氯吡格雷与替格瑞洛药动学影响因素的研究

发布时间：2018-06-23 18:10

本文选题：急性冠脉综合征 + LC-MS/MS　；参考：《河北医科大学》2017年硕士论文

【摘要】：第一部分分析急性冠脉综合征患者体内氯吡格雷活性代谢产物的影响因素目的:分析影响急性冠脉综合征(ACS)患者体内硫酸氢氯吡格雷活性代谢产物血浆浓度的主要因素。方法:105例ACS患者均给予阿司匹林100 mg·d-1及硫酸氢氯吡格雷75 mg·d-1,连续口服4 d;第5天晨起空腹给药1 h后,抽取静脉血4 m L,用乙二胺四乙酸抗凝。其中2 m L立即加入500 mmol·L-1的衍生化试剂2-溴-3’-甲氧基苯乙酮(MPB)溶液20μL,用于检测硫酸氢氯吡格雷活性代谢产物的浓度;另2 m L用于荧光检测细胞色素P450 2C19*2(CYP2C19*2)、*3和氧磷酶1(PON1)基因型。用单因素分析探索二分类变量及等级资料对硫酸氢氯吡格雷活性代谢产物血药浓度的影响,用Spearman分析探索硫酸氢氯吡格雷活性代谢产物血药浓度与连续性变量的相关性。结果:糖尿病、高脂血症、PON1携带、进行冠状动脉介入手术后与硫酸氢氯吡格雷活性代谢产物血药浓度呈负相关(P0.05),β受体阻滞药、他汀类药物、血清白蛋白(ALB)与硫酸氢氯吡格雷活性代谢产物的浓度呈正相关(P0.05)。结论:糖尿病、高脂血症、PON1携带及进行冠状动脉介入手术后等可减少硫酸氢氯吡格雷活性代谢产物血药浓度,而ALB以及合用β受体阻滞药或他汀类药物可增加其血药浓度。第二部分肠道菌群对硫酸氢氯吡格雷及其活性代谢产物在大鼠体内药动学的影响目的:建立大鼠血浆中硫酸氢氯吡格雷及其活性代谢产物的LC-MS/MS测定方法,并考察大鼠肠道菌群变化对其药代动力学的影响。方法:色谱及质谱条件:Diamonsil C18柱(150 mm×4.6 mm,5μm),流动相为乙腈-1 m M乙酸铵(含0.1‰的甲酸)(80∶20,V/V),流速1m L·min-1,柱温40℃,进样量10μL,内标为盐酸噻氯匹定。离子化方式:电喷雾离子化源(ESI);检测方式:正离子模式;扫描方式:多反应监测(MRM);离子源喷射电压:5500 V;离子源温度:550℃;气帘气(CUR)压力:25 psi;雾化气NEB(GS1,N2)压力:55 psi;辅助气AUX(GS2,N2)压力:50 psi;碰撞气(CAD)压力:4 psi;硫酸氢氯吡格雷的去簇电压(DP)和碰撞能(CE)值分别为68 V和21 e V,活性代谢产物衍生物(CAMD)的DP和CE值分别为114 V和26 e V,盐酸噻氯匹定的DP和CE值分别为60 V和23 e V;用于定量分析的离子对分别为m/z 322.2→212.0(硫酸氢氯吡格雷),504.4→354.1(CAMD),264.2→154.1(盐酸噻氯匹定)。动物实验:24只SD大鼠随机分为益生菌组、抗生素组和对照组,每组8只。分别灌胃双歧杆菌乳杆菌三联活菌(0.8 g·kg-1)、阿莫西林克拉维酸钾(125 mg·kg-1)和蒸馏水,连续7天,每天一次。第8天每只大鼠给予硫酸氢氯吡格雷片(10 mg·kg-1),并于给药前和给药后5、10、20、30、45 min、1、1.5、2、4、6、8、10 h眼内眦取血,于肝素抗凝离心管(含20μL 125 m M 2-溴-3’-甲氧基苯乙酮(MPB)衍生化试剂),立即涡旋30 s混匀,离心取上层血浆200μL,加入600μL含1.5 ng·m L-1内标的乙腈溶液,涡旋1 min,10900 r·min-1离心5 min,取上清液10μL进样。采用DAS 2.1.1软件拟合大鼠的药动学参数,使用SPSS 21.0软件对三组药动学参数进行统计学分析,P值小于0.05则认为有统计学差异。结果:益生菌组、抗生素组和对照组硫酸氢氯吡格雷的主要药动学参数如下:AUC0-t分别为(6.83±1.81)、(6.80±1.75)和(6.50±1.87)ng·h·m L-1;AUC0-∞分别为(12.11±4.90)、(11.67±5.75)和(13.94±6.68)ng·h·m L-1;Cmax分别为(3.25±1.73)、(2.87±0.73)和(2.76±1.15)ng·m L-1;t1/2分别为(9.07±4.57)、(7.75±4.64)和(12.5±10.95)h;Tmax分别为(0.30±0.19)、(0.33±0.08)和(0.43±0.23)h;CL分别为(966.61±418.40)、(1000.34±366.34)和(836.05±303.94)L·(h·kg)-1;V分别为(11261.73±4270.92)、(9437.67±2945.94)和(11655.10±5789.74)L·kg-1。益生菌组、抗生素组和对照组CAMD的主要药动学参数如下:AUC0-t分别为(362.26±126.43)、(491.68±169.72)和(404.85±92.71)ng·h·m L-1;AUC0-∞分别为(404.85±92.71)、(1524.19±176.40)和(422.45±91.08)ng·h·m L-1;Cmax分别为(258.12±121.92)、(272.00±72.04)和(258.55±112.03)ng·m L-1;t1/2分别为(2.19±0.76)、(3.59±1.95)和(2.42±0.83)h;Tmax分别为(0.52±0.16)、(0.84±0.18)和(0.78±0.16)h;CL分别为(30.45±13.39)、(20.76±5.95)和(24.67±5.40)L·(h·kg)-1;V分别为(100.37±60.28)、(110.66±73.57)和(87.10±35.88)L·kg-1。使用SPSS 21.0进行统计学分析发现,与对照组相比益生菌组和抗生素组各药动学参数均没有统计学差异。结论:本实验建立了大鼠血浆中硫酸氢氯吡格雷及其活性代谢产物的LC-MS/MS测定方法。药动学实验结果表明肠道菌群的变化对硫酸氢氯吡格雷及其代谢产物的药代动力学无影响。第三部分肠道菌群对替格瑞洛在大鼠体内药动学的影响目的:建立大鼠血浆中替格瑞洛的LC-MS/MS测定方法,并考察大鼠肠道菌群变化对替格瑞洛药代动力学的影响。