Study On Koumine Metabolism In Liver Microsomes Of Human,Pig,Rat,Monkey And Dog,as Well As The Effects Of Selective Inhibitors On The Metabolism In Vitro

Koumine is one of the highest content of Gelsemium alkaloids with high efficiency and low toxicity, and the research indicates that koumine has a great pharmaceutical potential. This research adopts the koumine hydrochloride?KM?,incubated with liver microsomes of virious species in vitro, and identified by UPLC method for determining koumine and its metabolites in the liver microsomes, to explore and compare the koumine metabolism by cytochrome P450 enzyme system of human and other various species, as well as the influence of enzyme inhibitors on the metabolism. Hopefully the research results will provide a scientific basis for selection of animal model in follow-up studies on the toxicology and pharmacology of koumine.Part one: The establishment of UPLC methoad for the metabolism of koumine in liver microsomes of various speciesChromatographic conditions was determined by comparing the number of theoretical plates and separation of the peak area and other indicators.The analysis was achieved by Agilent Poroshell HPH-C18 chromatogram column?2.1mm×100mm,2.7?m?.The column temperature was 30 ?, The mobile phase composed of acetonitrile-0.1% butylamine?25:75? at flow rate of 0.3 m L·min^-1. The detection wavelength was set at 256 nm. The injection volume was 1 ?L.Results:the methodological investigation showed that this methods was sensitive, koumine and its metabolites were effectively separated.The retention time of the major metabolites and koumine were 1.85 and 4.01 min, respectively.The linear calibration curve of koumine were obtained concentration range of?0.004⁰.1? g·L^-1 and?0.1².5? g·L^-1with detection limit of 0.08 ?g and detection limit of 0.04 ?g.Intra-day and inter-day were good, and their RSDs were less than 2.42% and 3.61%.The recovery is over90%.The measured sample of koumine under room temperature, and 4? was was stable within 8h.Part two: The optimization of incubation systems of koumine with liver microsomes of various species and the study on koumine metabolism in the systemsThe incubation systems of koumine with liver microsomes of virious species were optimized in terms of koumine concentration, microsomal protein content and incubation time.The results showed that the optimal incubation time was 4h for human and pig, 3 h for rat and dog, 2 h for monkey; the liver microsomal concentration was 0.5g·L^-1; the koumine concentration was 0.25 g·L^-1.Under the optimized reaction system, the koumine metabolites in liver microsomes of human,pig, rat, monkey and dog were observed respectively 2, 2, 6, 6, 7 kinds, and the major metabolite percentage in all metabolites based on chromatographic peak area were respectively 97.73 %, 58.09 %, 89.53 %, 90.40 % and 79.06 %. In addition, another metabolite in pig liver microsome accounted for 41.91%, and two in dog accounted for 6.69% and 7.35%. The metabolic ratio and speed in descending order were monkey, dog, rat, pig and human.The whole wavelength scanning?190-400nm? shows that the composition of each component and the distribution of the metabolites and the koumine were clearly separated.It indicates that the detection wavelength of 256 nm can be on behalf of all the distribution of metabolites.Part three: Affect of CYP450 enzyme inhibitors on the metabolism of koumineThe incubated system were added specific inhibitors such as ketoconazole?CYP3A?,quinidine?CYP2D?, sulfaphenazole?CYP2C? and ticlopidine?CYP2B? to obtain the metabolites of relative generation in reaction system, and evaluate the inhibitors on the influence of the metabolites formation. Results: When the concentrations of ketoconazole is 31.25 ? mol · L^-1, it can significantly inhibit the metabolism of koumine in liver microsomes of human and other species with the degree of inhibition in 78.31% 95.76%. Further tests under different concentrations of koumine showed that ketoconazole strongly inhibit the metabolism of koumine in liver microsomes of human(IC50< 0.91?mol·L^-1), while moderately in pig, rat,monkey and dog( IC50 between 1?mol·L^-1 to 10?mol·L^-1). The results indicated that CYP 3A is the major enzymes of koumine metabolism in vitro, and then the metabolic pathway was probably through Oxidation, N-demethylation and ?-hydroxylation.The inhibition of ticlopidine, quinidine and sulfaphenazole to the rat liver microsomes of koumine metabolism were 20.43% 44.31%, and the same as the ticlopidine to monkey or dog liver microsomes. As the IC50 of ticlopidine, quinidine and sulfaphenazole were higher than 10?mol·L^-1, CYP2 B, CYP2 D and CYP2 C are unlikely to participate koumine metabolism in vivo.In summary:1. The method of UPLC for determining the koumine metabolism in liver microsomes of various species was established;2. Drug-metabolizing enzymes in liver microsomes is obviously involved in the metabolism of koumine.3. There were some differences in koumine metabolism among various species.1) There were significant differences in the constitution of the major metabolite of koumine metabolism between human and pig.2) The rentention time of the major metabolite of koumine metabolism was same in humans, rats, dogs and monkeys; and the metabolic characteristics of koumine in various species were relatively similar. But the metabolic rate and the constitution of other metabolites of koumine metabolism were differences to some extent.4. CYP3 A is the major enzymes of koumine metabolism in vitro.And it maybe catalyze koumine metabolism through the pathway of oxidation, N-demethylation,?- hydroxylation.