Pharmacogenetics Research On Anti-platelet Drug Clopidogrel

Ethnic and individual differences in drug metabolism and response are very common in clinical drug treatment, which are always attributable by the co-effects of genetic variations and environmental factors, with the genetic variations as the determinant. Genetic variations of transportors, drug metabolizing enzymes and drug receptors are three major determinants affecting the drugs response.Clopidogrel, a new kind of anti-platelet drug, is a thieno-pyridine derivates and widely used in clinic during recent years. With the proved highly effective therapy and expensiveness, clopidogrel has been paid more attention. However clopidogrel anti-platelet effect is not uniform in all patients. The concept of clopidogrel resistance lies in patients taking clopidogrel and with no significant inhibition of ADP-induced platelet aggregation, leading to worse disease prognosis. Nowadays the mechanism of different effect of clopidogrel is still not clear, a famous Framingham epidemiological survey showed that the genetic factors was the main cause of the anti-platelet drug's different effects.Clopidogrel is the substrate of the transportor P-gp and metabolized by CYP450 to form the active product. This active metabolite can inhibit the platelet activation and aggregation by blocking the ADP interaction with P2Y₁₂ receptor on the platelet.MDR1 (multidrug resistance 1), the encoding gene of P-gp, has genetic polymorphisms affecting drugs response in population. Research has showed that MDR1 C3435T genetic polymorphism affected the pharmacokinetics of clopidogrel and there is no data on how the polymorphism affecting clopidogrel response.Clopidogrel, a pro-drug, is metabolismed by CYP450 to generate the active product. Early research showed CYP1A2 was the main CYP450 enzyme in liver metabliting clopidogrel in rat. But Clarke TA et al found that CYP3A4 and 3A5 were the major enzymes in rat liver microsomes and microsomes prepared from Spodoptera frugiperda insect cells that had been infected with a baculovirus containing the cDNA coding for P450 reductase and a single human P450 isozyme. At the same time CYP2B6 and 1A2 also play roles on clopidogrel metabolism. Other CYP450 including CYP2C19 showed no metabolizing activity on clopidogrel. CYP3A, mainly including CYP3A4 and CYP3A5 in adults, participate in metabolizing 50～60 percent of drugs used in clinic. Usually the activity of CYP3A4 is significantly higher than that of CYP3A5, and the known polymorphisms of CYP3A4 can not fully explain the individual difference of CYP3A4 activity. A recent study showed common gene polymorphisms of CYP2B6*5, CYP1A2*1F and CYP3A5*3 didn't affect the clopidogrel response, while CYP2C19*2 gene polymorphism significantly decreased the extent of inhibition on ADP-induced platelet aggregation after clopidogrel administration. The frequency of poor metabolites of CYP2C19*2/CYP2C19*2or*3 in Chinese is much higher than that in Caucasian. So it is worth doing some research on the effect of CYP2C9 genetic polymorphisms on clopidogrel response.The active metabolite of Clopidogrel forms a disulfide bridge with the two extracellular cysteine residues locating on the P2Y₁₂ receptor expressed on the platelet surface and causes an irreversible blockade of ADP binding for the platelet's life span. P2Y₁₂ receptor belongs to G-protein coupled receptor family. The genetic mutations in such receptors may result in significantly altered responses to drugs and endogenous substances mediated by relevant receptors. Fontana P et al firstly discovered that there are two synonymous mutations (G52T and C34T) in exon 2 and three mutations (-C139T, -T744C, -ins801A) in intron without shearing variation by PCR-sequencing method. The four mutations (-C139T, -T744C, -ins801A, G52T) is in complete linkage disequilibrium. But the subsequent clinic studies didn't show these mutations affecting clopidogrel response, and the research on P2Y₁₂ gene polymorphisms in oriental is still not investigated.Clopidogrel therapy is a long process in clinic. Studies both in vivo and in vitro showed clopidogrel has a potent inhibitional effect on CYP2B6 activity. Study in vitro also showed that the high concentration of clopidogrel could inhibit CYP2C19 activity significantly, but no further study is confirmed in vivo.Based on these data, our study was aimed to clarify the common genetic polymorphisms of MDR1, CYP2C19 and P2Y₁₂ on clopidogrel anti-platelet response in Chinese healthy population, and to determine whether clopidogrel administration for a period in healthy subjects could impact CYP2C19 activity or not.The present series studies found that:1. MDR1 C3435T gene polymorphism did not alter the drug clopidogrel anti-platelet aggregation response significantly in Chinese healthy population.2. The reduction degree of ADP-induced platelet aggregation in wild genotype subjects (CYP2C19*1/CYP2C19*1) was greater than that in homozygous mutation genotype subjects (CYP2C19*2/CYP2C19*2or*3) after clopidogrel administration. CYP2C19*2 and CYP2C19*3 gene polymorphisms significantly reduced the drug clopidogrel anti-platelet aggregation response with a gene-dosage effect in Chinese healthy population.3. The distribution characteristic of P2Y₁₂ was clarified in Chinese Han healthy subjects. The allele mutation frequencies of P2Y₁₂ receptor 52T, 34T and -744C were 24.18%, 15.42% and 17.92% respectively, which was consistent with those of Caucasian. But the G52T and -T744C were not completely linkage, which was not consistent with that of Caucasian. The C34T and G52T genetic polymorphisms had no significant impact on clopidogrel anti-platelet aggregation response.4. Taking clopidogrel for a period in Chinese healthy people could inhibit CYP2C19 activity, mainly in the homozygous EMs(CYP2C19*1/CYP2C19*1) and not in PMs(CYP2C19*2/CYP2C19*2or*3).In summary, the present series of studies clarified the effect of the common gene polymorphisms of MDR1, CYP2C19 and P2Y₁₂ on clopidogrel response. We also confirmed that taking clopidogrel for a period in Chinese healthy people could inhibit CYP2C19 activity in the CYP2C19*1/CYP2C19*1 genotype individuals. These studies could provide useful information for the clinical use of the drug clopidogrel.