Study On Molecular Mechanism Of OATP1B1and CYP2C9Genetic Polymorphism Effect On The Metabolism And Transport Of Fluvastatin

Background:Fluvastatin is the first of all chemical synthetic cholesterol-lowering drugs, ishydroxyl methyl glutaric acyl coenzyme A (HMG COA reductase inhibitors. Thelipid-lowering dose influenced by multiple genes polymorphisms. Enzyme CYP2C9fluoride cut statin as the main metabolic enzymes, statin metabolic pathways involvedin the fluorine. And fluorine cut statin is mainly by1b1（OATP1B1） polymorphismof organic anion transport into liver cells.Objective:This topic through the wild type and mutant enzyme CYP2C9reorganization, atthe same time by building OATP1B1*5,OATP1B1*1a and*15recombinant plasmidmodel, comprehensive research fluorine laval statin in different genotype individualstransfer in vitro metabolic differences and molecular mechanism; Fluorine cuttingthrough the analysis of the in vitro study of the test data above, statin in differentgenotype individuals likely to be caused by differences in metabolism and transportprocess of side effects or ineffective treatment. For in vivo tests and provides thetheory basis for clinical rational drug use.Methods:1.Grope for statin HPLC detection method and the establishment of fluorine.2.To set up the fluvastatin incubation method in the recombinant enzyme andsample processing methods.3.Fluorine laval statin in wild type and mutant enzyme CYP2C9reorganizationin vitro enzymatic dynamics analysis. Experiment is divided into blank control group,CYP2C9*1,CYP2C9*3recombinant enzyme group, through the hatch time,recombinant enzyme concentration, substrate fluorine cut statin to fluorineconcentration in the three aspects of simvastatin in wild type and mutant weight set ofcharacteristics on the kinetics of enzyme. Reaction system of fluorine weredetermined by HPLC method cut statins and their metabolic products, comparingfluorine laval statin in CYP2C9between wild type and mutant pharmacokinetic properties, and USES the mie equation model comparing fluorine laval statin group inwild type and mutant weight parameter V_max, K_m on the kinetics of enzyme, C_lint,etc.4.To set up the fluvastatin LC-MS and cell sample processing method5.HEK293T,OATP1B1*5and*15cells culture, to extend and determined byMTT test.6.Fluorine atorvastatin in expressing OATP1B1*1a,OATP1B1*15,OATP1B1*5and HEK293T cells absorb transfer experiments. Experiment is divided into blankcontrol group, with OATP1B1*5,OATP1B1*1a and OATP1B1*15plasmid group,through the study on intake process according to the different time points; By theinfluence of different enzyme concentration point investigation on intake process;Through respectively in each group to join series concentration of fluorine in statin,studying the effect of substrate concentration on intake process. Establish LC-MSmethod is used to determination of fluoride in HEK293T cell samples cut statinconcentration, at two different genotype fluorine in plasmid model laval uptakekinetics and dynamics parameters of statin (K_m,V_max,C_Lint), the analysis of twomutations influence on fluorine cut statin transshipment.7.The statistical analysis of data. All data are deviate by mean±standard （mean±SD）, using SPSS12.0software for data processing, the difference between the groupswith the design of the t-test, statistical results P<0.05was considered the difference issignificant.Results:1.To establish the HPLC method can completely meet the requirements of therecombinant enzyme samples analysis with high sensitivity.2.Establishment of fluorine in statin incubation method in the recombinantenzyme and samples processing method can completely meet the requirements of thissubject.3.Wild-type enzyme CYP2C9*1recombinant enzyme kinetic parameters were8.96K_m, V_max and C_Lint value plus or minus19.32uM and11.20±4.10pmol/min/pmol p450s,1.25; Mutant CYP2C9*3K_m,V_max and C_Lint enzymatic kineticparameters values were1.91±1.91uM and9.33pmol/min/pmol p450s,0.425.02 mm, the metabolic activity of fluorine cut statin CYP2C9*1slightly smaller than thewild type.4.Successfully established a fluorine statin cells sample treatment method andsample of LC-MS analysis detection method, has high sensitivity and specificity.5.HEK293T,OATP1B1*15and OATP1B1*5cells grew well, form of uniformsize, boundary outline clear, visible to the nucleus, cytoplasm and bright. MTT test todetermine atorvastatin no HEK293T cell toxicity in the range of1-150uM.6.OATP1B1*5-HEK293T for fluorine cut statin uptake kinetics parameters ofK_m, V_max and C_Lint were12.35±1.32uMand3.27±0.53pmol, min^-1, protein,0.26mg^-1; OATP1B1*15-HEK293T for fluorine cut statin uptake kinetics parameters ofK_m, V_max and C_Lint were18.21±1.72uM and6.88±2.01pmol, min^-1, protein,0.38mg^-1; OATP1B1*1a-HEK293T for fluorine cut statin uptake kinetics parametersof K_m, V_max and C_Lint were17.46±2.05uM and6.32±1.23pmol protein, min^-1,mg^-1,0.36. By comparing the intake of kinetic parameters, statistical analysis （P<0.01）difference was very significant. Results show that the statin OATP1B1*5genes forfluorine cut transport capacity significantly lower than the wild type OATP1B1*1a.Conclusion:This study shows that the CYP2C9*3（mutant） metabolic fluorine cut statinability is CYP2C9*1（wild type） decreased significantly, the genetic polymorphism ofCYP2C9in fluorine laval statin metabolic transformation effect significantly;OATP1B1*5（mutant） for fluorine cut statin transfer activity of the OATP1B1*1a（wild type）, a description of the OATP1B1genetic polymorphism of fluorine cutstatins affect exact transfer function. So the clinical application of fluorine cuttingtreatment, adjusting blood fat must be high attention by CYP2C9and OATP1B1genetic polymorphism of effectiveness safe hidden trouble.