Genetic Polymorphisms Of CYP2C19*2 And ABCB1 C3435T Affect The Pharmacokinetic And Pharmacodynamic Responses To Clopidogrel In 401 Patients With Acute Coronary Syndrome

Backgrounds and Objectives: As a novel inhibitor of platelet ADP P2Y12 receptors,Clopidogrel plays an important role in the treatment of acute coronary syndrome-an emergency involving active thrombosis.However,amongst the patients with the syndrome who have received standard administration of the very agent,some special suffers are still attacked by coronary thrombotic event.Clopidogrel resistance(CR),a condition in which the drug is less effective than normal in people who are treated with it,is mainly responsible for the not uncommon phenomenon.The exact explanation for CR is far from clear.However,accumulating data indicate that multiple factors including genetic component lead to the peculiar status.As a prodrug,clopidogrel has to first generate an active metabolite through biotransformation by liver and then irreversibly antagonizes ADP receptor P2Y12 on platelet after the processes of absorption in intestinal and biotransformation in liver.It has been clear that ATP-binding cassette subfamily B member1(ABCB1)and Cytochrome P450(CYP450)including CYP2C19 and CYP3A4 are the dominant factors in absorption and biotransformation of the agent respectively.Our research attempts to investigate the effect of gene polymorphism of ABCB1(10 genes),CYP450(5 genes for CYP2C19 and 3 genes for CYP3A4)and P2Y12 receptor(3 genes)on the plasma level of clopidogrel and its metabolites(active clopi-H4 and inactive CLPM)and platelet reactivity.Methods and Results: 401 acute coronary syndrome(ACS)patients received either a 300 mg loading dose following 75 mg maintenance dose daily or a 75 mg maintenance dose daily of clopidogrel.All patients received 100 mg asipirin.Demographic characteristics,clinical information,and medications were obtained from patient's files or face-to-face interview.These factors included age,sex,smoking,diabetes mellitus,hypertension(BP ? 140/90 mm Hg),hyperlipidemia,body mass index(BMI),family history of coronary artery disease(CAD),previous myocardial infarction(MI),and coronary stents.A total of eight blood samples from all 401 patients were collected on the 7th day after the last dose of clopidogrel taken for genotyping,platelet reactivity,and pharmacokinetic analyses.Inhibition of platelets was assessed using light transmittance aggregometry.Plasma concentrations of clopidogrel as well as its active(clopi-H4)and inactive(CLPM)metabolites were measured using HPLC-MS-MS method.A non-compartmental pharmacokinetic analysis of CLP,CLPM,clopi-H4 was performed using DAS(Drugs and Statistics,version 3.0)software.The peak plasma concentration(Cmax)and the corresponding time(Tmax)were directly obtained from the raw data.The elimination rate constant(ke)was obtained as the slope of the linear regression of the log-transformed concentration values versus time date in the terminal phase.The elimination half-life(t1/2)was calculated as 0.693/ke.The linear trapezoidal rule was used for the calculation of the area under the curve to the last measurable concentration(AUC0–t).The area under the curve to infinity(AUC0–?)was calculated as AUC0–? = AUC0–t + Ct/ke,where Ct is the last measurable concentration.A panel of 21 SNP of ABCB1,CYP2C19,CYP3A4,CYP3A5,P2RY12,P2Y1,and PON1 was selected based on tagged SNP from the International Hap Map Project website(http://hapmap.ncbi.nlm.nih.gov/)and previous investigations(Simon et al.,2009;Harmsze et al.,2010;Shalia et al.,2013;Zhang et al.,2013).Genotyping analysis of the SNP selected for fast-track validation analysis was performed using Sequenom Mass Array technology(San Diego,California,USA)A logistic regression analysis was used for the genotype distributions in the study groups.Hardy-Weinberg equilibrium(HWE),linkage disequilibria(LD),and allele frequency comparison were performed with SHEsis software,available online at http://analysis.bio-x.cn/my Analysis.php.In addition,the genetic models(dominant model and recessive model)of the two tag SNPs were compared in the study groups.Linear regression was used to calculate the mean difference in on-clopidogrel platelet reactivity(indicated as ?PA for LTA values)between carriers and non-carriers of a variant allele and to adjust for confounding factors.The following confounding factors were included in the multiple linear regression analysis: gender,age,body mass index,diabetes mellitus,previous MI,beta-blockers,proton pump inhibitors,and NSAIDs.Differences in clinical data of patients and controls were tested by either ANOVA when values were normally distributed or the non-parametric Kruskal–Wallis test when the distribution normality test failed.The P values reported in the study were based on two-sided probability test with a significance level of P < 0.05.Results Among 21 genes,the carriers of CYP2C19*2 were associated with lower exposure to its active(clopi-H4)and inactive(CLPM)metabolites(both P< 0.05 vs.non-carriers)and thus decreased platelet inhibition(P< 0.05 vs.non-carriers).Notably,the carriers of ABCB1 C3435 T were associated with lower levels of plasma clopidogrel and its active(clopi-H4)and inactive(CLPM)metabolites(all P< 0.05 vs.non-carriers)which also correlated with subsequently decreased platelet inhibition(P< 0.05 vs.non-carriers).There were no obvious effects of other studied genes on clopidogrel.We demonstrated that the maximal plasma concentrations(Cmax)of the inactive(CLPM)and active(clopi-H4)metabolites were significantly decreased in the carriers of CYP2C19*2 G681 A genotype when compared with that in the non-carriers(both P<0.05).However,the maximal plasma concentration of CLP was not statistically different between the carriers and non-carriers of CYP2C19*2 G681 A.Notably,the maximal plasma concentrations of CLP,inactive(CLPM),and active(clopi-H4)metabolites were markedly decreased in the carriers of ABCB1 C3435 T genotype when compared with that in the non-carriers(all P<0.05).In the patients receiving 75 mg maintenance dose daily of clopidogrel,platelet inhibition in response to clopidogrel was significantly attenuated in the carriers when compared to that in the non-carriers of the CYP2C19*2 allele(P< 0.05 at both 20 and 50 ?mol/l ADP).The carriers of the ABCB1 C3435 T allele showed decreased efficacy of clopidogrel on platelet aggregation when compared to the non-carriers of the ABCB1 allele(P< 0.05 at both 20 and 50 ?mol/l ADP).No differences between platelet reactivity and other genetic variants were observed.There were similar findings of platelet aggregation between patients receiving a 300 mg loading dose following 75 mg maintenance dose daily of clopidogrel and patients receiving a 75 mg maintenance dose daily of clopidogrel.In this study,we did find that patients receiving a 300 mg loading dose following 75 mg maintenance dose daily experienced more platelet reactivity at 50 ?mol/l ADP.Conclusions: CYP2C19*2 is a determinant for the formation of the active metabolite of clopidogrel and its antiplatelet effects.Meanwhile,ABCB1 C3435 T plays an important role in intestinal absorption of clopidogrel which further affects the exposure to the active metabolite of clopidogrel and platelet aggregation.