维拉帕米代谢的微生物模型研究
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摘要
本文的主要目的是以维拉帕米为模型药物，考察微生物模型和哺乳动物在药物代谢方面的异同，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；制备微生物微粒体并进行体外孵化实验，直接证明了维拉帕米在微生物模型中的Ⅰ相代谢反应是由菌体内的CYP450酶催化的，从亚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；利用微生物模型制备萘普生和甲苯磺丁脲的代谢产物，考察微生物模型用于制备药物代谢产物的通用性。
一、维拉帕米在微生物模型中的代谢研究
通过菌株筛选实验，考察了13株真菌对维拉帕米的转化能力，其中短刺小克银汉霉AS 3.153对维拉帕米的转化比较完全，且代谢产物种类多，因此将其选作模型微生物，用于维拉帕米的代谢研究。按照优化的条件(培养基初始pH 6.5、底物浓度0.75 mg／mL和转化时间72 h)进行制备规模转化，采用半制备反相高效液相色谱法分离纯化代谢产物。通过核磁共振光谱和电喷雾离子化质谱进行结构鉴定，制备得到的5种维拉帕米代谢产物的化学结构分别为30-O-去甲基维拉帕米、20-O-去甲基维拉帕米、N-去甲基维拉帕米、α-(3-甲氨基丙基)-3，4-二甲氧基-α-异丙基苯乙腈(N-去苯乙基部分代谢产物)和N-甲基-3，4-二甲氧基苯乙胺(N-去苯乙腈部分代谢产物)，可将它们作为代谢产物对照品用于维拉帕米的代谢研究。
采用液相色谱-多级质谱联用(LC／MS~n)方法对维拉帕米及其5种代谢产物进行质谱分析，总结得到以下规律：(1)结构中都含有一个碱性较强的氮原子，电喷雾离子化条件下采用正离子检测方式灵敏度较高；(2)一级全扫描质谱中只检测到准分子离子[M+H]~+，没有出现多聚体离子或其他加合离子，也没有出现明显的热裂解碎片离子；(3)化学键断裂主要集中在碱性氮原子附近，α键裂解只发生在较长的烷基侧链，没有出现脱去N-甲基的碎片离子；(4)碎片离子主要来源于一个键的断裂。也能检测到由两个键断裂所形成的碎片离子，但相应的离子强度较弱；(5)结构中苯乙基部分未发生变化的化合物，在二级全扫描质谱中的基峰离子为含有苯乙基部分的碎片离子；这部分结构如果发生改变，则基峰离子为含有苯乙腈部分的碎片离子；(6)发生N-去甲基反应和N-去烷基反应生成的代谢产物，均出现脱去异丙基自由基的碎片离子。以上质谱断裂规律可作为判断依据，由维拉帕米代谢产物的多级质谱碎片离子，确定其结构中的苯乙腈部分、苯乙基部分和N-烷基部分是否发生变化，以推测代谢产物的化学结构。
采用LC／MS~n方法首次研究了维拉帕米在微生物模型中的代谢情况。发现维
维拉帕米代谢的微生物模型研究
拉帕米在短刺小克银汉霉AS 3.153微生物模型中代谢广泛，共生成23种代谢产
物。综合分析每种代谢产物的准分子离子、多级碎片离子和色谱保留时间，并与
维拉帕米及制备的5种代谢产物对照品进行比较，鉴定代谢产物的化学结构分别
为口一去甲基代谢产物(VMI一VM3)，N-去甲基代谢产物(VM4)，双去甲基代
谢产物(VMS一VMS)，N-去烷基代谢产物(VMg和VM10)，N-去烷基后单去
甲基代谢产物(VMll和 VM12)，单去甲基代谢产物的硫酸结合物(VM13一
VM 15)和双去甲基代谢产物的硫酸结合物(VM16~VM23)。维拉帕米在微生
物模型中主要经历4种代谢途径，包括:O一去甲基、N-去甲基、N-去烷基和硫酸
结合。上述途径还可能发生交又，生成次级代谢产物。
二、维拉帕米在哺乳动物体内的代谢研究
在有关文献报道的基石出上，采用LC侧Sn方法研究了维拉帕米在人、比格犬
和大鼠体内的代谢情况。
健康受试者口服盐酸维拉帕米80 mg后，收集不同时间的血浆样品和尿样进
行分析，共发现了28种代谢产物。其中9种为已知代谢产物，巧种为首次发现
的代谢产物，还有4种代谢产物为文献中尚未确定结合物类型的H相代谢产物，
本文确定了它们的化学结构。维拉帕米在人体内代谢生成O-去甲基代谢产物
(VMI~VM3和VM24)，N-去甲基代谢产物(VM4)，双去甲基代谢产物
(VMS、VM7、VMS和VM25)，N-去烷基代谢产物(VMg和VM10)，N-去烷
基后单去甲基或双去甲基代谢产物(VM 11、VM12和VM26)，O一去甲基代谢产
物的葡萄糖醛酸结合物(VM27一VM35)和O-去甲基代谢产物的硫酸结合物
(VM 15、VM20、VM36一vM38)。综合以上研究结果，维拉帕米在人体内有5
种主要代谢途径，包括:0一去甲基、N-去甲基、N-去烷基、葡萄糖醛酸结合及硫
酸结合，而且这些代谢途径还可能发生交叉，生成次级代谢产物。比格犬与人体
