana could be a useful tool for generating the mammalian phase I metabolites of pantoprazole.     

Biotransformation of indomethacin by the fungus Cunninghamella blakesleeana

AIM: To investigate the biotransformation of indomethacin, the first of the newer nonsteroidal anti-inflammatory drugs, by filamentous fungus and to compare the similarities between microbial transformation and mammalian metabolism of indomethacin. METHODS: Five strains of Cunninghamella (C elegans AS 3.156, C elegans AS 3.2028, C blakesleeana AS 3.153, C blakesleeana AS 3.910 and C echinulata AS 3.2004) were screened for their ability to catalyze the biotransformation of indomethacin. Indomethacin was partially metabolized by five strains of Cunninghamella, and C blakesleeana AS 3.910 was selected for further investigation. Three metabolites produced by C blakesleeana AS 3.910 were isolated using semi-preparative HPLC, and their structures were identified by a combination analysis of LC/MS(n) and NMR spectra. These three metabolites were separated and quantitatively assayed by liquid chromatography-ion trap mass spectrometry. RESULTS: After 120 h of incubation with C blakesleeana AS 3.910, approximately 87.4% of indomethacin was metabolized to three metabolites: O-desmethylindomethacin (DMI, M1, 67.2%), N-deschlorobenzoylindomethacin (DBI, M2, 13.3%) and O-desmethyl-N-deschlorobenzoylindomethacin (DMBI, M3, 6.9%). Three phase I metabolites of indomethacin produced by C blakesleeana AS 3.910 were identical to those obtained in humans. CONCLUSION: C blakesleeana could be a useful tool for generating the mammalian phase I metabolites of indomethacin.     

Microbial models of mammalian metabolism: microbial transformation of naproxen

Preparative-scale fermentation of S-naproxen, the known antiinflammatory, analgesic and antipyretic drug, with Cunninghamella elegans ATCC 9245 afforded S-demethylnaproxen, the known human active metabolite of naproxen, in a 90% yield. Demethylnaproxen was also detected as the major metabolite of naproxen using Cunninghamella blakesleeana ATCC 8688a. A review of the previous microbial metabolism studies using the fungi Cunninghamella species suggested that it could be a plausible in vitro predictor for mammalian metabolism.     

Biotransformation of pantoprazole by the fungus Cunninghamella blakesleeana

To investigate the biotransformation of pantoprazole, a proton-pump inhibitor, by filamentous fungus and further to compare the similarities between microbial transformation and mammalian metabolism of pantoprazole, four strains of Cunninghamella (C. blakesleeana AS 3.153, C. echinulata AS 3.2004, C. elegans AS 3.156, and AS 3.2028) were screened for the ability to catalyze the biotransformation of pantoprazole. Pantoprazole was partially metabolized by four strains of Cunninghamella, and C. blakesleeana AS 3.153 was selected for further investigation. Three metabolites produced by C. blakesleeana AS 3.153 were isolated using semi-preparative HPLC, and their structures were identified by a combination analysis of LC/MSⁿ and NMR spectra. Two further metabolites were confirmed with the aid of synthetic reference compounds. The structure of a glucoside was tentatively assigned by its chromatographic behavior and mass spectroscopic data. These six metabolites were separated and quantitatively assayed by liquid chromatography-ion trap mass spectrometry. After 96?h of incubation with C. blakesleeana AS 3.153, approximately 92.5% of pantoprazole was metabolized to six metabolites: pantoprazole sulfone (M1, 1.7%), pantoprazole thioether (M2, 12.4%), 6-hydroxy-pantoprazole thioether (M3, 1.3%), 4′-O-demethyl-pantoprazole thioether (M4, 48.1%), pantoprazole thioether-1-N-β-glucoside (M5, 20.6%), and a glucoside conjugate of pantoprazole thioether (M6, 8.4%). Among them, M5 and M6 are novel metabolites. Four phase I metabolites of pantoprazole produced by C. blakesleeana were essentially similar to those obtained in mammals. C. blakesleeana could be a useful tool for generating the mammalian phase I metabolites of pantoprazole.