ABCB1、CES1 和 CYP2C19 基因多态性与 氯吡格雷抵抗的相关性研究进展

氯吡格雷是目前全球临床使用最为广泛的血小板受体抑制剂,但其抗血小板效应存在明显个体差异,部分病人服用常规剂量氯吡格 雷后存在抵抗现象,甚至发生不良临床事件。多项研究表明,ABCB1、CES1 和 CYP2C19 基因多态性对氯吡格雷抵抗的产生发挥重要作用。 简介氯吡格雷体内吸收与代谢机制和氯吡格雷抵抗的定义,综述 ABCB1、CES1 和 CYP2C19 基因多态性对氯吡格雷抵抗的影响。     

ABCB1 基因单核苷酸多态性rs2235048 与氯吡格雷抵抗的相关关系

目的:探讨ABCB1基因单核苷酸多态位点rs2235048在中国汉族人群中的分布及其与氯吡格雷抵抗(Clopidogrelresistance,CR)发生的相关关系。方法:采用光学比浊法测定20μmol/L ADP诱导的残余血小板聚集率(Residual plateletagglutination,RPA)。当RPA≥70%时,即为CR。所有入选患者分为CR组和氯吡格雷非抵抗组(Non-clopidogrel resistance,NCR)。采用焦磷酸测序法测定ABCB1基因rs2235048单核苷酸多态位点在CR组和NCR组的基因型及等位基因分布频率。结果:ABCB1基因rs2235048多态在CR组和NCR组基因型分布频率符合Hardy-Weinberg平衡。在CR组和NCR组中,ABCB1基因rs2235048多态的基因型频率分布无统计学差异(P=0.527,X2=1.281);T、C等位基因频率在两组间分布频率也没有统计学差异(P=0.740,OR=0.958,95%CI=0.742～1.236)。结论:对PCI术后服用氯吡格雷的冠心病患者进行分析发现,ABCB1基因单核苷酸多态位点rs2235048与冠心病患者CR的发生无相关关系。     

CYP3A5基因单核苷酸多态性rs3800959与氯吡格雷抵抗的相关性研究

目的:探讨细胞色素P450 3A5基因(CYP3A5)单核苷酸多态位点rs3800959与氯吡格雷抵抗(Clopidogrel resistance,CR)发生的关系。方法:于2010年3月至2011年10月期间,连续入选在沈阳军区总医院心内科住院的接受标准双联抗血小板治疗(阿司匹林+氯吡格雷)的冠心病患者共800例。以光学比浊法测定20μmol/L浓度ADP诱导的残余血小板聚集率(Residual plateletagglutination,RPA),并定义RPA≥70%为CR,所有入选患者分为CR组和氯吡格雷非抵抗组(Non-clopidogrel resistance,NCR)。所有入选病例提取血液白细胞基因组DNA后,采用直接测序的方法测定CYP3A5基因rs3800959单核苷酸多态位点的基因型及等位基因。结果:所入选的800例病人中,CR组为150例,NCR组为650例,CR发生率为18.75%。rs3800959基因型频率在CR组为TT型110例(73.3%)、CT型39例(26.0%)及CC型1例(0.7%);NCR组rs3800959基因型频率分别为477例、159例及14例(73.4%、24.5%及2.1%)。两组间各基因型频率分布无统计学差异(P=0.460,x2=1.554);T、C等位基因分布频率在两组间亦无明显差异(P=0.784,OR=0.942,95%CI=0.655~1.356)。结论:CYP3A5基因单核苷酸多态位点rs3800959与冠心病人CR的发生无相关关系。     

