【作者】 张晓星；

【导师】 李一石；

【作者基本信息】 北京协和医学院 ， 内科学， 2014， 博士

【摘要】 一、研究背景氯吡格雷作为血小板膜蛋白P2Y12受体的抑制剂,通过阻断其与二磷酸腺苷(ADP)的结合发挥抑制血小板聚集作用。该药与阿司匹林联合应用,预防冠状动脉粥样硬化性心脏病(冠心病,CHD)患者接受了经皮冠状动脉介入治疗(PCI)术后的心脏缺血性事件复发,已成为目前美国、欧洲及我国指南共同推荐的常规治疗策略。近年的研究表明,这种双联抗血小板的治疗策略尚不能完全避免冠状动脉支架植入术后心脏缺血性事件的发生,其剩余风险可能与抗血小板药物的个体反应性,特别是氯吡格雷的个体间差异存在一定程度的关联。目前已知一种由CYP2C19基因编码的代谢酶,在氯吡格雷经肝脏代谢转化为活性产物的过程中发挥着至关重要的作用。携带该功能丧失性等位基因的患者表现为对氯吡格雷反应性不佳,成为PCI术后支架内血栓形成及主要心血管不良事件发生的一个重要因素。血小板功能检测结果作为抗血小板药物疗效的一种表现形式,与CYP2C19基因型一并成为评价个体对氯吡格雷反应性的指标,在临床研究中作为个体化治疗策略调整的依据被广泛采用。然而,近期数个依据血小板活性对患者进行分组并给予不同治疗方案的前瞻性临床研究却未能取得预期的阳性结果。本研究拟以血小板聚集率为指标,评价CYP2C19基因型、不同剂量的氯吡格雷预处理策略和PCI介入操作本身对该指标的影响,了解其各自之间的关联性；并以冠状动脉支架植入术后心血管事件和出血事件的发生率为指标,评价根据CYP2C19的基因型或血小板聚集率的表现型两种分组方式对患者远期预后的预测价值,为进一步寻找最佳的氯吡格雷治疗模式提供相应的理论依据。二、研究内容及结果第一部分不同氯吡格雷预处理剂量CYP2C19基因型对冠心病患者PCI术前血小板聚集率、术后心肌酶学指标的影响1.研究目的评价PCI术前两种不同的氯吡格雷预处理策略：未曾服用该药的患者接受300mg负荷剂量,对比已长期服用该药75mg每日一次的维持剂量,在不同CYP2C19基因型的CHD患者中是否存在血小板抑制作用的差异。2.研究方法2.1研究对象：连续入选2012年5月至2013年5月间在阜外心血管病医院住院,并拟接受PCI治疗的CHD患者。入院前已连续服用氯吡格雷(波利维)75mg/日持续7天以上(维持剂量组),或未曾服用过氯吡格雷(负荷剂量组)。2.2CYP2C19基因型检测：采用Taqman(?)技术在全部患者中进行,测量位点包括CYP2C19*2、CYP2C19*3、CYP2C19*17。除外*17携带者后,依患者是否携带*2或*3任一功能丧失性(LOF)等位基因,将其分为携带者组与非携带者组。2.3血小板功能检测：负荷剂量组患者在服用300mg氯吡格雷后12小时,维持剂量组患者在上一次服用75mg氯吡格雷后24小时进行空腹采血,以ADP (10umol/L)诱导激活的光学比浊法对血小板聚集率进行测定。2.4依据上述氯吡格雷预处理策略和CYP2C19基因型两种方式对患者进行两两分组,比较在全部患者中PCI术前的血小板聚集率、以及在接受冠状动脉支架植入患者中术后的心肌酶学指标(cTNI和CK-MB)在不同组间是否存在差异。3.研究结果3.1在全部入选的840例患者中,根据氯吡格雷的预处理策略(负荷剂量组470例,维持剂量组370例),以及CYP2C19基因型(CYP2C19*2或*3LOF等位基因携带者494例,非携带者346例)进行了两两分组及进一步的2×2分组。3.2接受负荷剂量的患者与维持剂量组相比,表现出更高的血小板聚集率(59.22±11.67%vs.52.83±12.17%, P<0.001); CYP2C19功能丧失性等位基因携带者与非携带者相比,亦表现出与氯吡格雷预处理策略分组间相似幅度的血小板聚集率升高(59.41±10.91%vs.52.10%±12.90%,P<0.001)。3.3负荷剂量给药策略在CYP2C19功能丧失性等位基因携带者或非携带者中,均表现出比维持剂量策略更高的血小板聚集率(61.50±10.61%vs.56.84±10.74%,P<0.001；56.06±12.34%vs.46.88±11.78%,P<0.001),且在氯吡格雷给药策略和CYP2C19基因型之间存在对血小板聚集率影响的交互作用(P=0.001)。3.4在371例接受了冠状动脉支架植入的患者中,分别根据不同的氯吡格雷预处理策略和CYP2C19基因型进行分组,术后的cTNI和CK-MB均未观察到显著的组间差异(P均>0.05)。4.研究小结在接受PCI的中国患者中,氯吡格雷300mg负荷剂量组对血小板的抑制作用弱于75mg维持剂量组；该作用差异在CYP2C19功能丧失性等位基因非携带者中更为显著,但对PCI术后的心肌酶学水平无显著影响。第二部分连续服用氯吡格雷的冠心病患者中单纯冠状动脉造影术与支架植入术对血小板聚集率的影响差异研究1.研究目的评价冠状动脉造影检查术或冠状动脉支架植入术两种介入操作对连续服用氯吡格雷的CHD患者血小板聚集率的影响差异。2.研究方法2.1研究对象：连续入选2012年5月至2013年5月间在阜外心血管病医院住院,并拟接受PCI治疗的CHD患者,入院前已连续服用氯吡格雷(波利维)75mg/日持续7天以上。2.2血小板功能检测：分别采集PCI术前及术后12-24小时内的空腹血样本,以ADP(10umol/L)诱导激活的光学比浊法对血小板聚集率进行测定。2.3根据是否进行冠状动脉支架植入将患者分为冠脉造影组和支架植入组,分别比较两组内术后与术前的血小板聚集率是否存在差异,以及两组间术后与术前的血小板聚集率变化幅度是否存在差异。2.4在部分患者中探索性研究miR-223与miR-96水平是否与血小板聚集率存在关联。3.研究结果3.1总共入选343例接受冠状动脉介入操作的患者,其中单纯冠脉造影组173例,支架植入组170例。3.2与术前相比,冠脉造影组术后血小板聚集率无明显变化(54.21±11.44%vs.53.15±11.80%,P=0.062)；支架植入组术后血小板聚集率显著升高(55.59±10.47%vs.52.47±11.97%,P<0.001)。3.3与冠脉造影组相比,支架植入组术后的血小板聚集率升高幅度更为显著(3.12±8.31%vs.1.06±7.40%,P=0.010)。