Molecular Mechanism Of CYP2C9 And OATP1B1/1B3 Genetic Polymorphism Effect On The Metabolism And Transport Of Glimepiride,Glibenclamide,Glipizide

Background:Sulfonylurea hypoglycemic agents are one of the main hypoglycemic agents currently used in the treatment of non-insulin-dependent diabetes mellitus.Although widely used,their individual effects on hypoglycemic effect and toxic side effects have brought troubles and risks to clinical applications.The gene polymorphisms of OATP1B1 and OATP1B3 affect the functional activity of the transporter.The genetic polymorphism of CYP2C9 also leads to the change of enzyme activity.This is an important reason leading to individual differences in drugs clinical applications.It has been confirmed that glimepiride,glibenclamide and glipizide are substrates for CYP2C9,and our previous study showed that glimepiride is a transport substrate for OATP1B1 uptake,glibenclamide and glipizide are OATP1B3 transports substrates.The transport and metabolism of glimepiride glibenclamide and glipizide in vivo may be affected by the polymorphism of the gene,leading to individual differences in efficacy and toxic and side effects.The purpose of this paper is to investigate the effect of the genetic polymorphisms of transporter OATP1B1/1B3 and metabolic enzyme CYP2C9 on the transport and metabolism of glimepiride glibenclamide and glipizide.Provide theoretical basis and experimental basis for rational use of sulfonylurea hypoglycemic agents in clinical practice.Objectives:The CYP2C9*1,*2,*3 recombinase model and stable high expression of OATP1B1*1a,*5,*15 and OATP1B3,OATP1B3（344T>G）,OATP1B3（699G> A）recombinant plasmid model was established to study the effects of genetic polymorphisms of the recombinase CYP2C9 and transporter OATP1B1/1B3 on the transport and metabolism of glibenclamide and glipizide.Investigate the molecular mechanisms of transport and metabolism in individual treatment of glimepiride,glibenclamide and glipizide that produce individual differences in clinical treatment.Methods:1.The incubation system of CYP2C9 recombinase to glimepiride,glibenclamide and glipizide was established and a sample of LC-MS detection method was established.2.Enzymatic Kinetics analysis method that CYP2C9 recombinase at different mutation sites to glimepiride glibenclamide and glipizide was performed.The experiments were divided into CYP2C9*1（wild-type）,CYP2C9*2,and CYP2C9*3,blank control groups was set for every group.Incubate system including 2μL glimepiride glibenclamide and glipizide,recombinant enzyme 0.4 mg/ml.After 30 min of incubation,100 μl of ice acetonitrile was added to stop the reaction.LC-MS quantitative detection method was used to detect the remaining concentration in the incubation system.The enzymatic kinetic parameters V_max,Km,Clint of CYP2C9 recombinase at different mutation sites were compared.3.A method for culturing HEK293 T cells was established and LC-MS detection methods for glimepiride glibenclamide and glipizide in the cells were established.The cell samples were extraction with ethyl acetate.4.The cytotoxicity of glimepiride,glibenclamide and glipizide on HEK293 T cells was measured by MTT assay.On this basis,glimepiride in OATP1B1*5-HEK293 T,OATP1B1*1a-HEK293 T and OATP1B1*15-HEK293 T cells were studied.The experiments were divided into blank control groups OATP1B1,OATP1B1*5 and *15 plasmid groups.The effects of time（2,5,10,15,20,30 min）and the effects of different substrate concentrations（2.5,5,10,20,40,80μM）on the uptake of glimepiride were investigated.To investigate the transport of glibenclamide and glipizide in HEK293 T cells with OATP1B3 and 344T>G and 699G>A mutation sites the experiments were divided into control group OATP1B3,344T>G and 699G>A plasmid group.The effect of time（2,5,10,15,20,30 min）and different substrate concentrations（2.5,5,10,20,40,80μM）uptake of glibenclamide and glipizide were investigated.The uptake kinetic parameters V_max,Km,Clint in OATP1B1-HEK293 T cells of different genotypes were compared by measuring the concentration of glimepiride in the cell disruption liquid.By measuring the concentrations of glibenclamide and glipizide in the cell disruption fluid,the kinetic parameters V_max,Km,and Clint of glibenclamide and glipizide in the different genotype OATP1B3-HEK293 T cell models were compared.Results:1.The LC-MS analysis method established in this experiment was used to determine the metabolic enzyme incubation system and HEK293 T cells sample of glimepiride,glibenclamide and glipizide have specific properties,high precision,good stability,meet the requirements of biological sample analysis.2.Glimepiride（1,2,5,10,20,40,80 M）enzymatic kinetic parameters of Km,V_max and CLint value were 2.97±1.18 μM,21.58±7.78 nmol/min/ mg protein,7.45±3.02 μL/min/（mg proteins）in wild type CYP2C9*1 the recombinant enzyme incubation;In the mutant CYP2C9*2 enzyme kinetics parameters Km,V_max and CLint value were 3.06±1.76 