CYP3A5基因单核苷酸多态性rs3800959与氯吡格雷抵抗的相关性研究

目的:探讨细胞色素P450 3A5基因(CYP3A5)单核苷酸多态位点rs3800959与氯吡格雷抵抗(Clopidogrel resistance,CR)发生的关系.方法:于2010年3月至2011年10月期间,连续入选在沈阳军区总医院心内科住院的接受标准双联抗血小板治疗(阿司匹林+氯吡格雷)的冠心病患者共800例.以光学比浊法测定20μmol/L浓度ADP诱导的残余血小板聚集率(Residual platelet agglutination,RPA),并定义RPA＞ 70％为CR,所有入选患者分为CR组和氯吡格雷非抵抗组(Non-clopidogrel resistance,NCR).所有入选病例提取血液白细胞基因组DNA后,采用直接测序的方法测定CYP3A5基因rs3800959单核苷酸多态位点的基因型及等位基因.结果:所入选的800例病人中,CR组为150例,NCR组为650例,CR发生率为18.75％.rs3800959基因型频率在CR组为TT型110例(73.3％)、CT型39例(26.0％)及CC型1例(0.7％);NCR组rs3800959基因型频率分别为477例、159例及14例(73.4％、24.5％及 2.1％).两组间各基因型频率分布无统计学差异(P=0.460,x2=1.554);T、C等位基因分布频率在两组间亦无明显差异(P=0.784,OR=0.942,95％CI=0.655～1.356).结论:CYP3A5基因单核苷酸多态位点rs3800959与冠心病人CR的发生无相关关系.     

ABCB1 与氯吡格雷抵抗的研究进展

氯吡格雷和阿司匹林双联抗血小板已是急性冠状动脉综合征和经皮冠脉介入术后的标准治疗,因此氯吡格雷抵抗越来越受到人们的关注,但焦点更多的集中在氯吡格雷氧化代谢基因(P2Y12、CYP3A4等)多态性方面,而对氯吡格雷吸收方面基因多态性的关注相对较少,本文就调控氯吡格雷在肠道吸收的基因(ABCB1)进行综述,并探讨其多态性与氯吡格雷抵抗的关系。     

氯吡格雷抵抗及其相关因素

氯吡格雷在临床上被公认为最广泛的抗血小板药物,但最近研究发现,有些患者存在氯吡格雷抵抗,即接受氯吡格雷抗血小板药物治疗后而不能提供充分抗血小板作用的一种现象.氯吡格雷抵抗者血小板聚集高,容易发生不良心血管事件.在这里我们就氯吡格雷抵抗及其相关因素作一综述.     

应用HRM技术对CYP2C19*2和CYP2C19*3进行双重SNP分型

氯吡格雷是一种广泛用于预防静脉血栓形成的抗血小板药物。研究表明, 携带有CYP2C19基因功能缺失型等位基因CYP2C19*2、CYP2C19*3的病人, 其体内代谢氯吡格雷成为其活性形式的能力降低, 导致氯吡格雷抑制血小板聚集功能减弱。文章旨在建立一种利用高分辨率熔解曲线分析(High-resolution melting curve analysis,HRM)技术在闭合单管中同时对CYP2C19*2、CYP2C19*3两个多态性位点进行简便、准确分型的方法。本实验针对两个SNP位点分别设计特异性的HRM引物, 并在两个位点引物的5′端分别加上富含AT和GC的序列, 保证两个位点的扩增产物熔解峰无重叠。利用HRM技术, 快速、灵敏地对64例随机DNA样本的CYP2C19*2 、CYP2C19*3两个多态性位点进行了基因分型, 且HRM方法的分型结果与测序验证结果完全一致。因此, 利用HRM技术可以实现在闭合单管中简便、准确地对CYP2C19*2 、CYP2C19*3两个多态性位点同时进行基因分型。该方法有望应用于临床, 指导氯吡格雷的个体化用药。     