3.4接受冠脉支架植入的患者中,有66例在术后30天自愿参加了血小板聚集率复测的亚组研究。在该亚组患者中,术后30天的血小板聚集率显著低于术后24小时(54.71±11.64%vs.56.68±10.21%,P=0.019),且与术前基线值相比无显著差异(54.71±11.64%vs.54.26±12.23%,,P=0.901)。3.5miR-96水平与血小板聚集率存在一定程度关联(相关系数0.380,P=0.038),而miR-223则否(相关系数0.075,P=0.688)。4.研究小结冠状动脉支架植入操作会导致术后血小板活性的升高,该作用可在术后30天内消失,而单纯冠状动脉造影则无此影响。第三部分基于基因型或表现型分组方式对预测氯吡格雷在冠状动脉支架植入术后患者中的临床疗效和安全性效果评价1.研究目的评价以CYP2C19基因型或血小板功能表现型两种不同的分组方式,对冠状动脉支架植入术后持续服用氯吡格雷的患者临床预后及安全性的预测价值。2.研究方法2.1研究对象：连续入选2012年5月至2013年5月间在阜外心血管病医院住院、接受冠状动脉造影检查并至少植入一枚支架的患者,术后持续给予氯吡格雷75mg/日及阿司匹林100mg/日。2.2CPY2C19基因测序：采用Taqman(?)技术进行,测量位点包括CYP2C19*2、 CYP2C19*3、CYP2C19*17,根据检测结果将患者按三种不同的代谢型进行分组：超快或快代谢型(*1/*1、*1/*17、*17/*17)、中间代谢型(*1/*2、*1/*3、*2/*17、*3/*17)、慢代谢型(*2/*2、*2/*3、*3*3)。2.3血小板聚集率检测：以ADP (10umol/L)诱导激活的光学比浊法对血小板聚集率进行测定,根据检测结果将患者按三种不同的表现型进行分组：良好反应者、中度反应者、无反应者。2.4随访至最后1例入组患者术后6个月,观察主要心脏事件(心脏性死亡、急性心肌梗死、支架内血栓、以及预期外的支架植入血管再次血运重建)、心脏症状事件(上述主要心脏事件以及支架植入术后再次心绞痛发作)、和全部出血事件(GUSTO研究中定义的轻度出血、中度出血、以及致命性出血)的发生率。比较根据基因型或表现型对患者进行分组后,上述事件的发生率是否存在组间差异。3.研究结果3.1共入选380例接受了冠状动脉支架植入的患者,其中根据基因型分组,氯吡格雷超快代谢型或快代谢型共157例(41.3%),中间代谢型176例(46.3%),慢代谢型47例(12.4%)；根据表现型分组,氯吡格雷反应良好者98例(25.8%),中度反应者149例(39.2%),无反应者133例(35.0%)。3.2根据基因型进行分组时,心脏症状事件的发生率在慢代谢型患者中依次高于中间代谢型(34.0%vs.19.1%, HR=2.126,95%CI1.131～3.997, P=0.019)和超快或快代谢型患者(34.0%vs.15.4%, HR=2.772,95%CI1.359～5.655, P=0.005),主要心脏事件和出血事件的发生率无组间显著差异(P均>0.05)3.3根据表现型进行分组时,出血事件的发生率在反应良好者中依次高于中度反应者(32.7%vs.19.0%, HR=1.919,95%CI1.009～3.650, P=0.047)和无反应者(32.7%vs.18.3%, HR=2.119,95%CI1.045～4.292, P=0.037),主要心脏事件和心脏症状事件的发生率无组间显著差异(P均>0.05)4.研究小结氯吡格雷在人群中可以表现为不同的个体反应性,并导致患者的临床预后存在差异。CYP2C19基因型检测结果对冠状动脉支架植入术后心脏缺血性事件的预测更有意义,而血小板功能的表现型检测结果对出血事件的预测更有意义。三、研究结论1.300mg负荷剂量的氯吡格雷预处理策略、以及CYP2C19*2或*3LOF等位基因的携带,均是导致氯吡格雷抗血小板作用减弱的因素,且二者之间存在交互关系；这两种因素导致的血小板功能差异,尚不足以影响患者PCI术后的心肌酶学水平；2.冠状动脉支架植入术会导致血小板活性的一过性升高,在已接受长期抗血小板药物治疗的患者中,介入操作术前的血小板功能检测结果与药物的远期效应更趋于一致；3. CYP2C19基因型与冠状动脉支架植入术后心脏症状性事件的发生率相关,而血小板功能检测的表现型则与出血风险相关,在未来的研究设计和临床实践中应将两种检测方式结合考虑,用于患者远期预后的预测及抗血小板治疗策略的调整。更多还原

【Abstract】 BackgroundClopidogrel is an inhibitor of P2Y12receptor on the platelet membranin, which blocks its combination with adenosine diphosphate (ADP) to restrain the platelet aggregation. Clopidogrel is administered with aspirin to prevent the recurrent ischemia events in patients suffered from coronary heart disease after percutaneous coronary intervention. It has been demonstrated recently that the dual anti-platelet therapy strategy cannot completely avoid cardiac ischemia events after coronary stent implantation, the residual risk may be related to the insufficient response to the anti-platelet medication, especially because of the individual variability response of clopidogrel.The metabolic enzyme encoded