方法:色谱及质谱条件:Diamonsil C18柱(150 mm×4.6 mm,5μm),流动相为乙腈-1‰的甲酸(80∶20,V/V),流速1 m L·min-1,柱温35℃,进样量10μL,内标为布洛芬。离子化方式:电喷雾离子化源(ESI);检测方式:负离子模式;扫描方式:多反应监测(MRM);离子源喷射电压:5500 V;离子源温度:600℃;气帘气(CUR)压力:40 psi;雾化气NEB(GS1,N2)压力:55 psi;辅助气AUX(GS2,N2)压力:50 psi;碰撞气(CAD)压力:10 psi;替格瑞洛的解离电压(DP)和碰撞能(CE)值分别为-111.36 V和-32.43 e V,布洛芬的DP和CE值分别为-49.61 V和-10.27 e V;用于定量分析的离子对分别为m/z 521.2→361.3(替格瑞洛),205.1→161.1(布洛芬)。动物实验:45只SD大鼠随机分为益生菌组、抗生素组和对照组,每组15只。分别灌胃双歧杆菌乳杆菌三联活菌(0.8 g·kg-1)、阿莫西林克拉维酸钾(125 mg·kg-1)和蒸馏水,连续7天,每天一次。第8天每只大鼠给予替格瑞洛(18 mg·kg-1),并于给药前和给药后5、15、30 min、1、1.5、2、3、4、6、8、12、24 h眼内眦取血,于肝素抗凝离心管,离心取上层血浆200μL,加入600μL含150 ng·m L-1内标的乙腈溶液,涡旋1 min,10900 r·min-1离心10 min,取上清液10μL进样。采用DAS 2.1.1软件拟合大鼠的药动学参数,使用SPSS 21.0软件对三组药动学参数进行统计学分析,P值小于0.05则认为有统计学差异。。结果:益生菌组、抗生素组和对照组替格瑞洛的主要药动学参数如下:AUC0-t分别为(6336.24±1840.46)、(4444.05±1033.43)和(4469.32±928.47)ng·h·m L-1;AUC0-∞分别为(6841.98±1975.95)、(4656.66±1083.78)和(4736.47±897.42)ng·h·m L-1;Cmax分别为(858.65±275.98)、(648.81±215.59)和(617.49±168.95)ng·m L-1;t1/2分别为(6.40±2.18)、(5.25±1.39)和(5.68±2.08)h;Tmax分别为(0.88±0.23)、(0.90±0.21)和(1.30±0.59)h;CL分别为(2.82±0.72)、(4.07±0.99)和(3.95±0.91)L·(h·kg)-1;V分别为(26.07±12.00)、(31.45±14.65)和(32.95±14.17)L·kg-1。使用SPSS 21.0进行统计学分析发现,与对照组比较益生菌组的AUC0-t、AUC0-∞和Cmax增大,Tmax和CL减小(P值均小于0.05);抗生素组与对照组相比,药动学参数均没有统计学差异。结论:本研究建立了大鼠血浆中替格瑞洛的LC-MS/MS测定方法。药动学实验结果显示,口服益生菌使大鼠肠道菌群发生变化后,可增大替格瑞洛在大鼠体内的峰浓度,降低清除速率,增加替格瑞洛在大鼠体内的暴露量。
[Abstract]:The first part analyzed the influence factors of clopidogrel active metabolites in patients with acute coronary syndrome: the main factors affecting the plasma concentration of clopidogrel sulfate active metabolites in patients with acute coronary syndrome (ACS) were analyzed. Methods: 105 patients with ACS were given aspirin 100 mg. D-1 and 7 of clopidogrel sulfate. 5 mg. D-1, continuous oral 4 D, and 1 h on the fifth day morning, 4 m L and anticoagulant with ethylenediamine tetra acetic acid. 2 m L immediately added 500 mmol L-1 derivative reagent 2- bromide -3 '- methoxy acetophenone (20) to detect the concentration of the active metabolites of the hydrogen sulfate clopidogrel; and 2 for fluorescence detection. Cytochrome P450 2C19*2 (CYP2C19*2), *3 and oxyphosphatase 1 (PON1) genotypes. The effects of two classified variables and grade data on the blood drug concentration of the clopidogrel sulfate active metabolites were investigated by single factor analysis. The correlation between the blood concentration of the clopidogrel sulfate metabolites and the continuous variables was investigated by Spearman analysis. Disease, hyperlipidemia, PON1 carrying, after coronary intervention, was negatively correlated with the blood concentration