氯吡格雷对大鼠肝药物酶CYP3A4 和CYP1A2 表达的影响

目的：氯吡格雷主要由CYP3A4 催化使其激活,CYP1A2 也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外 文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4 和CYP1A2 的表达,探讨氯吡格雷对大鼠肝药物酶的影 响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13.5,6.75mg/kg/d）,雄性健康大鼠连续灌胃给药7天,脱臼处 死,取肝组织,通过western blot法检测大鼠肝脏CYP3A4 和CYP1A2 蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4 蛋白 表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4 蛋白表达量降低（P<0.05）;氯吡格雷低中高剂量组间进行比较,大 鼠CYP3A4 蛋白表达量呈梯度减少（P<0.05）;2）、氯吡格雷抑制大鼠CYP1A2 蛋白表达,氯吡格雷高中低剂量组分别比生理盐水 组大鼠CYP1A2 蛋白表达量降低（P<0.05）,氯吡格雷低中高剂量组间进行比较,大鼠CYP1A2 蛋白表达量呈梯度减少（P<0.05）。 结论：氯吡格雷使肝细胞色素氧化酶CYP3A4 和CYP1A2 的表达量减少,因此氯吡格雷高、中、低3 个剂量组均不同程度的抑制 大鼠肝脏CYP3A4 和CYP1A2 的表达,提示当氯吡格雷与某些主要经CYP3A4 和CYP1A2 代谢的药物合用时,发生代谢性相关 作用的可能性大。     

氯吡格雷对大鼠肝药物酶CYP3A4和CYP1A2表达的影响

目的：氯吡格雷主要由CYP3A4催化使其激活,CYPlA2也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4和CYPlA2的表达,探讨氯吡格雷对大鼠肝药物酶的影响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13．5,6．75mg／kg／d）,雄性健康大鼠连续灌胃给药7天,脱臼处死,取肝组织,通过westernblot法检测大鼠肝脏CYP3A4和CYPlA2蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4蛋白表达量降低（P〈0．05）;氯吡格雷低中高剂量组间进行比较,大鼠CYP3A4蛋白表达量呈梯度减少（P〈0．05）;2）、氯吡格雷抑制大鼠CYPlA2蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYPlA2蛋白表达量降低（P〈0．05）,氯吡格雷低中高剂量组间进行比较,大鼠CYPlA2蛋白表达量呈梯度减少（P〈0．05）。结论：氯吡格雷使肝细胞色素氧化酶CYP3A4和CYPlA2的表达量减少,因此氯吡格雷高、中、低3个剂量组均不同程度的抑制大鼠肝脏CYP3A4和CYPlA2的表达,提示当氯吡格雷与某些主要经CYP3A4和CYPlA2代谢的药物合用时,发生代谢性相关作用的可能性大。     

CYP2C19基因多态性PCR扩增体系的正交优化与验证

本实验通过建立CYP2C19*2、*3和*17基因多态性PCR反应体系和条件,筛选出4μL体系中各因素最佳水平。采用SNaPshot技术对CYP2C19基因3个SNP位点*2、*3和*17同时进行复合扩增检测,利用L9(34)正交实验设计,对影响PCR反应体系和条件的3个因素(PCR Mix, Taq DNA聚合酶,循环次数)在3个水平上进行优化,结果采用综合评分法和极差分析法进行分析。用3组已知样本对正交优化所得条件进行重复性和稳定性验证。结果表明CYP2C19*2、*3和*17基因PCR扩增体系的影响因素依次为:PCR Mix>循环数>Taq DNA聚合酶。最佳反应体系为PCR Mix 2.0μL、Taq DNA聚合酶0.2μL、循环次数32次。3组样本验证效果满意。优化的CYP2C19*2、*3和*17基因PCR反应体系稳定性高,重复性和经济性较好,为该基因多态性的大规模调查奠定了基础。     

Role of CYP2C9 and CYP2C19 polymorphisms in patients with atherosclerosis

The arachidonic acid metabolizing CYP enzymes with prominent roles in vascular regulation are epoxygenases of the two gene family which generate epoxyeicosatrienoic acids. Carriers of CYP2C9 mutant alleles exhibit a diminished CYP2C9 metabolic capacity leading to decreased endothelium-derived hyperpolarizing factors (EDHF) synthesis and an increased risk for atherosclerosis. We investigated whether the polymorphisms of CYP2C9/19 are related with atherosclerosis. We examined 108 patients having angioraphically > or =70 coronary artery narrowing and 90 healthy controls. CYPC2C9/19*2 and CYP2C9/19*3 alleles were investigated in both patients and controls by a real time PCR instrument. There was no significant difference in the distribution of the CYP2C9*2/*3 alleles between cases and the controls. We found that smoker patients having CYP2C9*2 heterozygote genotype have 3.7-fold risk of developing atherosclerosis. CYP2C19*3 heterozygote alleles are more frequent in patients than in controls (10.2%, 5.6% respectively) and it is related with a three-fold risk of atherosclerosis (odds ratio (OR) = 3.75, confidence interval (CI) = 0.75-18.65). It becomes clear that cigarette smoking can cause almost all major diseases prevalent today, such as cancer or heart disease. This inter-subject variability in cigarette-induced pathologies is partly mediated by genetic variants of genes that may participate in detoxification processes, e.g., cytochrome P450 (CYP), cellular susceptibility to toxins, such as p53, or disease development such as atherosclerosis.     

