【作者】 陈碧莲；

【导师】 周宏灏；

【作者基本信息】 中南大学 ， 药理学， 2007， 博士

【摘要】 药物代谢和反应的个体及种族间差异是临床用药中的普遍现象。这些差异的产生是遗传因素与环境因素共同作用的结果，其中遗传因素为决定因素。转运体、药物代谢酶和药物受体的遗传变异是产生药物反应个体差异的三个主要因素。氯吡格雷为噻吩并吡啶衍生物，是一类新型的抗血小板药物，临床应用才10余年，但显著的疗效和较高的价位使其备受关注。氯吡格雷疗效也存在明显的个体差异。临床“氯吡格雷抵抗”是指个体服用氯吡格雷后疗效不佳，实验室检测ADP诱导的血小板聚集率不能被明显抑制，并对疾病预后产生不良影响。目前氯吡格雷疗效个体差异的机制尚不清楚。著名的Framingham流行病学调查表明遗传因素在抗血小板药物疗效个体差异中起着主要作用。氯吡格雷为口服药物，是转运体P-糖蛋白（P-glycoprotein，P-gp）的底物，须经细胞色素P450代谢生成活性代谢产物；该活性代谢产物作用于血小板表面P2Y₁₂受体，阻断ADP（腺苷二磷酸）与该受体的结合，起到抗血小板活化和聚集的效应，最终产生抗血栓过度形成的临床疗效。编码转运体P-gp的MDR1（多药耐药基因）在人群中存在着影响药物疗效的基因多态性。研究表明MDR1 C3435T基因多态性影响氯吡格雷及活性代谢产物的血药浓度；但无进一步研究来阐明该基因多态性对氯吡格雷药物疗效的影响。氯吡格雷本身无治疗效应，它由细胞色素P450氧化生成活性代谢产物（一种硫醇衍生物）而发挥抗血小板作用。CYP450是超家族基因编码的同工酶，参与了许多外源性和内源性物质的代谢。最初研究表明大鼠体内主要是CYP1A2参与氯吡格雷的代谢。2002年Clarke等利用大鼠肝微粒体和转染人类单一CYP450基因的昆虫细胞制备肝微粒体进行研究，发现主要是CYP3A4、3A5参与了氯吡格雷的代谢，CYP2B6和CYP1A2也有代谢活性，而CYP2C19等不参与氯吡格雷的代谢。成人体内CYP3A主要是CYP3A4和CYP3A5，参与了人体内50～60％药物的代谢，通常CYP3A5的浓度明显低于CYP3A4，而有些个体则是相反。目前发现的CYP3A4基因多态性都不足以预测CYP3A4蛋白活性的正常变异。进一步研究表明人体内CYP2B6*5，CYP1A2*1F和CYP3A5*3常见的基因多态性并不影响氯吡格雷的疗效，而CYP2C19*2突变型杂合子服用氯吡格雷后血小板聚集率抑制程度减轻，即疗效降低。而中国人群中CYP2C19*2和CYP2C19*3基因突变所致弱代谢者的发生频率远高于白种人，所以值得深入系统地研究该酶的基因多态性对中国人群中氯吡格雷疗效的影响。氯吡格雷活性代谢产物与血小板表面P2Y₁₂受体形成稳定的二硫键，从而不可逆地阻断ADP与P2Y₁₂受体的结合，抑制ADP诱导的不可逆性血小板聚集。P2Y₁₂受体是G蛋白藕连的受体。发生在G蛋白藕连受体功能区的遗传多态性常常是受体生物学功能变异的结构基础，进而导致机体对药物和内源性活性物质的反应发生改变。2004年Fontana等首先在白种人中采用PCR-测序方法发现P2Y₁₂基因在2号外显子存在着2个同义突变（G52T，C34T）；内含子存在的3个基因突变（-C139T，-T744C，-ins801A）均未引起剪切变异。其中4个位点的基因突变（-C139T，-T744C，-ins801A，G52T）呈完全连锁状态。后续临床病人治疗过程中，并未发现这些突变对氯吡格雷疗效的影响。而东方人群中尚未开展P2Y₁₂受体基因多态性及对氯吡格雷疗效影响的研究。同时氯吡格雷临床治疗是一个长期的过程，体内外研究已证明氯吡格雷对CYP2B6有明显抑制作用。目前已有体外试验证明高浓度氯吡格雷对CYP2C19也有明显的抑制作用，但人体内尚无进一步的研究。基于以上研究背景，本课题旨在通过严格设计的临床实验，系统性研究在中国健康人群中MDR1、CYP2C19和P2Y₁₂受体常见基因多态性及对氯吡格雷疗效的影响，阐明氯吡格雷疗效个体差异的分子机制；同时探讨健康成人服用一段时间氯吡格雷对CYP2C19蛋白有无抑制或诱导作用。为临床实施基因导向性的合理的个体化用药提供指导和进一步的研究方向。本课题的主要研究结果如下：一、以健康受试者为研究对象，查明了MDR1 C3435T基因多态性对氯吡格雷抗血小板聚集的疗效无明显影响。二、以健康受试者为研究对象，查明了CYP2C19*1／CYP2C19*1野生型个体服用氯吡格雷后所产生的抑制血小板聚集的效应明显大于CYP2C19*2／CYP2C19*2or*3突变型纯合子个体，而CYP2C19*1／CYP2C19*2or*3突变型杂合子个体抑制血小板聚集的效应介于CYP2C19*1／CYP2C19*1野生型个体和CYP2C19*2／CYP2C19*2or*3突变型纯合子个体之间。系统地论证了CYP2C19*2和CYP2C19*3基因多态性降低了氯吡格雷抗血小板聚集的疗效，并呈现出基因剂量效应。三、查明了中国健康人群中P2Y₁₂外显子C34T、G52T和内含子T744C的基因多态性是存在的，三个位点基因突变频率分别为24.17％、15.42％和17.92％，与白种人报道频率无明显差异；但是外显子G52T与内含子T744C基因突变不是呈现完全连锁状态，这与白种人之间存在着明显的种族差异。前瞻性研究发现健康人群中P2Y₁₂外显子C34T和G52T的基因多态性对氯吡格雷抗血小板聚集的疗效无明显影响。四、查明了中国健康人群服用氯吡格雷后会对CYP2C19蛋白产生抑制作用，主要是对CYP2C19*1／*1野生型个体产生抑制作用，对CYP2C19*2／CYP2C19*2or*3突变型纯合子个体则无抑制作用。综上所述，本项目系统地研究了中国健康人群中MDR1、CYP2C19和P2Y₁₂受体常见基因多态性对氯吡格雷抗血小板聚集疗效的影响；同时阐明健康成人CYP2C19野生型个体服用氯吡格雷后对CYP2C19蛋白有抑制作用。这为临床合理使用抗血小板药物氯吡格雷提供了实验依据和理论指导。更多还原

【Abstract】 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.更多还原