by CYP2C19plays a pivotal role in active metabolite transformation of clopidogrel through CYP450enzymes in the liver. Patients carried loss-of-function (LOF) allele of CYP2C19do not response well to clopidogrel and may experience a higher risk of stent thrombosis and major adverse cardiovascular events than non-carriers. Results of platelet function test and CYP2C19genotype are considered to be manifestations of individual response to clopidogrel and widely used in clinical trials during anti-platelet therapy strategy decision making. However, none of recent prospective studies showed a positive result when individual anti-platelet strategy was randomly administered in patients with high on-treatment platelet reactivity.The RDPAC study aimed to evaluate the impact of CYP2C19genotype, clopidogrel pretreatment dose, and PCI procedure to the platelet aggregation, as well as the predict value of long-term clinical outcome by the genotyping of CYP2C19and phenotyping of platelet function, in order to find the evidence based medicine for optimal clopidogrel therapeutic model.Part1:Impact of clopidogrel pretreatment dose and CYP2C19genotype on platelet aggregation prior to PCI and cardiac enzymes after PCI in patients with coronary heart disease ObjectivesTo evaluate the platelet inhibition effect difference between the two clopidogrelpretreatment strategies:300-mg loading dose (LD) in clopidogrel naive patients or75-mg maintenance dose (MD) in patients received chronic clopidogrel therapy in CHD patients with different CYP2C19genotypes.MethodsCHD patients administered in Fuwai hospital undergoing elective PCI during May2012to May2013were assigned to2X2groups according to different clopidogrel pretreatment strategies (470patients in the LD group versus370patients in the MD group) and CYP2C19genotypes (494carriers of any CYP2C19*2or*3LOF allele versus346non-carriers). The primary outcome was platelet aggregation (PA) as measured by the10μmol/L ADP induced light transmission aggregation prior to PCI and cardiac enzymes (cTNl and CK-MB) after PCI between the different groups.Results1. Compared with the MD group, the LD strategy showed a significantly higher on-treatment PA (59.22±11.67%vs.52.83±12.17%, P<0.001), similar PA difference was observed in the CYP2C19loss-of-function carriers compared with the non-carriers (59.41110.91%vs.52.10%±12.90%P<0.001).2. Patients treated with the LD strategy in either the CYP2C19loss-of-function allele carrier or non-carrier group showed a significantly higher PA compared with the MD group (61.50±10.61%vs.56.84±10.74%, P<0.001;56.06±12.34%vs.46.88±11.78%, P<0.001, respectively), a quantitative