HPLC Method For The Study Of Kinetics Of Enzyme-catalyzed Reactions In Different Species Of Cytochrome P450 2C9 And Inhibitory Activity Evaluation Of Traditional Chinese Medicine Active Ingredients

Drug metabolizing enzymes belonging to the cytochrome P450 superfamily (CYP) influence the metabolic fate of most drugs and chemicals. Although surveys of the literature have been published concerning the role played by P450 isoforms in human drug metabolism, fewer and more contradictory data have been reported from drug metabolism studies in veterinary species.Cytochrome P450 2C (CYP2C) catalyses the biotransformation of several drugs received extensive attention.TB is an oral hypoglycaemic agent used in the treatment of adult-onset diabetes mellitus. Hydroxylation of the tolyl methyl group is the first and rate-limiting step in metabolism; the subsequent oxidation of hydroxytolbutamide (OHT) by alcohol and aldehyde dehydrogenases produces carboxytolbutamide. In humans, TB has been shown to be a suitable and sensitive substrate for CYP2C9 activity, which represents the CYP2C isoform mainly expressed in the human liver.Through establishment of hugh-pressure liquid chromatography to detect the obutamide in plasma, We found that tolbutamide and internal standard could separated completely throuth chromatographic detection condition. TB retention time was 17.8 min and IS retention time was 7.8 min. On the basis of this method, we used vitro simulated experiment to explore the tolbutamide metabolism pharmacokinetic parameters of different species of CYP 2C9. The Km of human, rabbit, rat were 0.3466?0.4861?0.3294 mol L-1,Vmax were 0.8492,0.6731,0.3787?g/min/mg protein,CLmet were 2.7606?1.4062?1.1497?L/min/mg protein?Also trying to find the microsomes metabolism of the cancer patients liver and normal intestinal and polyps intestinal microsomes.The results show that the amount of liver microsomal metabolites of the liver cancer patients was rarely, normal intestinal and polyps intestinal microsomes did not metabolites the tolbutamide.Inhibition experiments,we choose sulfaphenazolum-the best inhibitor of the vitro experiments.The results showed that the IC50 of human, rabbit, rat were 0.5678,0.5930,0.6226 ?g·mL-1, were less than 1?g·mL-1.That was proved it was strong inhibitor, In addition,10 kinds of traditional Chinese medicine were used in the vitro experiment of three species. We had found that a certain degree of inhibition, but the degree was not obvious.