羧酸酯酶1A2基因多态性对氯吡格雷代谢及抗血小板活性影响研究

发布时间：2018-01-28 05:47

  本文关键词： 氯吡格雷 基因多态性 肝微粒体 健康志愿者 CES1A2A(-816)C　出处：《苏州大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：通过羧酸酯酶1A2（CES1A2）对人肝微粒体体系中氯吡格雷体外代谢和中国健康志愿者口服氯吡格雷的药代动力学及药效学影响的研究，系统探讨CES1A2基因多态性在氯吡格雷代谢和抗血小板活性差异中的作用，以期为临床氯吡格雷合理使用提供依据。 方法：（1）基因型测定：用Promega试剂盒提取肝组织和EDTA抗凝的全血中的DNA，用PCR-RFLP法测定CYP2C19*2/*3基因型，并随机抽取样本率不少于10%用直接测序对方法学进行验证；用直接测序法测定CES1A2A(-816)C基因型。 （2）氯吡格雷及其活性和非活性代谢产物浓度测定：人肝微粒体孵育体系和人血浆中氯吡格雷及其活性和非活性代谢产物浓度均用超高效液相串联质谱(UPLC-MS/MS)法测定。 （3）人肝微粒体孵育体系中CES1A2不同基因型下氯吡格雷的体外代谢测定：根据CYP2C19*2及*3突变基因的数目将肝组织分为三种代谢表型：快代谢者[EMs(CYP2C19*1/*1)]，中间代谢者[IMs (CYP2C19*1/*2，*1/*3)]和弱代谢者[PMs(CYP2C19*2/*2，*2/*3或*3/*3)]。在根据CYP2C19分型后的肝组织中，分别随机抽取CES1A2不同基因型肝组织（CYP2C19EM型中CES1A2-816AA，AC和CC各6例；CYP2C19IM型中AA和AC各6例，未检出CC型）；同种基因型肝组织合并后进行氯吡格雷体外代谢试验，考察CES1A2催化氯吡咯雷生成非活性代谢产物的酶促反应动力学特征，同时测定CES1A2不同基因型氯吡格雷活性代谢产物生成的情况。利用Graphpad5.0软件拟合底物浓度与反应速率的非线性回归方程：单位点的Michaelis-Menten方程，计算氯吡咯雷非活性代谢产物的体外代谢酶促反应的表观动力学常数Vmax和Km，求得内在清除率Clint（Vmax/Km）。三组基因型肝微粒体中非活性代谢产物生成的酶动力学参数和活性代谢产物生成水平的比较采用单因素方差分析（ANOVA），三组基因型中的两两比较采用LSD（least significant difference）事后检验或t检验。P0.05认为具有统计学差异。 （4）中国健康志愿者药代动力学-药效学测定：11例符合入组标准并签署知情同意书的健康志愿者按照CES1A2不同基因型筛选分成两组：CYP2C19均为EM，一组为CES1A2AA型（n=6例），一组为CES1A2AC或CC型（n=5例），两组均给予氯吡格雷300mg后，分别于给药前（0h）和给药后0.25，0.5，0.75，1，1.25，1.5，2，3，4，5，6，8，12，24，36h采集血样，用于药代动力学（PK）研究；并于给药前（0h）和给药后1，2，4，12，24h采集血样，用于药效学（PD）研究。利用UPLC-MS/MS测定氯吡格雷及其非活性和活性代谢产物的浓度，利用非房室模型分析氯吡格雷原药、活性及非活性代谢产物药动学参数；利用四通道血小板聚集仪测定的ADP诱导的血小板聚集率，以血小板聚集抑制的变化程度（IPA%）作为氯吡格雷药效学指标。 结果：（1）基因型分布：86例人肝组织样本中进行CYP2C19*2/*3和CES1A2A(-816)C基因测定：○1CES1A2A(-816)C基因分布：AA型43例（50.0%），AC型37例（41.86%），CC型6例（8.14%）； ○2ECAYP2C19*2/*3基因表型分布：快速代谢型EM有37例（43.02%），中等代谢型有38例（44.19%），弱代谢型有11例（12.79%）； ○A3EA亚型分布：在37例CYP2C19EM的亚组中：CES1A2A(-816)C AA型15例（40.54%），，AC型16例（43.24%），CC型6例（16.22%）；在38例CYP2C19IM的中，AA型24例（63.16%），AC型14例（36.84%）；在11例CYP2C19PM中，AA型4例（36.36%），AC型7例（63.64%）。 （2）人肝微粒体孵育体系与人血浆中氯吡格雷原药、活性及非活性代谢产物浓度的方法学：氯吡格雷活性代谢产物在肝微粒体孵育体系中和人血浆中的线性范围均为1~200ng·mL-1，且线性良好（R2分别为0.9977和0.9930）；氯吡格雷非活性代谢产物在肝微粒体孵育体系中和人血浆中的线性范围分别为10~2000ng·mL-1和25~10000ng·mL-1，且线性良好（R2分别为0.9964和0.9945）；氯吡格雷原药在血浆中的线性范围为0.1~20ng·mL-1，且线性良好（R2为0.9961），方法的批内批间精密度、回收率、基质效应、稳定性等均符合生物样本检测要求。 （3）CES1A2不同基因型对人肝微粒体孵育体系中氯吡格雷非活性代谢产物生成酶动力学及活性代谢产物浓度的影响： ○A1ECAES1-816CC基因型的Vmax是-816AA型的1.34倍（CC vs AA：635.20±12.06vs475.10±26.55pmol·min-1·mg·protein-1，P0.05），相应的活性代谢产物水平在底物浓度为20、50μM时呈现出了降低趋势（P0.05）。 ○A2EA根据CYP2C19代谢表型分组后，在EM组中，CES1-816CC的Vmax是-816AA型的1.39倍（CC vs AA：669.80±20.72vs480.6±13.84pmol·min-1·mg·protein-1，P0.05），相应的氯吡格雷活性代谢产物水平只有-816AA型的60%或更低。