The Correlation Study Of The Activity Determination Of The Liver Microsome CYP2C19 And The Effect Of Drug Combination On Microsome CYP2C19 In Rat

Objective:To know and illuminate the mechanism and individual variation of medicine reaction found commonly in the clinical medication can improve therapeutic efficacy and the quality of life,which has become a subject of extensive studies concerning individual administration.The main reason of medicine reaction individual variation is the difference of drug metabolism,whose study is beneficial to clinical medication and new drug development.Liver is the main metabolic organ,in which cytopigment P450(CYP450) is the main metabolic enzyme system and one of the most drug-metabolizing apoenzymes.Cytopigment P450 that have been found 200 species take part in oxidation metabolism including not only drug but also cancerogen,steroid hormone,fatty acid.CYP2 having 15 subfamilies is the biggest family in the CYP450 family.CYP2A～2E reside in mammal,in which CYP2C is the biggest subfamilies.CYP2C9 and CYP2C19 are closely relevant to drug metabolism.During the past few years,the CYP2C19 has been studied and found that participate in hydroxylation reaction in vivo.It is well known that the activity of CYP2C19 shows individual and racial differences,also a genetic polymorphism.The individual activity of CYP2C9 induces the individual variation of drug metabolism and drug adverse reaction caused by the different blood drug level.The drug interactions using drugs metabolized by CYP2C19 together may affect therapeutic effect and creat adverse effect.Therefore,the study the relationship between the activity of CYP2C19 and rational administration is significant.The study establish rat liver CYP2C19 culture system in vitro,the method of activity determination,rat liver CYP2C19 esterase kinetics and the influence on the enzyme activity by other drugs used,in order to provide the study means to further study,to prevent drug adverse reaction,to offer theory reference to rational administration,and to comprehend the mechanism and substrate spectra of CYP450.This study is a RP-HPLC method to determine the activity of the rat liver CYP2C19 using omeprazole as exploring needle drug,to optimize rat liver cytomicrosome culture system in vitro,and to study the influence on the enzyme activity by the variation of metabolin content,also to summarize announcements and provide theory reference to drug combination.Methods:Male rates weight 190±10g were decapitated to prepare rat liver homogenate with calcium salts precipitation method,then rat liver homogenate was conserved in deep freezer(-70℃).After 2,4,6 mounths,the concentration of the protein and the total amount of CYP450 enzyme were determined.Orthogonal experimental design was used to optimize the incubating conditions of CYP2C 19 in vitro.CYP2C19 enzyme kinetics was researched using different conditions. RP-HPLC was used to determine the activity of CYP2C19 enzyme by the concentrationof omeprazole and its metabolic product 5-hydroxyl omeprazole, whose chromatographic condition:Phenomenex C₈ column(4.6 mm×150 mm,5μm), Shim-pack C₈ pre-column(4.6 mm×10 mm,5μm),the mobile phase was acetonitrile and 0.02 mmol·L^-1 phosphoric acid(26:74,v/v),the detective wavelength was at 302nm,The flow rate was 1mL·min^-1,and the column temperature was 30℃,the sample size was 20μL.The concentrations of omeprazole(OMZ) and 5- hydroxyl omeprazole(5-OHOMZ) in the culture system were determined by RP-HPLC with phenacetin as internal standard,using the ratio of omeprazole and 5-hydroxyl omeprazole(OMZ /5-OHOMZ) to express the activity of CYP2C19 esterase.The change of enzymatic active were determined with the drug usually used in combination with omeprazole using the culture condition established and the assay method of CYP2C19 enzyme,in order to investigate the influence caused by drug combination.Results:The RP-HPLC method of the activity determination of CYP2C19 enzyme by the concentrationof omeprazole and its metabolite 5-hydroxyl omeprazole is simple,rapid,and having the accuracy.The results show that omeprazole linear correlation was good among the concentration circumscription of 1～15μmol/L and 5-hydroxyl omeprazole linear correlation was good among the concentration circumscription of 0.1～5μg/L.The optimal incubating conditions is that 50 mmol/L NADPH 0.02 mL,rat liver cytomicrosome protein 0.05 mL,350μmol/L omeprazole 0.02 mL,100 mg/Lphenacetin 0.02 mL and buffer phosphate to 1 mL were incubated in the pH 7.4 incubating solution for 20 minutes.The Michaelis constant of CYP2C19 esterase is K_m=429.67 nmol/Land V_max=2.56μmol·min^-1·mL^-1,the optimum reaction temperature is 37℃and the optimum pH is 7.4.The enzyme is more sensitive to pH changes and is extremely unstable to the heat.Among the drugs used along with omeprazole,chloromycetin,Cimetidine, tanshinoneâ…¡_A,ammothamnine,paclitaxel,cyclophosphamide,cyclophosphamide have significant inhibiting effects on the activitiy of CYP2C19 esterase;phenytoin sodium,danshensu sodium have significant inducing effects;nifedipine,metoprolol, verapamil have no significant effects on the activity of CYP2C19 esterase.Conclusions:The determination method of CYP2C19 enzyme activity is simple, accurate and rapid,the simultaneous determination the concentration of omeprazole and 5-hydroxyl omeprazole is suitable for the study on CYP2C19 enzyme in vitro and vivo.Through the analysis of the CYP2C19 enzyme activity,it was found that the enzyme was more sensitive to pH changes and is extremely unstable to the heat.The study provided considerable reference value to clinical rational administration.