Influence Of Genetic And Non-genetic Factors On The Plasma Concentrations Of The Clopidogrel Metabolite (SR26334)

BackgroundClopidogrel, an orally administered thienopyridine antiplatelet agent, inhibits platelet aggregation induced by adenosine diphosphate (ADP) selectively and irreversibly. It is used widely in the prevention and treatment of major vascular diseases, including acute coronary syndrome, percutaneous coronary intervention and stroke. It was estimated that up to30%of patients do not achieve an adequate antiplatelet effect from clopidogrel, which is probably due to significant interindividual variability in the clopidogrel response. Clopidogrel resistance has been suggested as a possible cause, with putative mechanistic explanations involving genetic polymorphisms in the drug metabolizing enzyme involved in metabolizing clopidogrel. It is one of the major concerns in the management of high-risk vascular patients, especially those who have experienced acute coronary syndrome.Polymorphisms of CYP2C19affect both the pharmacodynamic and pharmacokinetic profiles of active metabolite of clopidogrel, and it has been determined that it is one of the major determinants of interindividual variability in clopidogrel pharmacodynamic and pharmacokinetic responsiveness. The US Food and Drug Administration recently recommended that CYP2C19genotyping be considered prior to prescribing clopidogrel.In spite of genetic factor, clinical and pharmacokinetic factors may also contribute to this wide variability in response. Thus, the drug doses need to be individualized, especially for patients who have genetic or non-genetic factors that affect the effect of clopidogrel.Because about85%of the drug is hydrolyzed to the clopidogrel metabolite (SR26334) by esterases and the active metabolite is difficult to be determined, the exposure and pharmacokinetic parameters for clopidogrel have been indirectly determined by quantifying the clopidogrel metabolite (SR26334). Determination of the clopidogrel metabolite (SR26334) has been used to monitor the therapeutic drug in clopidogrel-treated patients and to characterize the anti-platelet action of clopidogrel. ObjectiveThe objectives of the current study are as follows.1. To establish a rapid, accurate and reliable method for genetic screening of common SNPs in CYP2C19.2. To determine allele frequencies of CYP2C19*2,*3and*17in Chinese patients.3. To establish a method to determine the plasma concentrations of SR26334.4. To study the influence of genetic and non-genetic factors on the plasma concentrations of the clopidogrel metabolite (SR26334) among Chinese patients.MethodsThe study subjects consisted of150unrelated Chinese patients with different races (the Han nationality, the Uigur nationality, the Mongolian nationality, the Ozbek nationality and the Xibo nationality) who were18years old or above, received clopidogrel therapy. They were from Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and the First Affiliated Hospital of Xinjiang Medical University from November2011to June2012.Information on sex, race, age, body weight, height, hypertension, diabetes mellitus, smoking status, alcohol drinking, hyperlipidemia, hyperthyroidism and clopidogrel doses were obtained from the clinical records of patients.Blood samples (3mL) were drawn from each patient using the sodium heparin tubes for DNA analysis and the SR26334concentration measurements. Blood collection time (the time interval between taking clopidogrel and blood collection) was also recorded at that time.An allele-specific polymorphism chain reaction method was established to determine the genotypes in CYP2C19*2,*3and*17. Based on the establishment of those two genotyping methods, the allele frequencies at those two sites were determined in Chinese patients. Genomic DNAs were extracted from blood samples using phenol-chloroform method. According to CYP2C19genotypes, patients were divided into three groups (extensive metabolizers (EMs):CYP2C19*1/*1, intermediate metabolizers (IMs):CYP2C19*1/*2and CYP2C19*1/*3, and poor metabolizers (PMs):CYP2C19*2/*2, CYP2C19*2/*3and CYP2C19*2/*3).A HPLC-UV method was modified according to the previous reports and established under the experimental conditions in our lab to determine the plasma concentrations of SR26334. Following a simple protein precipitation using a mixture of methanolic solution of ZnSO4, the analyte and internal standard were separated using a mobile phase of water-acetonitrile (65:35, v/v) adjusted to pH3.5on an Inertsil ODS-SP (250mm×4.6mm,5μm) column at a flow rate of1mL/min. The method validation included the specificity and sensitivity and recovery, stability, accuracy and precision.A multiple regression analysis was performed to estimate the influence of genetic factor, gender, race, age, body weight, height, hypertension, diabetes mellitus, smoking status, alcohol drinking, hyperlipidemia, hyperthyroidism, clopidogrel doses and blood collection time of the patients on the plasma concentrations of SR26334. The block entry of variables in the stepwise (P entry<0.05, P removal>0.10) method was used for selecting the predictor variables.ResultsThe allele frequencies of CYP2C19*2,*3and*17in Chinese patients were29%,3%and0%, respectively. CYP2C19genotyping showed that68patients (45.33%) were extensive metabolizers (EMs),68(45.33%) were intermediate metabolizers (IMs) and14(9.33%) were poor metabolizers (PMs).The plasma concentrations of SR26334were determined with a HPLC-UV method. Protein was precipitated from the samples by mixing a200μL aliquot of plasma,10μL of IS (Ticlopidine), and200μL of methanolic solution of ZnSO4. The mobile phase was composed of water-acetonitrile (65:35, v/v) adjusted to pH3.5and the flow rate was1mL/min. Separation of SR26334and IS was achieved by using Inertsil ODS-SP (250mm×4.6mm,5μm) column. SR26334was linear from100-10,000ng/mL. All the results were complied with the Guidelines for Bioanalytical Method Validation of the Food and Drug Administration.Using multiple linear regression analysis, four variables, including blood collection time, smoking status, CYP2C19metabolizer groups and clopidogrel doses were selected as significant factors that influenced the plasma concentrations of SR26334. Sex, race, age, body weight, height, hypertension, diabetes mellitus, alcohol drinking, hyperlipidemia and hyperthyroidism were not included as predictors because of their weak association with the plasma concentrations of SR26334(P>0.05). The final model could explain63.1%of the variation in the observed plasma concentrations of SR26334. The final equation:the plasma concentrations of SR26334=exp [6.657-0.085(blood collection time)-0.382(smoking status)+0.251(CYP2C19metabolizer groups)+0.002(clopidogrel doses)], in which exp is the exponential function.ConclusionOur study is a first step to investigate the contribution of the genetic polymorphism of CYP2C19and non-genetic factors to the SR26334concentrations in Chinese patients. Blood collection time, smoking status, genetic polymorphism of CYP2C19and clopidogrel doses were highly associated with the plasma concentrations of SR26334. This model may be useful for therapeutic drug monitoring and reference for the clinical doses adjustments of clopidogrel when the information of the model is available in patients.