| 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 modelThirteen 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 wereinvestigated 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 mammalsMetabolites 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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