| Background:Gliclazide belongs to treat type 2 diabetes of sulfonylurea hypoglycemic drugs,mainly used for clinical treatment of non-insulin-dependent diabetes mellitus,Existing study has shown the sulfonylurea drug clinical application fall blood sugar function is individual difference,which is based on the genetic polymorphisms of metabolic enzymes and transporter is one of the important mechanisms.The research will lead to gliclazide and metabolic enzyme CYP2C9 and transporter OATP1B1intrinsic connection between the genetic polymorphism of the mechanism,clinical rational drug use of sulfonylurea drugs so as to realize individualized medication to provide theoretical basis and experimental basis.Objectives:Through the establishment of a stable high expression of wild-type and mutant CYP2C9 recombinase model and OATP1B1 recombinant plasmid model,effects of CYP2C9*1,*2 and*3 of gliclazide metabolism,OATP1B1*5,*1a,*15-HEK293T and the effect of gliclazide transport,in-depth study of CYP2C9 and OATP1B1genetic polymorphisms affecting gliclazide clinical efficacy of individual differences molecular mechanism.Methods:1.To establish a reliable and stable gliclazide in CYP2C9 recombinant enzyme in the incubation system,and establish a detection method of LC-MS quantitative cliclazide in this system,the residual concentration of gliclazide in detection of incubation system.2.Study on wild type CYP2C9*1 and mutant CYP2C9*2 and CYP2C9*3 recombinant enzyme incubation system,gliclazide concentration and incubation time,the correlation between concentration of enzyme.And through the determination of recombinant enzyme system of gliclazide,metabolism of gliclazide in comparison between wild type and CYP2C9 mutant,enzyme kinetics curve fitting,calculation and comparison of wild type and mutant incubation parameters of enzyme kinetics of Vmax in the system,Km,Clint.3.Cultivate HEK293T cells,LC-MS detection method for the establishment of gliclazide in cells,cell samples by liquid in the detection of gliclazide broken cell extracted by ethyl acetate.4.MTT was used to detect the cytotoxicity of gliclazide on HEK293T,based on OATP1B1*5-HEK293T,OATP1B1*1a-HEK293T and OATP1B1*15-HEK293T cells uptake time,substrate concentration on the uptake process of gliclazide.Through the determination of cell disruption of gliclazide in liquid uptake kinetics of Vmax,Km,Clint comparison of 3 different genotypes of plasmid in Gregory model of gliclazide.5.Statistical analysis using SPSS 12 software for data processing,all data are average standard deviation(Mean+SD)said,P<0.05 has significant difference,P<0.01 has a very significant difference,P>0.05 was no significant difference.Results:1.The determination of metabolic enzyme incubation system and HEK293T cells in the sample of cliclazide LC-MS analysis method has strong specificity,high precision,good stability,comply with the relevant requirements of biological sample analysis.2.Gliclazide at different concentrations(0.1,0.2,0.4,0.6,0.8 mg/ml)of wild type CYP2C9*1 and mutant CYP2C9*2 and CYP2C9*3 recombinant enzyme incubation system in 100 min after incubation showed gliclazide at 0.2 mg/ml CYP2C9*1,0.2 mg/ml CYP2C9*2,0.4 mg/ml 3 CYP2C9*incubation system in Gregory gliclazide metabolism rate reached more than 80%.3.Gliclazide in 0.2 mg/ml wild-type CYP2C9*1,mutant CYP2C9*2 and CYP2C9*3 recombinant enzyme incubation system were incubated for 0,10,20,40,80100120 min after termination of the reaction,the results show that the optimum incubation in different incubation system in the incubation time were 60 min,80 min,100 min.4.Gliclazide(1,2.5,5,10,20,40,80 M)systems in enzymatic kinetic parameters of Km,Vmax and CLint value were 4.97±0.56μM、197.31±3.11 pmol/min/pmol、39.69±2.51μL/min/(mg proteins)in wild type CYP2C9*1 the recombinant enzyme incubation(mg proteins);in the mutant CYP2C9*2 enzyme kinetics parameters Km,Vmax and CLint value were 5.18±0.21μM、135.35±1.06 pmol/min/pmol、26.12±1.89μL/min/(mg proteins)in the mutant CYP2C9*3 enzyme kinetics parameters Km,Vmax and CLint value were 6.57±1.82μM、102.21±4.94 pmol/min/pmol、15.55±1.71μL/min/(mg proteins).Compared with wild-type CYP2C9*1,mutant CYP2C9*3 enzyme reaction system CYP2C9*2,the gliclazide Km value increased,but Vmax and Clint were significantly decreased(P<0.05 or P<0.01),suggesting that gliclazide in mutant CYP2C9*2,the metabolic activity of CYP2C9*3 enzyme in the reaction system of CYP2C9*1 was significantly lower than the wild type.5.OATP1B1*1a-HEK293T,OATP1B1*5-HEK293T and OATP1B1*15-HEK293T cells grew well and the morphology was uniform.Determination of gliclazide on HEK293T cell toxicity has little effect in the 1-80 concentration range of M was detected by MTT method.6.Gliclazide uptake in OATP1B1*5-HEK293T cells,Vmax kinetic parameters of Km and CLint were 14.69±1.68μM,5.84±0.37pmol/min/mg/protein,0.39±0.27 l/min/(mg proteins).In the OATP1B1*15-HEK293Tcell uptake kinetics of Km,Vmax and CLint were 9.80±0.35μM 11.02±0.39 pmol/min/mg/protein,1.12±1.89 l/min/(mg proteins);and in the cellular uptake of OATP1B1*1a-HEK293T Km,Vmax CLint and the kinetic parameters were 8.19±0.86μM 12.97±0.79 pmol/min/mg/protein,1.58±0.25 l/min/(mg proteins).Compared with wild-type OATP1B1*1a-HEK293T,mutant Km and Vmax type of OATP1B1*15-HEK293T cells was not significantly different,but CLint decreased significantly(P<0.05);the mutant OATP1B1*5-HEK293T cells of gliclazide uptake of Km was significantly increased(P<0.05),but the Vmax and CLint values significantly reduced(P<0.01).That gliclazide in mutant OATP1B1*15-HEK293T,OATP1B1*5-HEK293T cells uptake ability than wild type OATP1B1*1a-HEK293T significantly decreased in OATP1B1*5-HEK293T cell uptake ability is weaker.Conclusions:1.Gliclazide can be wild-type CYP2C9*1,mutant CYP2C9*2 and mutant CYP2C9*3 metabolism,compared with wild-type CYP2C9*1,mutant CYP2C9*2,CYP2C9*3 mutant metabolic enzyme activity were significantly decreased.2.GliclazidecanbeOATP1B1*5-HEK293T,OATP1B1*15-HEK293T,OATP1B1*1a-HEK293T uptake and transport.Compared with the wild type OATP1B1*1a-HEK293T,gliclazideinmutantOATP1B1*5-HEK293T,OATP1B1*15-HEK293T cells in transport capacity decreased significantly,OATP1B1*5-HEK293T transport ability is weaker. |
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