在IM中同样观察到了类似的现象，但在PM组中，由于活性代谢产物生成量极低，并未观察到类似的结果。 （4）CES1A2基因多态性对健康志愿者氯吡格雷药代动力学影响：CES1A2-816AA组与CES1A2-816AC/CC组相比，氯吡格雷AUC0-∞（15.6±4.8vs9.7±1.7ng·mL-1·h-1），氯吡格雷活性代谢产物AUC0-last、AUC0-∞（122.4±38.1vs73.9±17.9ng·mL-1·h-1；124.2±37.6vs76.4±17.71ng·mL-1·h-1），显著升高（P0.05）；CCAMAUC0-last、AUC0-∞和Cmax（41368.46±12832.65vs64346.12±13740.94ng·mL-1·h-1；42173.30±13382.47vs65545.46±14166.2ng· mL-1· h-1；11050.06±3308.02vs20671.22±3469.58ng·mL-1）明显降低（P＜0.05）。 （5）CES1A2基因多态性对健康志愿者氯吡格雷药效学影响：两组基因型个体给药前的血小板聚集率的基础值均无差异（P＞0.05）。CES1A2-816AA型个体服药后2、4h测定的血小板聚集率变化程度显著高于-816AC/CC个体（62.05士7.13%、82.91士6.96%vs47.25士9.44%、61.60士21.51%，P＜0.05）。其相应的平均效应时间曲线下面积AUEC0-24-816AA型是AC/CC型的1.27倍(1738.54±162.11vs1368.16±384.67h·%，P0.05)。 结论：CES1A2活性显著影响氯吡格雷的代谢和抗血小板活性；通过对氯吡格雷活性代谢产物浓度的影响，从而影响其抗血小板活性是其可能的机制；临床氯吡格雷剂量个体化调整时除考虑CYP2C19外，还需要考虑该影响因素。
[Abstract]:Objective : To study the pharmacokinetics and pharmacodynamics of clopidogrel in human liver microsome system by carboxylesterase A2 , and to investigate its effect on clopidogrel metabolism and antiplatelet activity , with a view to providing evidence for the rational use of clopidogrel . Methods : ( 1 ) Genotypic measurement : DNA in whole blood from liver tissue and EDTA was extracted by PCR - RFLP method . The samples were randomly sampled for not less than 10 % . The method was verified by direct sequencing . The genotypes of CES 1A2A ( -816 ) C were determined by direct sequencing . ( 2 ) Determination of clopidogrel and its active and inactive metabolite concentrations : The concentration of clopidogrel and its active and inactive metabolites in human liver microsomes incubation system and human plasma were determined by ultra - high performance liquid chromatography tandem mass spectrometry ( UPLC - MS / MS ) . ( 3 ) In vitro metabolism determination of clopidogrel in human liver microsomes incubation system : The liver tissue is divided into three metabolic phenotype based on the number of mutations in both metabolizers and * 3 mutations : fast - metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers , which are metabolizers of metabolizers , and those with weak metabolizers . &bra; 2 , * 2 / * 3 or * 3 / * 3 ) &ket; . In this paper , we used Graphpad 5.0 software to calculate the apparent kinetic constants ( Vmax ) and Km ( Km ) of the non - active metabolite , and to determine the intrinsic clearance Clint ( Vmax / Km ) . ( 4 ) Pharmacokinetics - pharmacodynamic