CYP2C19遗传多态性的研究进展

S-美芬妥英羟化代谢多态性不仅存在个体差异,而且存在种族差异。CYP2C19基因是决定S-美芬妥英羟化代谢的决定基因,该基因的突变是导致S-美芬妥英羟化代谢多态性的分子机制。近年来对CYN50s基因型和表型相关性的研究越来越受到重视,人们希望利用基因型分析来了解个体中该药物代谢酶的活性,期望既能提高药物治疗水平同时又降低不良反应的发生。有关CYP2C19的研究在此方面已树立了一个成功的典范。     

Lipophilicity Relationships in Inhibitors of CYP2C9 and CYP2C19 Enzymes

Quantitative structure-activity relationships (QSARs) within a series of cytochrome P450 2C9 (CYP2C9) and cytochrome P450 2C19 (CYP2C19) inhibitors are reported. In particular, it is noted that compound lipophilicity, in the form of log P values (where P is the octanol/water partition coefficient), is an important factor in explaining the variation in inhibitory potency within these series of compounds, many of which also act as substrates for the respective enzymes. In addition, there is a role for hydrogen bonding and π-π stacking interactions within the P450 active site which represent secondary factors in the binding processes of these compounds.     

Rabeprazole Can Overcome the Impact of CYP2C19 Polymorphism on Quadruple Therapy

Objectives: The prospective study was designed to clarify the impact of CYP2C19 on quadruple therapies and survey the efficacies of rabeprazole‐based quadruple therapy for Helicobacter pylori infection after failure of standard triple therapies. Patients and Methods: From January 2007 to March 2009, 1055 H. pylori‐infected patients received standard triple regimens (proton‐pump inhibitor (PPI), clarithromycin, and amoxicillin). Helicobacter pylori eradication was achieved in 865 (81.9%) subjects. One hundred ninety eradication‐failure patients were enrolled and randomly assigned to receive a 7‐day eradication therapy. Ninety‐six patients were treated with esomeprazole‐based quadruple rescue therapies (EB), while 94 patients were treated with rabeprazole‐based quadruple rescue therapies (RB). Follow‐up endoscopy was done 16 weeks later to assess the treatment response. Patients’ responses, CYP2C19 genotypes, and antibiotics resistances were also examined. Results: Intention‐to‐treat analysis revealed that RB had better eradication rates than EB (EB: 72.9%; 95% CI: 64.9–80.9% and RB: 78.7%; 95% CI 72.5–84.9%) (p value = .543). Per‐protocol results were EB = 75.3%; 95% CI: 70.3–80.3% and RB = 85.1%; 95% CI: 80.6–89.6% (p value = .0401). Both regimens had similar compliance (p value = 0.155) and adverse events (p value = 0.219). We also surveyed those patients without resistance of any antibiotics. RB still showed better outcome than EB. Our data showed that esomeprazole‐based regimen and CYP2C19 Hom EM genotype were important predictors for eradication failure. Conclusions: In quadruple therapy, rabeprazole‐based regimens had better efficacy than esomeprazole‐based regimens. CYP2C19 polymorphism also played an important role in quadruple therapy. It seems advisable to change PPI to rabeprazole in second‐line quadruple therapy.     

