The Effect Of Cytochrome P450 2C9 Gene Polymorphism On Drug Metabolism

Cytochrome P450 2C9(CYP2C9)is an important phase I drug-metabolizing enzymes and participate more than 15%clearance of clinical drugs.CYP2C9 gene is highly polymorphic and 60 CYP2C9 variants were currently reported.Those genetic variants potentially influence the metabolic clearance rate and thus change the drug plasma concentration and increase the risk of drug-induced side effects.Studies of CYP2C9 gene polymorphism are of significance for clinical safe medication,prediction for drug-induced side effects and optimizing drug therapy.Our meta-analysis found that CYP2C9 gene polymorphism is closely associated with the variety of phenytoin maintaince dose.However,current studies mainly focus on CYP2C9*2 and CYP2C9*3 rather than other genetic variants.CYP2C9*8 and CYP2C9*27 both mutate in 150 arginine of CYP2C9 gene and have a higher mutation frequency than CYP2C9*2 in their own population,respectively.To futher explore the CYP2C9 150 arginine mutation,we construct HEK293T cells that stably express CYP2C9,CYP2C9*8 and CYP2C9*27,respectively.Enzyme activities in vitro is adopted to analysis the effect of CYP2C9*8/*27 on CYP2C9 substrate metabolism.Our study found that the effect of CYP2C9*8/*27 on substrates is associated impaired interaction with P450 oxidoreductase(POR).Therefore,we also respectively constructed expression systems of POR and Cytochrome b5.Those cell lines are powerful tool to explore the effect CYP2C9 150 arginine mutation on interaction between CYP2C9 and catalytic coenzymes.Our study consists of three sections.SECTION I The association between CYP2C9/2C19 polymorphisms and phenytoin maintenance doses in Asian epileptic patients:a systematic review and meta-analysisObjectiveCYP2C9 gene polymorphism potential changes the pharmacokinetic behavior of CYP2C9 substrates and thus potential effect their drug safety.Therapeutic response to phenytoin(PHT),a first-line antiepileptic drug(AED),is highly variable,in part likely due to C YP2C9 genetic factors.Genetic polymorphisms in Cytochrome P450(CYP)2C9 and CYP2C19 are expected to affect the metabolism of PHT and consequently affect its maintenance doses.We aimed to clarify the effects of genetic polymorphisms in both enzymes on daily PHT maintenance dosage in Asian epileptic patients by meta-analysis.MethodsA systematic literature search was conducted in PubMed and EMBASE for relevant studies published prior to April 14,2017.RevMan 5.2.3 software was used to analyze the relationship between CYP2C9/2C19 polymorphisms and PHT maintenance doses.ResultsA total of 6 studies with 993 patients fulfilling the inclusion criteria were included in our meta-analysis.Data showed that patients carried CYP2C9 mutations need PHT dosage adjustments.Heterozygous CYP2C9 carriers should reduce PHT maintenance dose by 22%.When patient carried CYP2C9 wide type,heterozygous CYP2C19 mutation group(i.e.CYP2C19*1/*2 or CYP2C19*1/*3 group)did not require PHT dosage adjustments but homozygote CYP2C19 mutation group(i.e.CYP2C19*2/*2,CYP2C19*2/*3 or CYP2C19*3/*3 group)required significant decrease of PHT maintenance dose by 11%.ConclusionsThe meta-analysis indicates that CYP2C9 and CYP2C19 polymorphisms are associated with lower PHT maintenance dosage in Asian epileptic patients.Ethnic differences can influence the PHT maintenance dose.However,current studies focus on CYP2C9*2 and CYP2C9*3 rather than other genetic variants and the mechanism and effect of CYP2C9 150 arginine mutation on substrate metabolism is still insufficient.SECTION II Expression of Cytochrome P450 2C9*8/*27 and their catalytic activityObjectiveIn first section,we found that current studies mainly focus on CYP2C9*2 and CYP2C9*3 rather than other genetic variants.CYP2C9*27(499G>T)and CYP2C9*8(449G>A)both mutate in 150 arginine of CYP2C9 gene and have a higher mutation frequency than CYP2C9*2 in their own population.We constructed lentiviral expression plasmid of CYP2C9,CYP2C9*8 and CYP2C9*27,and stably expressed CYP2C9,CYP2C9*8 and CYP2C9*27 in HEK293T cells.To explore the effect of CYP2C9 150 arginine mutation on catalytic activity,we adopted cumene hydroperoxide as an electron donor to substitute PORMethodsC YP2C9 coding region was obtained by reverse transcription PCR from the human liver total RNA,and then cloned into the mammalian expression vector MigR1.CYP2C9*8 or CYP2C9*27 coding sequences were obtained from MigRl-CYP2C9 by overlapping PCR and then cloned into MigRl.The coding sequences were introduced to HEK293 cells by lentivirus,and 293T-2C9,293T-2C9*8 and 293T-2C9*27 were screened by flow cytometry and monoclonal picking.Cellular fluorescence,qRT-PCR,western blot and the metabolic activity assays were employed to identify the cells.To investigate the changes between CYP2C9 wild type and CYP2C9*8/*27,CYP2C9 specific substrate,diclofenac,was used as a probe and cumene hydroperoxide was adopt as an electron donor to substitute POR.ResultsMigRl-CYP2C9,MigRl-CYP2C9*8 and MigRl-CYP2C9*2293T-2C9*8 plasmids were constructed.All monoclonal cells 293T-2C9,293T-2C9*8 and 293T-2C9*27 cell lines expressed GFP after 30 passages.The qRT-PCR and western blot assay both showed CYP2C9 expression in the cells.Microsomes of the three cell lines were capable of catalyzing diclofenac to 4-OH diclofenac.Catalytic activity of CYP2C9*8 was lower than CYP2C9 when using POR as an electron donor.When using cumene hydroperoxide as an electron donor,the catalytic activity between CYP2C9 and CYP2C9*8 did not exhibit significant difference.Conclusion293T-2C9,293T-2C9*8 and 293T-2C9*27 cell lines stably expressed CYP2C9,CYP2C9*8 and CYP2C9*27,respectively.The cell lines provide useful tools for the function investigation of CYP2C9*8 and CYP2C9*27.Reduced catalytic activity of CYP2C9*8 is potentially associated with the impaired interaction with POR.SECTION III Expression of Cytochrome P450 Reductase and their effect on the catalytic activity of CYP2C9 150 arginine mutationObjectiveOur second section found that CYP2C9 150 arginine mutation potentially influence the interaction between CYP2C9 and POR.In this section,we constructed lentiviral expression plasmid of POR,stably expressed POR in HEK293T cells and explore the effect of POR on the catalytic activity of 150 arginine mutation.MethodsPOR coding region was obtained by reverse transcription PCR from the human liver total RNA,then cloned into the mammalian expression vector MigRl.The coding sequences were introduced to HEK293 cells by lentivirus.Flow cytometry and monoclonal picking were employed for screening the positive cells and named as 293T-POR.qRT-PCR and western blot were employed to identify the POR expression in 293T-POR cell line.In vitr,the changes of catalytic activity of CYP2C9 wide type and variants on diclofenac were investigated when adding POR.ResultsMigRl-POR plasmids were constructed.All monoclonal cells 293T-POR cell lines expressed GFP after 30 passages.The qRT-PCR and western blot assay both showed POR expression in the cells.The catalytic activity of CYP2C9 wide type change slightly when adding POR but POR may result in the decrease of enzymatic activity of CYP2C9*8.Conclusion293T-POR cell lines stably expressed POR.Adding additional POR showed higher influence on CYP2C9*8 activity than that of the wide type.