| Coronary heart disease is the most important causes of death, also consumes huge medical resources of disease. Percutaneous coronary intervention (PCI) is one of the significant progress in the treatment of coronary heart disease. PCI has been widely used in the treatment of patients with coronary heart disease, which clinical curative effect is precise and significantly improve patients’ quality of life and survival by improved the symptoms of ischemia and hypoxia of patients.Due to the stents implantation in the PCI operation, it can promote platelet activated, and thrombosis formation, raised bracket within thrombosis, myocardial infarction and death, clinical end event. In clinical end events,10%-15%of patients in1years will have heart brain vascular event, including brain stroke and major adverse cardiac events (MACE including heart death, myocardial infarction and reconstruction). Therefore, in patients with PCI need antiplatelet therapy-double resistance to therapy (aspirin and clopidogrel), which has become a routine treatment in patients with coronary heart disease, and improves the prognosis situation of patients. But there are individual differences in the curative effect, if clinical efficacy is bad, which may cause myocardial infarction, hospitalization and even death. At present, many studies have shown that the gene polymorphism of transporter and metabolic enzymes of absorption of drug in the body, involved in the process of transformation, disposal is the main reason. CYP3A is one of the most important enzymes in the drug metabolism system, including CYP3A4, CYP3A5and CYP3A7, the expression of them is very high in the body, but not the only, which distributes in the liver and intestinal tissue, and participate in metabolism of45-60%clinical drug. There is a big difference between the expression and activity of CYP3A in different individuals, as high as60times, which may lead to treatment failure, unpredictable adverse reaction and drug toxicity, to a certain extent, which is the result of genetic and nongenetic factors (hormones/health/environmental factors). The differences of CYP3A activity can explain90%clinical curative effect in individuals. And clopidogrel can be converted into active metabolites through two steps in the body mainly, the first step is the oxidation reaction, which generates2-oxygen-clopidogrel, the second step is hydrolysis reaction, which generates active metabolites. In vitro studies, which show that the intermediate is catalyzed by CYP2C19, CYP1A2and CYP2B6, and then by the CYP3A4/5, CYP2B6, CYP2C19and CYP2C9. In vivo studies have shown that CYP3A4, CYP2C19CYP1A2are the important drug metabolic enzymes, the metabolic enzymes decide the amount of active metabolites of clopidogrel in organism, therefore, there is different clinical effects.Therefore, the experiment intends to expand in southern Chinese Han population in our study, first of all, establishing the method to detect the activity of CYP3A enzyme; Secondly building the metabolic model of clopidogrel in vitro; Finally, discussing the influence of CYP3A4/5genotypes on blood drug concentration of clopidogrel active metabolites, and the relevance to the platelet aggregation rate.The first chapter To establish high-performance liquid chromatography method for the determination of CYP3A4activity in human liver S9with testosterone as a probe and its applicationObjective:To establish high-performance liquid chromatography method for the determination of CYP3A activity in human liver S9with testosterone as a probe, and detect different patients of liver tissue CYP3A activity.Methods:Select hydrocortisone as an internal standard, and performed on Hypersil BDS C18column (4.6mm×150mm,5μm) with mobile phase consisted of methanol2mM KH2PO4(52:48, V/V), detect the6β-hydroxy testosterone production volume of major metabolite of testosterone by human liver S9in245nm, and the enzyme kinetics was evaluated by graphical analysis with Lineweaver-Burk double reciprocal plots.Results:The linear range of6β-hydroxy-testosterone and testosterone was0.5-32μg/mL and0.5-40μg/mL, respectively,(respectively R2=0.9993, R2=0.9988), The LOQ was0.5ug/ml. The recoveries were96.8%-99.1%and96%-98.6%, respectively, and RSD%<4.2; extraction recoveries were90.3%-95.5%and91.5%-94.7%, respectively, and RSD%<4.89; and intra-day precision degree RSD%<5, the samples were stable at room temperature for24h, good stability, RSD%<5.2. The parameters of CYP3A in the liver S9for Km and Vmax were41.19nmol/L and625pmol/min*mg, respectively.Conclusion:The established HPLC method was simple, and has a good stability, can be used to assess CYP3A enzyme activity in vitro.The second chapter The correlation between CYP3A4/5gene mutation and the activity of CYP3A in liver S9componentsObjective:Cytochrome P450are important drug metabolic enzymes, and which participate in the biological transformation of a variety of clinical medicines. But there are big differences of metabolic enzyme activity in individuals, and with different genotype, which has different rate of drug metabolism. Explore the influence of CYP3A5*3genetic mutations and CYP3A4promoter region gene variants on the CYP3A enzyme activity of the liver S9components.Methods:from September2012to September2013, the small normal liver tissues of40patients were collected from sun yat-sen memorial hospital, TaqMan probe method is used to detect CYP3A5*3genotype, and the genetic variability of