趋化因子CX3CL1与冠心病合并2型糖尿病发病的相关关系

目的:探讨趋化因子CX3CL1与冠心病合并2型糖尿病发病的相关关系。方法:采用病例-对照的研究方法,收集冠心病合并2型糖尿病患者400例,收集对照组400例,利用免疫组化检测冠心病合并2型糖尿病患者颈外动脉旋切术后斑块组织中CX3CL1表达水平,分别检测上述2组不同人群血清中的CX3CL1表达水平,同时采用直接测序方法检测CX3CL1基因rs170364位点基因型及等位基因的分布频率在对照组和冠心病合并2型糖尿病人群的分布差异。结果:冠心病合并2型糖尿病患者颈外动脉斑块组织中CX3CL1表达明显增高,冠心病合并2型糖尿病患者血清中CX3CL1的表达水平明显高于对照人群中CX3CL1的表达。CX3CL1基因rs170364单核苷酸多态位点的三种基因型分布频率(GG型,GT型和TT型)在冠心病合并2型糖尿病患者的分布频率为42.7%,40.0%和17.2%,在对照组分布频率为50.2%,39.6%和10.2%,CX3CL1基因rs170364位点T等位基因是冠心病合并2型糖尿病发病的一个独立危险因素(P0.05)。Logistic回归校正性别、年龄、体重指数、吸烟、高血压、高脂血症等冠心病合并2型糖尿病的易患因素后,CX3CL1基因rs170364 T等位基因仍是冠心病合并2型糖尿病发病的一个独立的危险因素。结论:CX3CL1在冠心病合并2型糖尿病的患者血清和颈外动脉动脉血管组织中表达明显增高,CX3CL1基因rs170364T等位基因可能是冠心病合并2型糖尿病患者发病的独立危险因素。     

Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms

Cytochrome P450 (CYP) 2C8 and 2C9 are polymorphic enzymes. The CYP2C8*3 and CYP2C9*2 are the major variant alleles in Caucasian populations. The enzymes encoded by these variant alleles have impaired function for the metabolism of several drug substrates. In the present study 1468 subjects that were used as population-based controls in the Stockholm Heart Epidemiology Program (SHEEP) were genotyped by allelic discrimination using a 5'-nuclease assay for CYP2C8*1, 2C8*3, 2C9*1, 2C9*2, and 2C9*3 variant alleles in which the frequencies appeared to be 0.91, 0.095, 0.83, 0.11, and 0.066, respectively. Approximately, 96% of the subjects with CYP2C8*3 allele also carried a CYP2C9*2 and 85% of the subjects that had CYP2C9*2 variant also carried a CYP2C8*3. The number of subjects carrying both of the CYP2C8*1*3 and CYP2C9*1*2 was 4.5-fold higher than expected. This strong association may be of importance especially for the metabolism of common substrates of CYP2C8 and CYP2C9 like arachidonic acid that produces physiologically active metabolites.     

Active-site characteristics of CYP2C19 and CYP2C9 probed with hydantoin and barbiturate inhibitors

Three series of N-3 alkyl substituted phenytoin, nirvanol, and barbiturate derivatives were synthesized and their inhibitor potencies were tested against recombinant CYP2C19 and CYP2C9 to probe the interaction of these ligands with the active sites of these enzymes. All compounds were found to be competitive inhibitors of both enzymes, although the degree of inhibitory potency was generally much greater towards CYP2C19. Inhibitor stereochemistry did not markedly influence K(i) towards CYP2C9, and log P adequately predicted inhibitor potency for this enzyme. In contrast, stereochemistry was an important factor in determining inhibitor potency towards CYP2C19. (S)-(+)-N-3-Benzylnirvanol and (R)-(-)-N-3-benzylphenobarbital emerged as the most potent and selective CYP2C19 inhibitors, with K(i) values of < 250nM--at least two orders of magnitude greater inhibitor potency than towards CYP2C9. Both inhibitors were metabolized preferentially at their C-5 phenyl substituents, indicating that CYP2C19 prefers to orient the N-3 substituents away from the active oxygen species. These features were incorporated into expanded CoMFA models for CYP2C9, and a new, validated CoMFA model for CYP2C19.