interaction effect was observed between the clopidogrel pre-treatment strategy and CYP2C19genotype (P=0.001).3.371patients received coronary stent implantation, both cTNl and CK-MB levels were not significantly different between groups defined by clopidogrel pretreatment strategy or CYP2C19genotype (P>0.05). Part2:Impact of coronary angiography and stent implantation on platelet aggregation in CHD patients under chronic clopidogrel therapyObjectives To evaluate the impact of coronary angiography and stent implantation procedureon platelet aggregation in CHD patients under chronic clopidogrel therapy.Methods CHD patients administered in Fuwai hospital who had been treated with clopidogrel (75mg/day) and aspirin (100mg/day) for at least7consecutive days undergoing elective PCI were prospectively enrolled during May2012to May2013. Based on whether a coronary stent was implanted, all of the included patients were divided into two groups:the coronary angiography (CAG) group and the coronary stent implantation (CSI) group. The differences in PA as measured by the10μmol/L ADP induced light transmission aggregation between the preoperative baseline level and the level at24hours after the procedures in both patient groups were determined.Results1. A total of343patients receiving coronary intervention were enrolled in this study, including173patients in the CAG group and170patients in the CSI group.2. Compared with the preoperative baseline, there was no significant difference in PA after the operation in the CAG group (54.21111.44%vs.53.15±11.80%, P=0.062), while a significant postoperative PA increase was observed in the CSI group (55.59110.47%vs.52.47111.97%, P<0.001).3. Compared with the CAG group, the PA increase in the CSI group was significantly greater (the mean PA increase was2.07%,95%CI:0.40%to3.74%; P=0.010).4.66patients received repetitive PA testing30days after coronary stent implantation, which is significantly lower than the PA24hours after PCI (54.71±11.64%vs.56.68±10.21%, P=0.019) and comparable with the baseline PA before procedure (54.71±11.64%vs.54.26±12.23%, P=0.901) in this subgroup.Part3:Grouping by the phenotype vs. genotype for prediction of clinical efficacy and safety in patients received clopidogrel underwent PCIObjectivesTo evaluate the predictive value of both CYP2C19genotype and platelet function phenotype grouping in clinical outcome and bleeding events of patients received clopidogrel underwent PCI.MethodsCHD patients administered in Fuwai hospital underwent elective PCI and received coronary stent implantation were prospectively enrolled during October2012to May2013. Patients were assigned