μM,15.69±5.95 nmol/min/mg protein,4.11±1.57 μL/min/（mg proteins）;In the mutant CYP2C9*3 enzyme kinetics parameters Km,V_max and CLint value were 3.70±2.34 μM,9.17±3.03 nmol/min/mg protein,2.42±1.48 μL/min/（mg proteins）.The metabolic activity of glimepiride in the mutant CYP2C9*2,*3 recombinase was significantly lower than that in the wild type.3.Glibenclamide（1,2,5,10,20,40,80 M）kinetic parameters of Km,V_max and CLint value were 3.17 ±1.02 μM,1.58±0.71 nmol/min/ mg protein,0.45 ±0.12 μL/min/（mg proteins）in wild type CYP2C9*1 the recombinant enzyme incubation;In the mutant CYP2C9*2 enzyme kinetics parameters Km,V_max and CLint value were 3.46±1.56 μM,0.69±0.25 nmol/min/mg protein,0.20±0.17 μL/min/（mg proteins）;In the mutant CYP2C9*3 enzyme kinetics parameters Km,V_max and CLint value were 3.90±2.34 μM,0.41±0.13 nmol/min/mg protein,0.12±0.08 μL/min/（mg proteins）.The metabolic activity of glibenclamide in the mutant CYP2C9*2,*3 recombinase was significantly decreased than that in the wild type.4.Glipizide（1,2,5,10,20,40,80 M）enzymatic kinetic parameters of Km,V_max and CLint value were 3.07±1.31μM,8.82±2.78 nmol/min/mg protein,2.95±1.61 μL/min/（mg proteins）in wild type CYP2C9*1 the recombinant enzyme incubation;In the mutant CYP2C9*2 enzyme kinetics parameters Km,V_max and CLint value were 3.51±1.76 μM,5.99±1.95nmol/ min/mg protein,1.69±0.47 μL/min/（mg proteins）;In the mutant CYP2C9*3 enzyme kinetics parameters Km,V_max and CLint value were 4.10±2.66μM,2.87±1.03nmol/min/mg protein,0.72±0.48μL/min/（mg proteins）.The metabolic activity of glipizide in the mutant CYP2C9*2,*3 recombinase was significantly lower than that of the wild type.5.Glimepiride uptake in OATP1B1*1a-HEK293 T cells,kinetic parameters of Km,V_max and CLint were 7.20±1.35μM,155.0±8.72pmol/min/mg protein,21.63±5.84μl/min/（mg proteins）.In the OATP1B1*5-HEK293 T cell uptake kinetics of Km,V_max and CLint were 11.32±2.71μM,80.5±9.61pmol/min/mg protein,7.95±2.46 l/min/（mg proteins）;in the OATP1B1*15-HEK293 T cell Km,V_max,CLint and the kinetic parameters were 10.90±3.75μM 84.56±8.20pmol/min/ mg protein,8.24±1.80μl/min/（mg proteins）.Compared with wild-type OATP1B1*1a-HEK293 T,mutant OATP1B1*5,*15 the Km was increased,V_max and CLint decreased significantly（P<0.01）;That Glimepiride in mutant OATP1B1*15-HEK293 T,OATP1B1*5-HEK293 T cells uptake ability than wild type OATP1B1*1a-HEK293 T was significantly decreased.6.Glibenclamide uptake in OATP1B3-HEK293 T cells kinetic parameters of Km V_max and CLint were 18.91±5.31 μM,44.91±7.97 pmol /min/ mg protein,2.39±1.06 μl/min/（mg proteins）.In the OATP1B3（344T>G）-HEK293 T cells,uptake kinetics of Km,V_max and CLint were 21.55±3.06 μM,46.08±8.69 pmol /min/ mg protein,2.13±0.84μl/min/（mg proteins）;In OATP1B3（699G>A）-HEK293 T cell kinetic parameters of Km,V_max CLint were 25.91±6.79μM 37.31±5.04pmol/min/mg protein,1.51±0.70μl/min/（mg proteins）.Compared with the wild-type OATP1B3-HEK293 T cell,there was no difference in the uptake of glibenclamide in the mutant OATP1B3（344T>G）-HEK293 T cells.However,at OATP1B3（699G>A）-HEK293 T cell Km is increased than wild-type,V_max and CLint are decreased than wild-type（P<0.05）.It was suggested that the transport capacity of glibenazide in mutant OATP1B3（699G>A）-HEK293 T cells was decreased than that in the wild-type OATP1B1* 1a-HEK293 T.7.Glipizide uptake in OATP1B3-HEK293 T cells kinetic parameters of Km,V_max and CLint were 28.02±6.91μM,16.50±3.64pmol/min/mg protein,0.59± 0.16μl/min/（mg proteins）.In the OATP1B3（344T>G）-HEK293 T cells uptake kinetics of Km,V_max and CLint were 30.52±5.36μM,16.87±4.23 pmol /min/ mg protein,0.55±0.24 μl/min/（mg proteins）;and in OATP1B3（699G>A）-HEK293 T cells Km,V_max CLint the kinetic parameters were 33.55±6.63μM;13.42± 2.79pmol/min/mg protein,0.38±0.17μl/min/（mg proteins）.Compared with the wild-type OATP1B3-HEK293 T,there was no difference in the uptake of glipizide in the mutant OATP1B3（344T>G）-HEK293 T cells.However,at OATP1B3（699G>A）-HEK293 T cell Km was increased than wild-type,V_max and CLint are decreased than wild-type（P < 0.05）.It was suggested that the transport capacity of glibenazide in mutant OATP1B3（699G>A）-HEK293 T cells was decreased than that in the wild-type OATP1B1* 1a-HEK293 T.Conclusions:1.The metabolic rates of glimepiride,glibenclamide and glipizide in the mutant CYP2C9*2,*3 recombinase were significantly decreased than that in wild-type CYP2C9*1.2.Glimepiride can be uptaked and transported by OATP1B1*5-HEK293 T,OATP1B1*15-HEK293 T and OATP1B1*1a-HEK293 T cells.Compared with the wild type OATP1B1*1a-HEK293 T cells glimepiride in mutant OATP1B1*5,*15-HEK293 T cells the transport capacity decreased significantly.3.The glibenclamide and glipizide can be transported by the OATP1B3,OATP1B3（344T>G）and OATP1B3（699G>A）-HEK293 T cells.Compared with the wild-type OATP1B3-HEK293 T cells,the transport activity in the mutant 699G>A was decreased.But the transport activity of the mutant 344T>G was not affected.