内的代谢情况相似，只缺少了1种O一去甲基代谢产物(VM24)和3种硫酸结合
物(VMZO、VM36和VM38)。
在大鼠体内发现了5种人和比格犬体内未检测到的代谢产物，分别是22，30-
O一双去甲基维拉帕米(VM6)、32一口一去甲基子人去甲基维拉帕米( VM39)以及它
们的葡萄糖醛酸结合物(VM41和VM42)和32一O-去甲基维拉帕米的葡萄糖醛
酸结合物(VM40)，均为首次发现。
三、维拉帕米在微生物模型和哺乳动物体内的代谢产物比较
维拉帕米在短刺小克银汉霉AS 3.153微生物模型和哺乳动物(包括人、比
格犬和大鼠)体内都进行了广泛代谢，分别生
Verapamil was used as a structural probe to compare the similarities and differences between microbial models and mammals on drug metabolism, and to investigate the feasibility to partly replace mammals by microbial models in drug metabolism studies. Microbial microsomes were prepared using ultracentrifuge method, and in vitro incubation proved that the phase I metabolic reactions in microbial models were catalyzed by CYP450 en2ymes, which was the material base at subcellular level of the abilities of microbial models to mimic mammalian metabolism of drug. The generality of using microbial models to prepare drug metabolites was indicated by the preparation of the metabolites of naproxen and tolbutamide.
1. The metabolism of verapamil in the microbial model
Thirteen fungus were used to evaluate the abilities to biotransform verapamil, Cunninghamella blakesleeana AS 3.153 gave the best biotransformation yield at 92.6% and produced various metabolites of verapamil, so it was chosen as the model microorganism for further investigation. In order to isolate major metabolites of verapamil in sufficient quantities for structural elucidation, the biotransformation of verapamil by C. blakesleeana AS 3.153 was carried out on preparative scale according to the optimization experiments (initial medium pH 6.5, substrate concentration 0.75 mg/mL, biotransformation time 72 h). Five major metabolites were isolated by the semipreparative HPLC and identified by NMR and ESI-MS. They were 30-0-demethyl-verapamil, 20-O-demethyl-verapamil, N-demethyl-verapamil, N-methyl-4-(3,4-dimethoxyphenyl)-4-cyano-5-methyl-hexylamine and N-methyl-2-(3,4-dimethoxyphenyl)ethyl amine, which could be used as reference substances in verapamil metabolism studies.