of the clopidogrel sulfate active metabolites (P0.05). Beta blockers, statins, serum albumin (ALB) were positively correlated with the concentration of the active metabolites of hydrogen sulfate clopidogrel (P0.05). Conclusion: diabetes, hyperlipidemia, PON1 The blood drug concentration of the clopidogrel sulfate active metabolites can be reduced with and after coronary intervention, while ALB and the combination of beta blockers or statins can increase the concentration of blood drugs. The effect of the second part of the intestinal flora on the pharmacokinetics of clopidogrel and its active metabolites in rats LC-MS/MS method for the determination of clopidogrel and its active metabolites in rat plasma and the effect of intestinal microflora change on its pharmacokinetics in rats. Methods: chromatographic and mass spectrometry conditions: Diamonsil C18 column (150 mm x 4.6 mm, 5 u m), mobile phase of acetonitrile -1 m M acetate (including 0.1% formic acid) (80: 20, V/V), flow rate 1m L. The column temperature is 40 C, the injection amount is 10 L, the internal standard is tilopidine hydrochloride. Ionization mode: electrospray ionization source (ESI); detection mode: positive ion mode; scanning mode: multi reaction monitoring (MRM); ion source injection voltage: 5500 V; ion source temperature: 550; CUR pressure: 25 psi; atomization gas NEB (GS1, N2) pressure: 55 psi; auxiliary gas AUX (GS2,) pressure Force: 50 psi; collision gas (CAD) pressure: 4 psi; the de cluster voltage (DP) and collision energy (CE) value of clopidogrel sulfate (CE) are 68 V and 21 e V respectively. The DP and CE values of the active metabolite derivatives (CAMD) are 114 V and 26 respectively, respectively, 60 and 23 respectively, respectively. The ion pairs for quantitative analysis are 322.2 to 212., respectively. 0 (clopidogrel sulfate), 504.4 to 354.1 (CAMD), 264.2 to 154.1 (ticlopidine hydrochloride). Animal experiment: 24 SD rats were randomly divided into probiotics group, antibiotic group and control group, 8 rats in each group. They were fed with Lactobacillus Bifidobacterium triad (0.8 g. Kg-1), amoxicillin clavulanate potassium (125 mg. Kg-1) and distilled water for 7 days, one day for 7 days. At the next eighth days, each rat was given Clopidogrel Bisulfate Tablets (10 mg. Kg-1), and blood was taken from the eye canthus in 5,10,20,30,45 min and 1,1.5,2,4,6,8,10 h before and after administration, and the heparin anticoagulant centrifuge tube (including 20 mu L 125 m M 2- bromine -3 'methoxy acetophenone (MPB) derivatization reagent), and immediately the vortex 30 was mixed, and the upper plasma was