Chiral Separation And Study On Metabolite Profiles Of Sixteen Clausenamide Enantiomers In Vitro

Clausenamide is a chiral nootropic drug extracted from Clausena lansium(Lourd.)Skeels. It has been synthesized in the Institute of Materia Medica and has been studied in the clinical trials of Phase II. The nootropic effect of (-)-clausenamide is much better than that of (+)-clausenamide and the latter has stronger toxicity than the former. In addition, including the (-)clausenamide and (+)clausenamide, all clausenamide enantiomers have different effects on colony form frequency in human embryonic neural stem cells, and as well as on induction of LTP in hippocampal of rat, and on concentration of nerve cell cytosolic free calcium.The biotransformations of sixteen clausenamide enantiomers have been investigated in vitro in this study. The stereo-selectivity on metabolism and differences of sixteen clausenamide enantiomers has been elucidated by means of identification of metabolites and explanation of pathways. The research was mainly concerned with the following subjects:1. High performance liquid chromatographic separation of sixteen clausenamide enantiomers on chiral stationary phaseThe separation of sixteen clausenamide enantiomers had been optimized on different chiral stationary phases such as α1-AGP acid glycoprotein, chiralpak AD, chiralcel OJ, and chiralcel OD by means of reversed phase (RP) or normal phase (NP) high performance liquid chromatographic (HPLC) methods. All the clausenamide enantiomers have showed good resolution (R>1.5) at different chromatographic conditions. The possible separation mechanism of each chiral column had been discussed.2. The optimization of microsomal incubation system and the development of HPLC method for analysis of clausenamide enantiomers and their metabolites.The microsomal system was optimized by the NADPH-generating system. The RP-HPLC-PDA method for analysis of sixteen clausenamide enantiomers and their metabolites has been investigated. More than150metabolites of the enantiomers have been detected by UV detector and about120metabolites by IT-TOF MS detector from microsomal incubate system and the differences of the biotransformations of the enantiomers were displayed. 3. The structure identification of the metabolites and the proposition of the metabolic pathways of the sixteen clausenamide enantiomers.The sixteen clausenamide enantiomers were incubated in the optimized microsomal incubate system respectively. High performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC-ESI-IT-TOF) was applied for identification of metabolites of the sixteen clausenamide enantiomers. The reaction of hydroxylation, demethylation, dehydration or dehydroxylation occurred in the incubate system. Accroding to the information of the IT-TOF MS, the number of each enantiomer’s metabolites as follow.9metabolites were identified in (-)clausenamide incubation system, and9metabolites were identified in (+)clausenamide incubation system;7metabolites were identified in (-)neoclausenamide incubation system, and8metabolites were identified in (+)neoclausenamide incubation system;5metabolites were identified in (-)epiclausenamide incubation system, and8metabolites were identified in (+)epiclausenamide incubation system;6metabolites were identified in (-)epineoclausenamide system, and4metabolites were identified in (+)epineoclausenamide incubation system;8metabolites were identified in (-)csiclausenamide incubation system, and7metabolites were identified in (+)csiclausenamide incubation system;8metabolites were identified in (-)csiepiclausenamide incubation system, and8metabolites were identified in (+)csiepiclausenamide incubation system;6metabolites were identified in (-)csineoclausenamide incubation system, and7metabolites were identified in (+)csineoclauseanmide;7metabolites were identified in (-)csiepineoclausenamide incubation system, and8metabolites were identified in (+)csiepineoclauseanmide incubation system. The metabolic pathway of the sixteen clausenamide enantiomers in rat microsomal incubate were proposed based on the result.4. The stereo-selectivity of the biotransformations of the sixteen clausenamide enantiomersThe stereo-selectivity of the biotransformations of the sixteen clausenamide enantiomers had been explored by means of comparison of the metabolic results of the enantiomers in rat liver microsomal incubate under the same condition. The significant differences of stereo-selectivity and possible reason were discussed according to the types of metabolites which was produced by Cytochrome P-450isozymes. Several metabolic principles were concluded for these enantiomers of clausenamide.Therefore, the results of this study showed that the metabolism of sixteen enantiomers of clausenamides in vitro had significant difference based on their different stereo-structure. These differences had been probably produced from Cytochrome P-450isozymes which exhibit substrate enantio-selectivity to clausenamide. The differences or enantio-selectivity have provided a scientific clue for the study on pharmacological or toxicological action of the enantiomers of clausenamide.