本文關(guān)鍵詞： 氯吡格雷 基因多態(tài)性 肝微粒體 健康志愿者 CES1A2A(-816)C　出處：《蘇州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：通過羧酸酯酶1A2（CES1A2）對(duì)人肝微粒體體系中氯吡格雷體外代謝和中國健康志愿者口服氯吡格雷的藥代動(dòng)力學(xué)及藥效學(xué)影響的研究，系統(tǒng)探討CES1A2基因多態(tài)性在氯吡格雷代謝和抗血小板活性差異中的作用，以期為臨床氯吡格雷合理使用提供依據(jù)。 方法：（1）基因型測(cè)定：用Promega試劑盒提取肝組織和EDTA抗凝的全血中的DNA，用PCR-RFLP法測(cè)定CYP2C19*2/*3基因型，并隨機(jī)抽取樣本率不少于10%用直接測(cè)序?qū)Ψ椒▽W(xué)進(jìn)行驗(yàn)證；用直接測(cè)序法測(cè)定CES1A2A(-816)C基因型。 （2）氯吡格雷及其活性和非活性代謝產(chǎn)物濃度測(cè)定：人肝微粒體孵育體系和人血漿中氯吡格雷及其活性和非活性代謝產(chǎn)物濃度均用超高效液相串聯(lián)質(zhì)譜(UPLC-MS/MS)法測(cè)定。 （3）人肝微粒體孵育體系中CES1A2不同基因型下氯吡格雷的體外代謝測(cè)定：根據(jù)CYP2C19*2及*3突變基因的數(shù)目將肝組織分為三種代謝表型：快代謝者[EMs(CYP2C19*1/*1)]，中間代謝者[IMs (CYP2C19*1/*2，*1/*3)]和弱代謝者[PMs(CYP2C19*2/*2，*2/*3或*3/*3)]。在根據(jù)CYP2C19分型后的肝組織中，分別隨機(jī)抽取CES1A2不同基因型肝組織（CYP2C19EM型中CES1A2-816AA，AC和CC各6例；CYP2C19IM型中AA和AC各6例，未檢出CC型）；同種基因型肝組織合并后進(jìn)行氯吡格雷體外代謝試驗(yàn)，考察CES1A2催化氯吡咯雷生成非活性代謝產(chǎn)物的酶促反應(yīng)動(dòng)力學(xué)特征，同時(shí)測(cè)定CES1A2不同基因型氯吡格雷活性代謝產(chǎn)物生成的情況。利用Graphpad5.0軟件擬合底物濃度與反應(yīng)速率的非線性回歸方程：?jiǎn)挝稽c(diǎn)的Michaelis-Menten方程，計(jì)算氯吡咯雷非活性代謝產(chǎn)物的體外代謝酶促反應(yīng)的表觀動(dòng)力學(xué)常數(shù)Vmax和Km，求得內(nèi)在清除率Clint（Vmax/Km）。三組基因型肝微粒體中非活性代謝產(chǎn)物生成的酶動(dòng)力學(xué)參數(shù)和活性代謝產(chǎn)物生成水平的比較采用單因素方差分析（ANOVA），三組基因型中的兩兩比較采用LSD（least significant difference）事后檢驗(yàn)或t檢驗(yàn)。P0.05認(rèn)為具有統(tǒng)計(jì)學(xué)差異。 （4）中國健康志愿者藥代動(dòng)力學(xué)-藥效學(xué)測(cè)定：11例符合入組標(biāo)準(zhǔn)并簽署知情同意書的健康志愿者按照CES1A2不同基因型篩選分成兩組：CYP2C19均為EM，一組為CES1A2AA型（n=6例），一組為CES1A2AC或CC型（n=5例），兩組均給予氯吡格雷300mg后，分別于給藥前（0h）和給藥后0.25，0.5，0.75，1，1.25，1.5，2，3，4，5，6，8，12，24，36h采集血樣，用于藥代動(dòng)力學(xué)（PK）研究；并于給藥前（0h）和給藥后1，2，4，12，24h采集血樣，用于藥效學(xué)（PD）研究。利用UPLC-MS/MS測(cè)定氯吡格雷及其非活性和活性代謝產(chǎn)物的濃度，利用非房室模型分析氯吡格雷原藥、活性及非活性代謝產(chǎn)物藥動(dòng)學(xué)參數(shù)；利用四通道血小板聚集儀測(cè)定的ADP誘導(dǎo)的血小板聚集率，以血小板聚集抑制的變化程度（IPA%）作為氯吡格雷藥效學(xué)指標(biāo)。 