centrifuged to 200 mu, and 600 was added to 600. L containing 1.5 ng. M L-1 internal standard acetonitrile solution, vortex 1 min, 10900 r min-1 centrifuge 5 min, taking the supernatant 10 micron samples. The pharmacokinetic parameters of rats were fitted with DAS 2.1.1 software. Three groups of pharmacokinetic parameters were statistically analyzed using SPSS 21 software. The values were less than 0.05. Results: probiotics group, antibiotic group and The main pharmacokinetic parameters of the control group were as follows: AUC0-t was (6.83 + 1.81), (6.80 + 1.75) and (6.50 + 1.87) ng. H. M L-1, AUC0- infinity (12.11 + 4.90), (11.67 + 5.75) and (13.94 + 6.68) ng. H. M L-1 respectively, Cmax (3.25 + 1.73) and ng. .64) and (12.5 + 10.95) H; Tmax (0.30 + 0.19), (0.33 + 0.08) and (0.43 + 0.23) h, CL respectively (966.61 + 418.40), (1000.34 + 366.34) and (836.05 + 0.30) L. (H. Kg) -1; V respectively, the main pharmacokinetic parameters of the antibiotic and the control group. As follows: AUC0-t (362.26 + 126.43), (491.68 + 169.72) and (404.85 + 92.71) ng. H. M L-1, AUC0- infinity respectively (404.85 + 92.71), (1524.19 + 176.40) and (422.45 + 91.08) ng. H. M L-1 respectively, Cmax are respectively (258.12 + 121.92) and ng. X (0.52 + 0.16), (0.84 + 0.18) and (0.78 + 0.16) h, CL respectively (30.45 + 13.39), (20.76 + 5.95) and (24.67 + 5.40) L. (H. Kg) -1, V respectively (100.37 + 60.28), and L kg-1. using SPSS statistical analysis, found that the pharmacokinetic parameters of probiotics group and antibiotic group were not compared with the control group. There were statistical differences. Conclusion: this experiment established a LC-MS/MS method for the determination of clopidogrel and its active metabolites in rat plasma. The results of pharmacokinetic experiment showed that the changes in intestinal flora had no effect on the pharmacokinetics of clopidogrel and its metabolites. The third part of intestinal microflora was in the rat body of tigreloo. The objective of internal pharmacokinetic study: to establish a method for the determination of Grillo's LC-MS/MS in rat plasma and to investigate the effect of the change of intestinal flora on the pharmacokinetics of tegloro. Methods: chromatographic and mass spectrometry conditions: Diamonsil C18 column (150 mm x 4.6 mm, 5 u m), mobile phase (80: 20, V/V), 1 m L. Min-1, and 35 column temperature. Centigrade is 10 mu L, internal standard is ibuprofen. Ionization mode: electrospray ionization source (ESI); detection mode: negative ion mode; scanning mode: multi reaction monitoring (MRM); ion source injection voltage: 5500 V; ion source temperature: 600; gas