冠心病患者氯吡格雷低反应性药物基因组学及药物效应动力学的研究

【作者】 唐晓芳；

【导师】 杨跃进；

【作者基本信息】 北京协和医学院 ， 内科学， 2014， 博士

【摘要】 研究背景氯吡格雷抑制血小板聚集具有个体差异性。研究发现部分患者使用氯吡格雷后仍有死亡、心肌梗死、中风或支架内血栓形成等严重不良缺血心血管事件发生,该现象称之为氯吡格雷低反应性。遗传因素是氯吡格雷低反应性的发生机制之一。目前尚不明确国人冠心病患者氯吡格雷代谢通路上相关基因的功能多态性位点与服用氯吡格雷后的血小板功能及不良临床预后的相关性。研究目的本研究拟验证国人冠心病患者氯吡格雷代谢通路上相关基因(ABCB1、CYP2C19、CYP3A、CYP3A5和口P2Y12)的功能多态性位点与服用氯吡格雷后的血小板功能及不良临床预后的相关性。对象和方法2012年1月到2013年2月入选收住我院拟行经皮冠状动脉介入治疗并置入支架的冠心病患者1408例。多重高温连接酶检测反应技术检测ABCB1、CYP2C19、 CYP3A4、CYP3A5和P2Y12基因上的15个功能多态性位点。血栓弹力图检测支架置入术后服用氯吡格雷24小时以内的血小板功能。所有入选患者均正规服用氯吡格雷12个月,并进行为期一年的临床随访。主要终点事件是指严重不良心血管事件,包括死亡、非致死性心肌梗死、靶血管血运重建或支架内血栓形成等复合终点事件；次要终点事件是指支架置入术后一年随访期间的死亡、非致死性心肌梗死、靶血管血运重建和支架内血栓形成。研究结果本研究入选国人冠心病患者携带CYP2C19功能缺失等位基因(*2和*3)的频率高达59.3%。携带两个CYP2C19功能缺失等位基因的患者与携带一个CYP2C19功能缺失等位基因或未携带CYP2C19功能缺失等位基因的患者相比,服用氯吡格雷后的血小板抑制率最低(44.6±31.1vs.52.0±30.0vs.55.4±30.5;P=0.001)。62位(4.4%)患者支架置入术后一年随访期间发生严重不良心血管事件。携带两个CYP2C19功能缺失等位基因的患者与携带一个CYP2C19功能缺失等位基因或未携带CYP2C19功能缺失等位基因的患者相比,严重不良心血管事件的发生率最高(7.5%vs.4.9%vs.2.8%；P=0.016)。多变量Cox回归分析发现：携带两个CYP2C19功能缺失等位基因的患者与未携带CYP2C19功能缺失等位基因的患者相比,支架置入术后一年随访期间严重不良心血管事件的发生率升高近3倍(HR=2.962；95%CI：1.426-6.152；P=0.004)：携带一个CYP2C19功能缺失等位基因的患者与未携带CYP2C19功能缺失等位基因的患者相比,严重不良缺血心血管事件的发生率没有明显升高(HR=1.833；95%CI：0.986-3.406；P=0.055)。氯吡格雷代谢通路上其他基因(ABCB1、CYP3A4、CYP3A5、和P2Y12)的功能多态性位点与国人冠心病患者服用氯吡格雷后的血小板反应性和支架置入术后一年随访期间严重不良心血管事件无明显相关性(P>0.05)。结论携带两个CYP2C19功能缺失等位基因的国人冠心病患者与服用氯吡格雷后的血小板功能和支架置入术后一年随访期间的严重不良心血管事件有明确相关性；携带两个CYP2C19功能缺失等位基因是国人冠心病患者支架置入术后一年随访期间严重不良心血管事件的独立预测因素；氯吡格雷代谢通路上其他基因(ABCB1、CYP3A4、CYP3A5和P2Y12)的功能多态性位点与国人冠心病患者服用氯吡格雷后的血小板功能和支架置入术后一年随访期间严重不良心血管事件无显著相关性。研究背景研究证实服用抗血小板药物后的残存血小板高反应性可能与不良的临床预后有相关性。血小板功能检测方法是评价冠心病患者体内血小板活性和服用抗血小板药物后残存血小板反应性的实验室检测手段。目前血小板功能检测方法众多,例如光学比浊法,血栓弹力图,VerifyNow快速分析仪,活化舒血管磷蛋白磷酸化的检测等。哪种检测方法能更为准确特异的反应血小板功能和预测临床预后结果尚无明确结论,尤其是中国冠心病患者服用氯吡格雷后的血小板高反应性更无统一的诊断标准。研究目的本研究拟比较光学比浊法和血栓弹力图两种方法检测服用氯吡格雷后血小板高反应性的相关性及探讨对国人冠心病患者支架置入术后不良临床预后的预测价值。对象和方法2012年1月到2013年2月入选收住我院已行经皮冠状动脉介入治疗并置入支架的冠心病患者789例。服用氯吡格雷24小时以内,所有患者均采用光学比浊法和血栓弹力图两种方法检测血小板功能。所有入选患者支架置入术后均进行为期一年的临床随访。严重不良心血管事件是指支架置入术后一年随访期间死亡、非致死性心肌梗死、靶血管血运重建或支架内血栓形成等复合终点事件。研究结果支架置入术后一年随访期间,有32例患者(4.1%)发生严重不良心血管事件,其中有5例发生非致死性心肌梗死,3例患者发生支架内血栓,26例患者行靶血管血运重建。光学比浊法和血栓弹力图两种方法检测服用氯吡格雷后的残余血小板反应有相关性(Spearman r=0.733,P<0.001)。ROC曲线分析表明,支架置入术后一年的随访期间,光学比浊法(曲线下面积：0.677；95%可信区间：0.643-0.710；P=0.0009)和血栓弹力图(曲线下面积：0.684；95%可信区间：0.650-0.716；P=0.0001)两种检测方法均能一定程度的预测中国冠心病患者支架置入术后的严重不良心血管事件。光学比浊法评估服用氯吡格雷后血小板高反应性的患者严重不良心血管事件的发生率较血小板正常反应的患者明显升高(7.4%vs.2.7%；P=0.003)；血栓弹力图评估服用氯吡格雷后血小板高反应性的患者严重不良心血管事件的发生率较血小板正常反应的患者亦明显升高(6.7%vs.2.6%；P=0.005)。Kaplan-Meier分析表明,光学比浊法或血栓弹力图诊断为服用氯吡格雷后血小板高反应性的患者与血小板正常反应的患者相比,支架置入术后一年随访期间严重不良心血管事件的发生率明显升高(HR：2.752,95%CI：1.360-5.569,P=0.002；或HR：2.601；95%CI：1.279-5.290,P=0.005)结论光学比浊法和血栓弹力图两种血小板功能检测方法检测国人冠心病患者支架置入术后服用氯吡格雷的血小板功能,相关性较好；两种血小板功能检测方法均能一定程度上预测支架置入术后服用氯吡格雷的血小板高反应性患者发生严重不良心血管事件的风险明显升高。研究背景支架置入术后服用氯吡格雷的冠心病患者,是否应该常规检测CYP2C19基因分型和血小板功能以评估其临床预后尚不明确。本研究假设：整合CYP2C19基因分型和血小板功能检测两项结果是支架置入术后再发不良缺血心血管事件强有力的预测因素。研究目的本研究拟探讨CYP2C19基因分型联合血小板功能检测的两项结果预测国人冠心病患者支架置入术后一年随访期间的严重不良心血管事件。对象和方法2012年1月到2013年2月入选收住我院拟行经皮冠状动脉介入治疗并置入支架的冠心病患者1104例。多重高温连接酶检测反应技术进行CYP2C19基因分型(rs4244285和rs4986893)。服用氯吡格雷24小时以内,血栓弹力图检测入选患者的血小板功能。