The Effect Of The CES1A2Polymorphism On Clopidogrel Metabolism And Platelet Response

Objective: To assess the impact of carboxylesterase1A2(CES1A2) polymorphismon clopidogrel in vitro metabolism and explore its role in variability in clopidogrelpharmacokinetics and platelet response in healthy Chinese volunteers，expecting to provideuseful information for reasonable clinical usage of clopidogrel.Methods:(1) Genotyping: Genomic DNA from human liver samples andEDTA-treated blood was extracted with Wizard Genomic DNA Purification kit.CYP2C19*2and CYP2C19*3were genotyped by polymerase chain reaction-restrictionfragment length polymorphism (PCR-RFLP) and validated by direct sequencing of morethan10%of the total randomly selected samples. CES1A2A(-816)C was genotyped bydirect sequencing.(2) Determination of the concentrations of clopidogrel, its active and inactivemetabolite: The concentrations of clopidogrel, its active and inactive metabolite in humanliver microsome (HLM) incubation system and plasma were determined byUPLC-MS/MS.(3) Exploration of the effect of different CES1A2genotypes on clopidogrel invitro metabolism: Human liver tissues were stratified into three predicted phenotypegroups: extensive metabolizers [EMs (*1/*1)], intermediate metabolizers [IMs (*1/*2,*1/*3)] and poor metabolizers [PMs (*2/*2,*2/*3or*3/*3)] according to the number ofCYP2C19variant. Liver samples carrying CES1A2-816AA, AC or CC genotypes wererandomly selected according to the phenotype groups above (in CYP2C19EM, n=6foreach CES1A2genotype; in CYP2C19IM, n=6for-816AA and AC genotype, no-816CCgenotype were detected) and pooled for clopidogrel in vitro incubation experiments aimedto investigate the effect of different CES1A2genotypes on the formation of clopidogrelinactive and active metabolite. Apparent kinetic constants (Kmand Vmax) were estimated byfitting formation rates of clopidogrel inactive metabolite versus substrate concentrations tosimple single-site Michaelis-Menten equation by nonlinear regression analysis using Prismsoftware version5.0. In vitro intrinsic clearance (Clint) was given as Vmax/Km. Statisticalcomparisons of metabolism kinetic parameters and the level of active metabolite among genotypes were performed using one-way analysis of variance (ANOVA) with the leastsignificant difference (LSD) post-hoc test or t-test. Two-sided P-value <0.05wasconsidered statistically significant.(4) Study of clopidogrel pharmacokinetics and pharmacodynamics in healthyChinese volunteers:11healthy Chinese male volunteers without CYP2C19*2or*3loss-of-function variants were enrolled in this study after providing written informedconsent. They were categorized into2groups according to CES1A2-816A/C genotypes:AA for6subjects and AC/CC for5, and then received an oral dose of300mg clopidogrel.Blood samples for pharmacokinetics analysis were collected immediately before and0.25,0.5,0.75,1,1.25,1.5,2,3,4,5,6,8,12,24and36h after drug administration, whilepharmacodynamics samples were collected immediately before and1,2,4,12and24h afterdrug administration. The concentrations of clopidogrel, its active and inactive metabolitewere determined by UPLC-MS/MS and the pharmacokinetics analysis was evaluated bynonparametric methods. Pharmacologic specific index of the inhibition of plateletaggregation (IPA) was determined by a platelet aggregation profiler-4opticalaggregometer.Results:(1) Distribution of each genotype: A total of86human liver samples weregenotyped for CYP2C19*2/*3and CES1A2A (-816) C. Results are as follows:○A1ECAES1A2A(-816)C genotyping showed that43liver samples were AA genotype;37samples were AC genotype and6samples were AC genotype.