curtain gas (CUR) pressure: 40 psi; NEB (GS1, N2) pressure of atomization gas: 55 psi; auxiliary gas AUX (GS2, N2) pressure: 50; collision The gas (CAD) pressure: 10 psi; the dissociation voltage (DP) and the collision energy (CE) value of the DP and -32.43 e V respectively. The DP and CE values for the quantitative analysis are respectively 521.2 to 361.3 (Ti Gray Lo) and 205.1 to 161.1 (Bloven). Animal experiment: 45 rats were randomly divided into probiotics. The bacteria group, the antibiotic group and the control group, 15 in each group, were fed with Lactobacillus Bifidobacterium triad (0.8 g. Kg-1), amoxicillin potassium clavulanate potassium (125 mg. Kg-1) and distilled water for 7 days, once a day. Each rat was given to gilligillo (18 mg. Kg-1) for eighth days, and 5,15,30 min and 1,1.5,2,3,4,6,8,12,24 h eyes were given before and after administration. When the inner canthus was taken, the heparin anticoagulant centrifuge tube was centrifuged and the upper plasma plasma was centrifuged to 200 mu L, and the acetonitrile solution containing 150 ng. M L-1 was added to 600 mu. The vortex 1 min and 10900 R. Min-1 were centrifuged 10 min and 10 micron L of the supernatant. The pharmacokinetic parameters of the rats were fitted with DAS 2.1.1 software. Three groups of pharmacokinetic parameters were statistically analyzed with 21 software. The values were less than 0.05. Results: the main pharmacokinetic parameters of the probiotics group, the antibiotic group and the control group for Grillo were as follows: AUC0-t (6336.24 + 1840.46), (4444.05 + 1033.43) and (4469.32 + 928.47) ng. H. M L-1, AUC0- infinity (6841.98 + 1975.95), (4656.66 + 1083.78) and (4736.47 + 897.42) ng. H. M L- respectively 1; Cmax (858.65 + 275.98), (648.81 + 215.59) and (617.49 + 168.95) ng. M L-1, t1/2 respectively (6.40 + 2.18), (5.25 + 1.39) and (5.68 + 2.08) h, Tmax respectively (6.40 + 617.49) and h, respectively, CL and L. + 14.17) L. Kg-1. used SPSS 21 to make statistical analysis. Compared with the control group, the AUC0-t, AUC0- and Cmax increased, Tmax and CL decreased (P less than 0.05), and the pharmacokinetic parameters were not statistically different between the antibiotic group and the control group. Conclusion: This study established the LC-MS/MS determination of Grillo in the rat plasma. The results of pharmacokinetic study showed that after oral probiotics changed the intestinal flora of rats, the peak concentration of Grillo in rats could be increased, the clearance rate was reduced and the exposure of tiprolol in rats was increased.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R969.1

【参考文献】