CYP3A4promoter region is detected by the PCR-sequencing method. The activity of CYP3A enzyme is assessed by testosterone probe of high performance liquid chromatography (HPLC), the ratio of peak area of6β-hydroxy testosterone and the internal standard of hydrocortisone represents CYP3A enzyme activity. Analysis the relationship of CYP3A5*3genotype with the ratio of the peak area of6β-hydroxy testosterone and the internal standard of hydrocortisone.Results:40cases of liver tissues, male is26cases, and female is14cases, the median age is47.5. CYP3A5wild homozygous type (*1/*1) is27cases (67.5%), and mutation hybrid type (*1/*3) is10cases (25%), and mutation homozygous type (*3/*3) is3cases (7.5%). The activity of CYP3A enzyme of wild homozygous type (*1/*1) and mutation hybrid type (*1/*3) were higher than homozygous mutations (*3/*3) in liver S9, and wild homozygous type (*1/*1) was belower than the mutation hybrid type (*1/*3). After nonparametric test analysis:there was statistically difference of CYP3A enzyme activity between the mutation hybrid type (*1/*3) and homozygous mutations (*3/*3) in liver S9component (P<0.05). The analysis of non-genetic factors:The gender, age, hypertension or taking proton pump inhibitors of patients have no significant influence on the activity of CYP3A enzyme in liver S9components (P>0.05). There is no gene mutation in1kb of the CYP3A4promoter region.Conclusion:The study shows that CYP3A5*3affects the activity of CYP3A enzyme in liver S9components significantly, and the gender, age, hypertension or taking proton pump inhibitors of patients have no significant influence on the activity of CYP3A enzyme in liver S9components; This part of sample size of the study is less, which has certain limitation, the results still need further samples to validate.The third chapter The correlation between CYP3A5gene mutation and the antiplatelet effect of clopidogrelObjective:Antiplatelet therapy was based on Clopidogrel was an important measure to prevent thromboembolic events after percutaneous coronary intervention (PCI), but there were large individual differences. Many studies found that clopidogrel is mainly metabolized by CYP3A enzymes, which can account for40%. This part of the experiment to investigate CYP3A5gene variants on antiplatelet effect of clopidogrel, which can provide theoretical basis for antiplatelet drugs of gene target for the individualized medication.Methods:106cases was selected from guangdong genenal hospital, who took300mg loading clopidogrel with coronary heart disease for the first time. Using TaqMan probe to detect CYP3A5*3genotyping, and using liquid Chromatograph Mass Spectrometer (LC/MS) method to detect blood drug concentration of clopidogrel active metabolites (MP-H3/MP-H4); applying the VerifyNow P2Y12system to detect the rate of platelet aggregation, to evaluate clopidogrel on platelet inhibition.Results:1) In106cases, wild homozygous type (*1/*1) is59patients (55.67%), mutation type hybrid (*1/*3) is40cases (37.73%), mutation homozygous type (*3/*3) is7cases (6.60%), who carry allele*1is99cases (93.40%), not carry allele*1is7cases (6.60%).2) Establish LC/MS method:acetonitrile protein precipitation method and the extraction of MP-H3/MP-H4recovery rate is more than80%, and the quantitative limit is0.2ng/mL, the linear and stability are good.3)The metabolism experiment in vitro:40cases of liver tissue samples, the drug concentration of MP-H3/MP-H4of CYP3A5wild homozygous type (*1/*1) and mutation hybrid type (*1/*3) are higher than homozygous mutations (*3/*3), by nonparametric test analysis, there was statistically difference between the genetic type and blood drug concentration (P<0.05). There was a significant positive correlation between the drug concentration of MP-H3/MP-H4and the CYP3A enzyme activity in liver S9components, the correlation coefficient is0.413and0.435, respectively.4) The metabolism experiment in vivo:106cases of patients with coronary heart disease (CHD), the blood drug concentration of MP-H3/MP-H4of CYP3A5wild homozygous type (*1/*1) and mutation hybrid type (*1/*3) is slightly higher than homozygous mutations (*3/*3), by nonparametric test analysis, there was no statistically difference between the genetic type and blood drug concentration (P>0.05).5) Platelet inhibition:106cases of patients with CHD, CYP3A5wild homozygous type (*1/*1) is190±85, mutation type hybrid (*1/*3) is207±86, mutation homozygous type (*3/*3) is193±42, there is no statistical differences between them. There is a significant negative correlation between platelet response values and the blood concentrations of MP-H4in2h (P<0.05), the correlation coefficient is minus0.327.6) The clinical data of patients:There is a significant positive correlation between the age and triglycerides of patient and platelet response values PRU (P<0.05), the correlation coefficient is0.256and0.202, respectively; and other clinical datas (high density lipoprotein cholesterol, total cholesterol) does not interfere with the PRU.Conclusion:Established LC/MS method successfully, can accurately detect clopidogrel active metabolites. CYP3A5*3gene variant has important effects on the metabolism of clopidogrel activation, but the antiplatelet effect were diluted by clinical complex factors, and age, high blood lipid will increase the rate of platelet aggregation. The size of sample of the experiment is less, further validation. |
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