Using multi-stage ion trap mass spectrometric analysis, the characteristic fragment ions of verapamil and its metabolites were obtained. There was a basic nitrogen in their chemical structures, so they were all sensitive in ESI positive-ion mode. Most of the abundant fragment ions originated from single bond cleaves near the nitrogen. The metabolites with unchanged phenylethyl moiety gave the base ion including the phenylethyl moiety, whereas the metabolites with changed phenylethyl moiety showed the base ion including the phenylacetonitrile moiety. The N-demethylated or N-dealkylated metabolites gave the fragment ion formed by the loss of isopropyl group in MS/MS spectra.
Metabolites of verapamil in the microbial model of C. blakesleeana AS 3.153 were
investigated by LC/MSn method. A total of 23 metabolites were found and identified by comparisons of their chromatographic behaviors, ESI-MS, and MS/MS spectra to those of verapamil and five isolated standards, including O-demethylated metabolites (VM1 -VM3), N-demethylated metabolites (VM4), di-demethylated metabolites (VM5 - VM8), iV-dealkylated metabolites (VM9 and VM10), N-dealkylated and demethylated metabolites (VM11 and VM12), sulfate conjugates of mono-demethylated metabolites (VM13 - VM15) and sulfate conjugates of di-demethylated metabolites (VM16 - VM23). The major metabolic pathways of verapamil in the microbial model were O-demethylation, N-demethylation, N-dealkylation and sulfate conjugation. Secondary metabolism via these pathways was also evidenced.
2. The metabolism of verapamil in mammals
Metabolites of verapamil in mammals (human, dog and rat) were investigated by LC/MSn method.
A total of 28 metabolites were found in urine and plasma of healthy volunteers following single oral doses of 80 mg verapamil, among which 9 metabolites were known, 15 metabolites were novel, and 4 phase II metabolites were first gave definite structures. The metabolites of verapamil in human were O-demethylated metabolites (VM1 - VM3, VM24), N-demethylated metabolites (VM4), di-demethylated metabolites (VM5, VM7, VM8 and VM25), N-dealkylated metabolites (VM9 and VM10), TV-dealkylated and mono- or di-demethylated metabolites (VM111, VM12 and VM26), glucuronides of O-demethylated metabolites (VM27 - VM35), sulfate conjugates of O
一、维拉帕米在微生物模型中的代谢研究
通过菌株筛选实验，考察了13株真菌对维拉帕米的转化能力，其中短刺小克银汉霉AS 3.153对维拉帕米的转化比较完全，且代谢产物种类多，因此将其选作模型微生物，用于维拉帕米的代谢研究。按照优化的条件(培养基初始pH 6.5、底物浓度0.75 mg／mL和转化时间72 h)进行制备规模转化，采用半制备反相高效液相色谱法分离纯化代谢产物。通过核磁共振光谱和电喷雾离子化质谱进行结构鉴定，制备得到的5种维拉帕米代谢产物的化学结构分别为30-O-去甲基维拉帕米、20-O-去甲基维拉帕米、N-去甲基维拉帕米、α-(3-甲氨基丙基)-3，4-二甲氧基-α-异丙基苯乙腈(N-去苯乙基部分代谢产物)和N-甲基-3，4-二甲氧基苯乙胺(N-去苯乙腈部分代谢产物)，可将它们作为代谢产物对照品用于维拉帕米的代谢研究。