服用氯吡格雷后血小板高反应性定义为血栓弹力图检测二磷酸腺苷诱导的血小板抑制率≤30%。所有入选患者均正规服用氯吡格雷一年,并进行为期一年的临床随访。严重不良心血管事件包括死亡,非致死性心肌梗死,靶血管血运重建或支架内血栓形成等复合终点事件。研究结果47例(4.3%)患者支架置入术后一年随访期间发生严重不良心血管事件。多变量Cox回归模型分析CYP2C19基因分型的结果显示：CYP2C19功能缺失等位基因携带者(*1/*2、*1/*3、*2/*2、*2/*3和*3/*3)与未携带CYP2C19功能缺失等位基因的患者相比,支架置入术后一年随访期间严重不良心血管事件的发生风险升高近2.2倍(HR：2.195,95%CI：1.035-4.658,P=0.04)。多变量Cox回归模型分析服用氯吡格雷后的血小板功能,结果表明：服用氯吡格雷后血小板高反应性的患者与血小板正常反应的患者相比,支架置入术后一年随访期间严重不良心血管事件的发生风险也升高2.2倍左右(HR：2.218,95%CI：1.131-4.351,P=0.02)。通过多变量Cox回归模型分析CYP2C19基因分型联合血小板功能检测两项结果显示：携带CYP2C19功能缺失等位基因且服用氯吡格雷后血小板仍呈高反应性的患者,即2个阳性结果与无阳性结果(未携带CYP2C19功能缺失等位基因且服用氯吡格雷后血小板正常反应)的患者相比,PCI术后一年随访期间严重不良心血管事件(死亡非致死性心肌梗死、靶血管血运重建或支架内血栓形成等复合终点事件)的发生率升高近3.8倍(HR：3.777,95%CI：1.508-9.461,P=0.005)。CYP2C19基因分型联合血小板功能检测两项结果加入常规临床预测模型后,ROC曲线评估国人冠心病患者支架置入术后一年随访期间严重不良心血管事件的预测能力有所升高(AUC,from0.67to0.73；P=0.074),全部患者的净重新分类指数高达27.7%(P=0.003)整体鉴别指数是0.016(P=0.013)结论CYP2C19基因分型联合血小板功能检测均呈阳性结果的国人冠心病患者,支架置入术后一年随访期间严重不良心血管事件的发生率最高；CYP2C19基因分型联合血小板功能检测均呈阳性结果者,较单一CYP2C19基因分型或血小板功能检测,能更有效的预测国人冠心病患者PCI术后一年随访期间的严重不良心血管事件。更多还原

【Abstract】 BackgroundSeveral studies have demonstrated wide inter-individual variability in the pharmacodynamic response to clopidogrel. Observational studies have linked a poor pharmacodynamic response to cardiovascular events after percutaneous coronary intervention (PCI), including death, recurrent myocardial infarction, stroke, or stent thrombosis. Genetic factors were considered as one of the mechanisms for clopidogrel hyporesposiveness. Effect of the ABCB1, CYP2C19, CYP3A4, CYP3A5, and P2Y12variants on platelet reactivity and clinical outcomes of clopidogrel has not been reported in Chinese patients undergoing PCI.ObjectivesThe aim of this study was to investigate the effect of the ABCB1, CYP3A4, CYP3A5, and P2Y12variants on clopidogrel pharmacodynamics and clinical outcomes in these patients.Methods1408patients undergoing stent implantation were enrolled in a single-center registry. The ABCB1, CYP2C19, CYP3A4, CYP3A5, and P2Y12genotypes were detected by the improved multiple ligase detection reaction, and the antiplatelet effect of clopidogrel were assessed by thrombelastography. Primary clinical endpoint was defined as major adverse cardiovascular events (MACE), which included the composite of all-cause death, nonfatal myocardial infarction, target vessel revascularization, or stent thrombosis. The secondary clinical endpoints were all-cause death, nonfatal myocardial infarction, target vessel revascularization, and stent thrombosis during12-month follow-up.ResultsThe carriage of the CYP2C19loss-of-function alleles was relatively high (59.3%).62(4.4%) patients had MACE during12-month follow-up. The lowest risk of response to clopidogrel (55.4±30.5vs.52.0±30.0vs.44.6±31.1; P=0.001) and the highest incidence of MACE (2.8%vs.4.9%vs.7.5%; P=0.016) increased with the number of CYP2C19loss-of-function alleles. In the multivariate Cox regression analysis, two CYP2C19LOF alleles (HR=2.962,95%CI:1.426-6.152, P=0.004) were associated with MACE compared with noncarriers; the carriers of one CYP2C19LOF allele (HR=1.833;95%CI:0.986-3.406; P=0.055) were not associated with MACE compared with noncarriers. However, there were no significant differences in clopidogrel pharmacodynamics and adverse ischemic events across the ABCB1, CYP3A4, CYP3A5and P2Y12genotype groups (P>0.05).ConclusionsThe CYP2C19loss-of-function alleles had a gene dose effect on the clopidogrel pharmacodynamics and major adverse cardiovascular events in our population. The carrage of two CYP2C19LOF alleles was an independent predictor of major adverse cardiovascular events in patients undergoing PCI during follow-up period. The antiplatelet effect and adverse clinical outcomes of clopidogrel were not significantly influenced by ABCB1, CYP3A4, CYP3A5and P2Y12genotype in these patients. BackgroundPatients exhibiting heightened platelet reactivity to adenosine diphosphate (ADP) might be at increased risk for recurrent ischemic events after coronary stenting. Several platelet function tests are currently used to measure responsiveness to antiplatelet therapy.However, which test can be best to detect platelet function specifically and predict clincial outcomes was still uncertain. High on-treatment platelet reactivity of Chinese patients is still no uniform diagnostic criteria.ObjectivesThe aim of our study was to whether patients receiving clopidogrel undergoing stenting who display high on-treatment platelet reactivity measured by light transmittance aggregometry (LTA) and thrombelastography (TEG) will be at increased risk for poststenting ischemic events.MethodsProspective, observational, single-center study of789Chinese patients underwent stent implantation. This study was investigated the correlations between the2tests (LTA and TEG) and performed receiver operating characteristic curve (ROC) analysis for major adverse cardiovascular events (MACE), a composite of death, myocardial infarction (MI), stent thrombosis, or target vessel revascularization, at1-year follow-up.ResultsMajor adverse cardiovascular events occurred in32patients (4.1%), including5patients with recurrent MI,3patients with stent thrombosis, and26patients with target vessel revascularization. Correlations were strong between the2tests in the adenosine diphosphate induced platelet reactivity (Spearman r=0.733, P<0.001). ROC-curve analysis demonstrated that LTA (area under the curve [AUC],0.677;95%confidence interval [CI],0.643-0.710; P=0.0009), and TEG assay (AUC,0.684;95%CI,0.650-0.716; P=0.0001) had moderate ability to discriminate between patients with and without MACE at1-year follow-up. MACE occurred more frequently in patients with high on-treatment platelet reactivity (HTPR) when assessed by LTA (7.4%vs.2.7%; P=0.003), and by TEG (6.7%vs.2.6%; P=0.005). Kaplan-Meier analysis demonstrated that HTPR based on the LTA and modified TEG was associated with almost4-fold increased risk of MACE at1-year follow-up (HR:2.752,95%CI:1.360-5.569, P=0.005; HR:2.601;95%CI:1.279-5.290, P=0.008; respectively).Conclusions The ADP-induced platelet aggregation by LTA and TEG showed a strong correlation. Hypo-responsiveness