結(jié)果：（1）基因型分布：86例人肝組織樣本中進(jìn)行CYP2C19*2/*3和CES1A2A(-816)C基因測(cè)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：快速代謝型EM有37例（43.02%），中等代謝型有38例（44.19%），弱代謝型有11例（12.79%）； ○A3EA亞型分布：在37例CYP2C19EM的亞組中：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）人肝微粒體孵育體系與人血漿中氯吡格雷原藥、活性及非活性代謝產(chǎn)物濃度的方法學(xué)：氯吡格雷活性代謝產(chǎn)物在肝微粒體孵育體系中和人血漿中的線性范圍均為1~200ng·mL-1，且線性良好（R2分別為0.9977和0.9930）；氯吡格雷非活性代謝產(chǎn)物在肝微粒體孵育體系中和人血漿中的線性范圍分別為10~2000ng·mL-1和25~10000ng·mL-1，且線性良好（R2分別為0.9964和0.9945）；氯吡格雷原藥在血漿中的線性范圍為0.1~20ng·mL-1，且線性良好（R2為0.9961），方法的批內(nèi)批間精密度、回收率、基質(zhì)效應(yīng)、穩(wěn)定性等均符合生物樣本檢測(cè)要求。 （3）CES1A2不同基因型對(duì)人肝微粒體孵育體系中氯吡格雷非活性代謝產(chǎn)物生成酶動(dòng)力學(xué)及活性代謝產(chǎn)物濃度的影響： ○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），相應(yīng)的活性代謝產(chǎn)物水平在底物濃度為20、50μM時(shí)呈現(xiàn)出了降低趨勢(shì)（P0.05）。 ○A2EA根據(jù)CYP2C19代謝表型分組后，在EM組中，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），相應(yīng)的氯吡格雷活性代謝產(chǎn)物水平只有-816AA型的60%或更低。在IM中同樣觀察到了類似的現(xiàn)象，但在PM組中，由于活性代謝產(chǎn)物生成量極低，并未觀察到類似的結(jié)果。 （4）CES1A2基因多態(tài)性對(duì)健康志愿者氯吡格雷藥代動(dòng)力學(xué)影響：CES1A2-816AA組與CES1A2-816AC/CC組相比，氯吡格雷AUC0-∞（15.6±4.8vs9.7±1.7ng·mL-1·h-1），氯吡格雷活性代謝產(chǎn)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），顯著升高（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）明顯降低（P＜0.05）。 （5）CES1A2基因多態(tài)性對(duì)健康志愿者氯吡格雷藥效學(xué)影響：兩組基因型個(gè)體給藥前的血小板聚集率的基礎(chǔ)值均無差異（P＞0.05）。CES1A2-816AA型個(gè)體服藥后2、4h測(cè)定的血小板聚集率變化程度顯著高于-816AC/CC個(gè)體（62.05士7.13%、82.91士6.96%vs47.25士9.44%、61.60士21.51%，P＜0.05）。其相應(yīng)的平均效應(yīng)時(shí)間曲線下面積AUEC0-24-816AA型是AC/CC型的1.27倍(1738.54±162.11vs1368.16±384.67h·%，P0.05)。 結(jié)論：CES1A2活性顯著影響氯吡格雷的代謝和抗血小板活性；通過對(duì)氯吡格雷活性代謝產(chǎn)物濃度的影響，從而影響其抗血小板活性是其可能的機(jī)制；臨床氯吡格雷劑量個(gè)體化調(diào)整時(shí)除考慮CYP2C19外，還需要考慮該影響因素。
[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 鹵 12.06vs470.10 鹵 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 鹵 20.72vs 48.6 鹵 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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