采用液相色谱-多级质谱联用(LC／MS~n)方法对维拉帕米及其5种代谢产物进行质谱分析，总结得到以下规律：(1)结构中都含有一个碱性较强的氮原子，电喷雾离子化条件下采用正离子检测方式灵敏度较高；(2)一级全扫描质谱中只检测到准分子离子[M+H]~+，没有出现多聚体离子或其他加合离子，也没有出现明显的热裂解碎片离子；(3)化学键断裂主要集中在碱性氮原子附近，α键裂解只发生在较长的烷基侧链，没有出现脱去N-甲基的碎片离子；(4)碎片离子主要来源于一个键的断裂。也能检测到由两个键断裂所形成的碎片离子，但相应的离子强度较弱；(5)结构中苯乙基部分未发生变化的化合物，在二级全扫描质谱中的基峰离子为含有苯乙基部分的碎片离子；这部分结构如果发生改变，则基峰离子为含有苯乙腈部分的碎片离子；(6)发生N-去甲基反应和N-去烷基反应生成的代谢产物，均出现脱去异丙基自由基的碎片离子。以上质谱断裂规律可作为判断依据，由维拉帕米代谢产物的多级质谱碎片离子，确定其结构中的苯乙腈部分、苯乙基部分和N-烷基部分是否发生变化，以推测代谢产物的化学结构。
采用LC／MS~n方法首次研究了维拉帕米在微生物模型中的代谢情况。发现维
维拉帕米代谢的微生物模型研究
拉帕米在短刺小克银汉霉AS 3.153微生物模型中代谢广泛，共生成23种代谢产
物。综合分析每种代谢产物的准分子离子、多级碎片离子和色谱保留时间，并与
维拉帕米及制备的5种代谢产物对照品进行比较，鉴定代谢产物的化学结构分别
为口一去甲基代谢产物(VMI一VM3)，N-去甲基代谢产物(VM4)，双去甲基代
谢产物(VMS一VMS)，N-去烷基代谢产物(VMg和VM10)，N-去烷基后单去
甲基代谢产物(VMll和 VM12)，单去甲基代谢产物的硫酸结合物(VM13一
VM 15)和双去甲基代谢产物的硫酸结合物(VM16~VM23)。维拉帕米在微生
物模型中主要经历4种代谢途径，包括:O一去甲基、N-去甲基、N-去烷基和硫酸
结合。上述途径还可能发生交又，生成次级代谢产物。
二、维拉帕米在哺乳动物体内的代谢研究
在有关文献报道的基石出上，采用LC侧Sn方法研究了维拉帕米在人、比格犬
和大鼠体内的代谢情况。
健康受试者口服盐酸维拉帕米80 mg后，收集不同时间的血浆样品和尿样进
行分析，共发现了28种代谢产物。其中9种为已知代谢产物，巧种为首次发现
的代谢产物，还有4种代谢产物为文献中尚未确定结合物类型的H相代谢产物，
本文确定了它们的化学结构。维拉帕米在人体内代谢生成O-去甲基代谢产物
(VMI~VM3和VM24)，N-去甲基代谢产物(VM4)，双去甲基代谢产物
(VMS、VM7、VMS和VM25)，N-去烷基代谢产物(VMg和VM10)，N-去烷
基后单去甲基或双去甲基代谢产物(VM 11、VM12和VM26)，O一去甲基代谢产
物的葡萄糖醛酸结合物(VM27一VM35)和O-去甲基代谢产物的硫酸结合物
(VM 15、VM20、VM36一vM38)。综合以上研究结果，维拉帕米在人体内有5
种主要代谢途径，包括:0一去甲基、N-去甲基、N-去烷基、葡萄糖醛酸结合及硫
酸结合，而且这些代谢途径还可能发生交叉，生成次级代谢产物。比格犬与人体
内的代谢情况相似，只缺少了1种O一去甲基代谢产物(VM24)和3种硫酸结合
物(VMZO、VM36和VM38)。
在大鼠体内发现了5种人和比格犬体内未检测到的代谢产物，分别是22，30-
O一双去甲基维拉帕米(VM6)、32一口一去甲基子人去甲基维拉帕米( VM39)以及它
们的葡萄糖醛酸结合物(VM41和VM42)和32一O-去甲基维拉帕米的葡萄糖醛
酸结合物(VM40)，均为首次发现。
三、维拉帕米在微生物模型和哺乳动物体内的代谢产物比较
维拉帕米在短刺小克银汉霉AS 3.153微生物模型和哺乳动物(包括人、比
格犬和大鼠)体内都进行了广泛代谢，分别生
Verapamil was used as a structural probe to compare the similarities and differences between microbial models and mammals on drug metabolism, and to investigate the feasibility to partly replace mammals by microbial models in drug metabolism studies. Microbial microsomes were prepared using ultracentrifuge method, and in vitro incubation proved that the phase I metabolic reactions in microbial models were catalyzed by CYP450 en2ymes, which was the material base at subcellular level of the abilities of microbial models to mimic mammalian metabolism of drug. The generality of using microbial models to prepare drug metabolites was indicated by the preparation of the metabolites of naproxen and tolbutamide.