○A2ECAYP2C19*2/*3genotyping showed that37samples were extensive metabolizers;38samples were intensive metabolizers and11samples were poor metabolizers.○A3ESAubgroup analysis showed that in CYP2C19EM subgroup (n=37):15sampleswere AA genotype;16samples were AC genotype and6samples were CC genotype. InCYP2C19IM subgroup (n=38):24samples were AA genotype;14samples were ACgenotype. In CYP2C19PM subgroup (n=11):4samples were AA genotype and7sampleswere AC genotype.(2) UPLC-MS/MS methods for the simultaneous determination of clopidogrel, itsinactive and active metabolite in HLM incubation system or plasma: The calibrationcurves for clopidogrel active metabolite were linear during the range of1～200ng·mL-1(R2=0.9977and0.9930respectively) in both HLM incubation system and plasma. Thecalibration curves for clopidogrel inactive metabolite were linear during the range of10～ 2000ng·mL-1(R2=0.9964) and25~10000ng·mL-1(R2=0.9945) in HLM incubationsystem and plasma respectively. The calibration curves for clopidogrel were linear duringthe range of0.1～20ng·mL-1(R2=0.9961) in plasma. A good linearity, precision, andaccuracy were found in the methods respectively. The matrix effect and stability met therequirements of biological sample analysis as well.(3) Effect of different CES1A2genotype on the formation of clopidogrel inactiveand active metabolite:○A1EAThe-816CC genotype showed a1.34-fold higher maximalvelocity (Vmax) than the-816AA(CC vs AA：635.20±12.06vs475.10±26.55pmol·min-1·mg protein-1,P<0.05).Despite lack of statistical significance, it is noteworthy that the amount of activemetabolite exhibited a decresing trend in pool of-816CC genotype compared with-816AAwhere the substrate concentrations were20μM and50μM(P>0.05).○A2ECAoncerning CYP2C19EM phenotypes, the pooled-816CC genotype showed a1.39-fold higher maximal velocity (Vmax) for clopidogrel inactive metabolite (CC vs AA:669.80±20.72vs480.6±13.84pmol·min-1·mg·protein-1, P<0.05), coupled with almosta level of40%lower of active metabolite than-816AA genotype under the chosensubstrate concentrations. A similar result was also noted in CYP2C19IM phenotype, butnot in PM phenotype because the formation of active metabolite in PM phenotype wasextremely low.(4) Result of the effect of CES1A2polymorphism on clopidogrel metabolism inhealthy volunteers: Comparing with homozygous wild-type genotype-816AA, themutated genotype–816C resulted in higher mean clopidogrel AUC0-∞(15.6±4.8vs9.7±1.7ng·mL-1·h-1, P＜0.05), mean MP-AM AUC0-lastand AUC0-∞(122.4±38.1vs73.9±17.9ng·mL-1·h-1, P＜0.05;124.2±37.6vs76.4±17.71ng·mL-1·h-1, P＜0.05) and lower meanCCAM AUC0-last, AUC0-∞, and Cmax(41368.46±12832.65vs64346.12±13740.94ng·mL-1·h-1, P＜0.05;42173.30±13382.47vs65545.46±14166.21ng·mL-1·h-1, P＜0.05;11050.06±3308.02vs20671.22±3469.58ng·mL-1).(5) Result of the effect of CES1A2polymorphism on clopidogrel anti-plateletresponse in healthy volunteers: There was no significant difference in the basic MPAlevel between the two genotypes before drug administration（P＞0.05）. After2and4hoursof taking300mg loading dose of clopidogrel, CES1A2C mutant genotype showed a less reduction in IPA (47.25士9.44%,61.60士21.51%vs62.05士7.13%,82.91士6.96%, P＜0.05) compared to CES1A2AA genotype. The-816AA genotype showed a1.27-foldhigher aera under the time-effect curve (AUEC) than the AC/CC genotype(1738.54±162.11vs1368.16±384.67h·%, P<0.05).Conclusion: The metabolism and anti-platelet activity of clopidogrel weresignificantly influenced by CES1A2genotype. A possible mechanism for the differences inanti-platelet effect is the variability of active metabolite formation caused by CES1A2polymorphisms. Therefore, in addition to CYP2C19, CES1A2genotype should also betaken into account when considering individualized clopidogrel administration in clinic.