to clopidogrel measured by light transmittance aggregometry, and modified thrombelastography were significantly associated with MACE in Chinese patients undergoing stenting. However, the predictive accuracy of these two tests was moderate. BackgroundRoutine assessment of CYP2C19genotyping or platelet function after clopidogrel, which can be used to risk-stratify patients with coronary artery disease receiving stenting implantation, is still debated. We hypothesized that an aggregate risk evaluated by CYP2C19genotyping combined with platelet reactivity by thrombelastograph (TEG) platelet-mapping assay would be a powerful predictor of recurrent cardiovascular ischemic events.ObjectiveThis study sought to determine an integrate results evaluated by CYP2C19genotyping combined with platelet reactivity by TEG for enhanced prediction of major adverse cardiovascular events in Chinese patients undergoing coronary stenting with clopidogrel.Methods1104Chinese patients undergoing coronary stenting were enrolled in a single-center registry. CYP2C19genotyping (s4244285and rs4986893) were detected by the improved multiple ligase detection reaction. Platelet function testing were performed by thrombelastograph platelet-mapping assay. High on-treatment platelet reactivity (HTPR) was defined as cutoff values of ADP inhibition≤30%assessed by the TEG. Major adverse cardiovascular events (MACE) included the composite of all-cause death, nonfatal myocardial infarction, target vessel revascularization, or stent thrombosis. The follow-up period was12months.Results47patients (4.3%) had major adverse cardiovascular events. In the multivariate Cox regression analysis, carriers of CYP2C19loss-of-function alleles (*1/*2,*1/*3,*2/*2,*2/*3, and*3/*3) were increased nearly2.2-fold risk of MACE compared with noncarriers (HR:2.195,95%CI:1.035-4.658, P=0.04); HTPR were increased almost2.2-fold risk of MACE compared with normal on-treatment platelet reactivity (HR:2.218,95%CI:1.131-4.351, P=0.02). In the multivariate Cox regression analysis after an integrate results evaluated by CYP2C19genotyping combined with platelet reactivity by TEG,2positive results (carriers of CYP2C19loss-of-function alleles and HTPR) were significantly increased almost3.8-fold of the risk of MACE compared with0positive results (noncarriers of CYP2C19loss-of-function alleles and normal on-treatment platelet reactivity)(HR:3.777,95%CI:1.508-9.461, P=0.005). Adding the integrate results to conventional clinical predictors improved prediction trend for1-year MACE as measured by the area under the ROC curve (AUC, from0.67to0.73; P=0.074), and the net reclassification improvement (NRI=27.7%; P=0.003)/integrated discrimination improvement (IDI=0.016; P=0.013).ConclusionsThe integrate results evaluated by CYP2C19genotyping combined with platelet reactivity by TEG is an independent and additive predictor of1-year MACE in Chinese patients undergoing stenting with clopidogrel, which is better than the either single testing (CYP2C19genotyping or platelet function testing).更多还原