determination of Chinese healthy volunteers : 11 healthy volunteers who met the criteria of enrollment and signed the informed consent form were divided into two groups according to the different genotypes of CES A2 . Blood samples were collected before and at 0.25 , 0.5 , 0.75 , 1 , 1.25 , 1.5 , 2 , 3 , 4 , 5 , 6 , 8 , 12 , 24 , and 36 hours after administration . The platelet aggregation rate induced by ADP was determined by UPLC - MS / MS , and the change of platelet aggregation inhibition ( IPA % ) was used as the pharmacodynamic index of clopidogrel . Results : ( 1 ) The genotype distribution : 86 cases of human liver tissue samples were determined by the C gene : A A of 43 cases ( 50.0 % ) , AC type 37 cases ( 41.86 % ) and CC type 6 cases ( 8.14 % ) . There were 37 cases ( 43.02 % ) , 38 cases ( 44.19 % ) and 11 cases of weak metabolism ( 12.79 % ) . A3EA subtype distribution : Among the 37 cases of CYP2C19EM , there were 15 cases ( 40.54 % ) , 16 cases ( 43.24 % ) AC type and 6 cases of CC type ( 16.22 % ) . Among 38 cases of CYP2C19IM , 24 cases ( 63.16 % ) and 14 cases ( 36.84 % ) were AC type ; in 11 cases of CYP2C19PM , type A was 4 cases ( 36.36 % ) and AC type 7 cases ( 63.64 % ) . ( 2 ) The concentration of clopidogrel active metabolite in human plasma was 1 - 200 ng 路 mL - 1 and linear good ( R2 = 0.9964 and 0.9945 ) . The linear range of clopidogrel non - active metabolite in human plasma was 10 ~ 2000 ng 路 mL ~ ( -1 ) and 25 ~ 10000ng 路 mL ~ ( -1 ) , and the linearity was good ( R2 = 0.9964 and 0.9945 ) . The linear range of clopidogrel non - active metabolite was 0.1 ~ 20ng 路 mL ~ ( -1 ) , and the linearity was good ( R2 = 0.9961 ) . ( 3 ) The effects of different genotypes on the enzyme kinetics and the concentration of active metabolites of clopidogrel non - active metabolite in human liver microsomes incubation system : The Vmax of A1ECAES1 - 816CC genotype was 1.34times of - 816A A ( CC vs AA : 635.20 卤 12.06vs470.10 卤 26.55 pmol 路 min -1 路 mg 路 protein - 1 , P0.05 ) . A similar phenomenon was observed in the EM group compared with that of - 816A A ( CC vs AA : 669.80 卤 20.72vs 48.6 卤 13.84pmol 路 min -1 路 mg 路 protein - 1 , P0.05 ) . Similar phenomena were observed in IM , but similar results were observed in the PM group due to the very low production of active metabolites . ( 4 ) There was a significant increase in clopidogrel AUC0 - 鈭

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