into groups by genotype of CYP2C19(extensive metabolizers, intermediate metabolizers, and poor metabolizers) and phenotype of platelet function (clopidogrel responders, semi-responders, and non-responders). The rates of major adverse cardiovascular events, cardiovascular symptom events, and bleeding events were recorded during a follow-up period until6months after the last patient first visit and compared among the groups defined previously.Results1.380patients received coronary stent implantation were enrolled in this study, including157(41.3%) clopidogrel extensive metabolizers,176(46.3%) intermediate metabolizers, and47(12.4%) poor metabolizers according to the genotype grouping;98(25.8%) were responders to clopidogrel,149(39.2%) were semi-responders, and133(35.0%) were non-responders according to the phenotype grouping.2. The highest cardiovascular symptom events rate was observed in the poor metabolizers (34.0%) as compared to the intermediate metabolizers (19.1%, HR=2.126,95%CI1.131～3.997, P=0.019) and the extensive metabolizers (15.4%, HR=2.772,95%CI1.359～5.655, P=0.005) when grouping by the CYP2C19genotype, without a statistical difference of the major adverse cardiovascular events rate or bleeding events rate.3. The highest bleeding events rate was observed in the clopidogrel responders (32.7%) as compared to the semi-responders (19.0%, HR=1.919,95%CI1.009～3.650, P=0.047) and non-responders (18.3%, HR=2.119,95%CI1.045～4.292, P=0.037) when grouping by the platelet function phenotype, without a statistical difference of the major adverse cardiovascular events rate or cardiovascular symptom events rate.Conclusions1. The300-mg LD strategy results in a decreased effect on platelet inhibition compared with the75-mg MD in Chinese patients receiving clopidogrel prior to PCI, interacted with the CYP2C19*2or*3LOF allele carriage. The platelet function difference due to clopidogrel pretreatment strategy cannot affect the cardiac enzyme level after PCI.2. Coronary stent implantation causes a transient increased ADP-induced PA, while the coronary angiography procedure does not. The result of platelet function test before PCI is in accordance with the effect of long term anti-platelet medication in patients received chronic clopidogrel therapy.3. Genotyping of CYP2C19is a better predictor of cardiovascular symptom events, while phenotyping of platelet response to clopidogrel is a better predictor of bleeding events in CHD patients received coronary stent implantation. Further study design and clinical practice should take into account both in anti-platelet therapy strategy decision and long term prognosis prediction.更多还原