1. The metabolism of verapamil in the microbial model
Thirteen fungus were used to evaluate the abilities to biotransform verapamil, Cunninghamella blakesleeana AS 3.153 gave the best biotransformation yield at 92.6% and produced various metabolites of verapamil, so it was chosen as the model microorganism for further investigation. In order to isolate major metabolites of verapamil in sufficient quantities for structural elucidation, the biotransformation of verapamil by C. blakesleeana AS 3.153 was carried out on preparative scale according to the optimization experiments (initial medium pH 6.5, substrate concentration 0.75 mg/mL, biotransformation time 72 h). Five major metabolites were isolated by the semipreparative HPLC and identified by NMR and ESI-MS. They were 30-0-demethyl-verapamil, 20-O-demethyl-verapamil, N-demethyl-verapamil, N-methyl-4-(3,4-dimethoxyphenyl)-4-cyano-5-methyl-hexylamine and N-methyl-2-(3,4-dimethoxyphenyl)ethyl amine, which could be used as reference substances in verapamil metabolism studies.
Using multi-stage ion trap mass spectrometric analysis, the characteristic fragment ions of verapamil and its metabolites were obtained. There was a basic nitrogen in their chemical structures, so they were all sensitive in ESI positive-ion mode. Most of the abundant fragment ions originated from single bond cleaves near the nitrogen. The metabolites with unchanged phenylethyl moiety gave the base ion including the phenylethyl moiety, whereas the metabolites with changed phenylethyl moiety showed the base ion including the phenylacetonitrile moiety. The N-demethylated or N-dealkylated metabolites gave the fragment ion formed by the loss of isopropyl group in MS/MS spectra.
Metabolites of verapamil in the microbial model of C. blakesleeana AS 3.153 were
investigated by LC/MSn method. A total of 23 metabolites were found and identified by comparisons of their chromatographic behaviors, ESI-MS, and MS/MS spectra to those of verapamil and five isolated standards, including O-demethylated metabolites (VM1 -VM3), N-demethylated metabolites (VM4), di-demethylated metabolites (VM5 - VM8), iV-dealkylated metabolites (VM9 and VM10), N-dealkylated and demethylated metabolites (VM11 and VM12), sulfate conjugates of mono-demethylated metabolites (VM13 - VM15) and sulfate conjugates of di-demethylated metabolites (VM16 - VM23). The major metabolic pathways of verapamil in the microbial model were O-demethylation, N-demethylation, N-dealkylation and sulfate conjugation. Secondary metabolism via these pathways was also evidenced.
2. The metabolism of verapamil in mammals
Metabolites of verapamil in mammals (human, dog and rat) were investigated by LC/MSn method.
A total of 28 metabolites were found in urine and plasma of healthy volunteers following single oral doses of 80 mg verapamil, among which 9 metabolites were known, 15 metabolites were novel, and 4 phase II metabolites were first gave definite structures. The metabolites of verapamil in human were O-demethylated metabolites (VM1 - VM3, VM24), N-demethylated metabolites (VM4), di-demethylated metabolites (VM5, VM7, VM8 and VM25), N-dealkylated metabolites (VM9 and VM10), TV-dealkylated and mono- or di-demethylated metabolites (VM111, VM12 and VM26), glucuronides of O-demethylated metabolites (VM27 - VM35), sulfate conjugates of O
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