【作者】 李纳；

【导师】 施孝金；

【作者基本信息】 复旦大学 ， 药剂学， 2011， 硕士

【摘要】 细胞色素P450酶(cytochrome P450, CYP450),主要存在于肝微粒体中,在外源性化合物和内源性物质的生物转化中起着十分重要的作用,根据酶蛋白一级结构中氨基酸的同源度,CYP分为18个家族和44个亚家族,临床上90%以上的药物氧化代谢主要是通过CYP1A2、CYP2C9、CYP2C19、CYP2D6、CYP3A4这5种同工酶介导的。影响药物代谢酶活性的因素有很多,如食物和环境毒物等环境因素；年龄和性别等生理性因素；肝、肾或心脏疾病等病理性因素以及遗传因素。其中影响酶活性的重要因素是遗传因素,基因多态性是造成药物代谢种族和个体差异的主要来源。治疗剂量下的药物在弱代谢者中有可能发生严重的不良反应；在强代谢者中则可能无效,起不到治疗疾病的作用。因此,临床上需要预测患者的药物代谢酶活性,实行个体化的治疗方案。研究预测酶活性的方法主要有两种：探针底物法和基因分析法。混合探针底物法的应用较多,其优点有多个代谢途径的信息可在单个试验过程获得,可以降低个体问和个体内药物代谢酶活性变异的影响,同时缩短分析周期,降低实验费用,但是此方法不能应用于肝肾功能不良、对探针底物有不良反应的人群,且存在伦理道德、分析方法要求高等问题。而基因型检测只需很少的血浆,价格便宜,操作简单,可以更方便的应用于临床,但是需要预先鉴定影响代谢的基因分型。开展以基因型为指导的个体化用药是必需的,可以减少药物不良反应的发生,提高药物治疗效果,节约资源。各个亚型酶具有多个突变等位基因,需要探寻基因多态性和酶活性之间的关系,确定各种基因突变型引起的酶功能性的变化,实现通过检测个体的基因型来预测亚型酶对药物代谢的影响,从而优化给药方案,做到个体化给药。同时对临床药物试验筛选受试者也提供了可行的方法,临床试验中弱代谢者的排除,可以提高临床药物试验的安全性和有效性。本研究第一部分首先建立了一种同时测定血浆中CYP450酶的探针底物及其代谢物甲苯磺丁脲/4-羟基甲苯磺丁脲(CYP2C9)、奥美拉唑/5-羟基奥美拉唑(CYP2C19)右美沙芬/右啡烷(CYP2D6)、咪达唑仑/1’-羟基咪达唑仑(CYP3A4)浓度的LC-MS/MS分析方法。血浆样品预处理方法采用乙腈直接沉淀蛋白后用氮气吹干重组的方法。色谱条件为：色谱柱：CAPCELL PAK C18柱(MGⅢ、100mm×2.0mmID,5μm)流动相A：水/甲酸(100：0.05,v/v),流动相B：乙腈/甲酸(100：0.05,v/v),采用梯度洗脱程序：0～5 min,B%(10%～90%)；5～5.5 min,B%(90%)；5.5～6 min B%(90%-10%)；6-11 min,B%(90%)。流速0.3 mL/min,柱温为室温,进样体积为10μL电喷雾离子源(ESI),三重四级杆串联质谱,多级反应监测进行定量,正负离子分开扫描。奥美拉唑/5-羟基奥美拉唑、右美沙芬/右啡烷、咪达唑仑/1’-羟基咪达唑仑的浓度采用正离子监测,内标为非那西丁；甲苯磺丁脲/4-羟基甲苯磺丁脲采用负离子监测,内标为氯唑沙宗。结果奥美拉唑在0.4-40 ng/mL,5-羟基奥美拉唑、右美沙芬、1’-羟基咪达唑仑、4-羟基甲苯磺丁脲在0.2～20 ng/mL,右啡烷、甲苯磺丁脲在O.1-10 ng/mL,咪达唑仑在0.3-30 ng/mL范围内线性关系良好(r>0.999)。各分析物的批内及批问精密度均小于15%,准确度均在85%-115%范围内。建立的方法简便、快速、准确、灵敏,同时测定4种CYP450亚型酶的探针底物及其代谢物,可促进混合探针底物法的广泛应用,满足高通量的分析要求。第二部分进行了采用混合探针底物法研究CYP1A2、CYP2C9、CYP2C19、CYP2D6、CYP3A4的酶活性。选择咖啡因、奥美拉唑、右美沙芬、甲苯磺丁脲和咪达唑仑为探针底物,分别用于CYP1A2、CYP2C19、CYP2D6、CYP2C9、CYP3A4酶的活性研究与表型分析。筛选出50名健康受试者,同时给予健康受试者上述5种探针底物后3h,5h,24h采集血样,然后用第一部分建立的LC-MS/MS分析方法,测定血浆中的探针底物及其代谢物的浓度,来预测相应的药物代谢酶活性。结果这5种药物之间无相互作用,受试者在整个试验期间各项监测指标均在正常范围内,无不良事件的发生。血样测定中除咖啡因与副黄嘌呤由于空白血浆中干扰大没有进行准确定量测定外,其余各分析物都进行了精确的测定,测出了50名受试者体内各分析物的血浆浓度,为下一步的研究奠定基础。本研究采用的探针底物组合咖啡因+奥美拉畔+右美沙芬+甲苯磺丁脲+咪达唑仑来同时测定体内CYP1A2、CYP2C19、CYP2D6、CYP2C9、CYP3A4的活性,采样方便快捷,可用于同工酶活性和表型的研究以及用于评价药物之间的相互作用等方面。第三部分进行了CYP1A2、CYP2C9、CYP2C19、CYP2D6、CYP3A(?)的基因多态性与探针底物代谢差异的相关性研究。采用直接测序法对50名健康受试者进行了CYP1A2、CYP2C9、CYP2C19、CYP2D6、CYP3A4各亚型酶在中国人群中主要突变位点的基因分型研究。同时考察了这些突变位点在本研究人群中的频率分布,并研究了这些亚型酶突变位点的不同基因型对各自的探针底物在体内代谢的影响。所研究的基因多态性位点在本研究人群中的突变基因分布频率分别为CYP1A2*1B(10%)、CYP1A2*1C (31.9%)、CYP1A2*1D(16.4%)、CYP1A2*1F(59%)、CYP2C9*3(6%)CYP2C9*13 (0%)、CYP2C19*2(20.8%)、CYP2C19*3(8.2%)、CYP2D6*10(64.1%)、CYP3A4*1G (26.7%)、CYP3A4*15(0%)、CYP3A4*18(2%)。CYP2C9*3、CYP2C19*2、CYP2D6*10的突变位点可以引起相应的酶活性的降低,携带有这些位点突变基因的人群在临床用药中要减少剂量,才能减少不良反应的发生。CYP1A2*1B、CYP1A2*1C、CYP1A2*1D、CYP1A2*1F、CYP2C19*3、CYP3A4*1G、CYP3A4*18位点的突变基因则对各自的酶活性无影响,CYP2C9*13、CYP3A4*15没有检测到突变基因,无法确定其对酶活性的影响。研究结果将为实现以基因型为指导的临床个体化给药提供依据,有利于促进临床上的合理用药,保证用药的安全性。更多还原

【Abstract】 Cytochrome P450 enzymes (CYP450) are mainly in the liver microsome. They play an important role in the biotransformation of exogenous and endogenous compounds. CYP450 are divided into 18 families and 44 subfamilies, including CYP1、CYP2、CYP3 families which have 8 to 10 isozymes, respectively. More than 90% drugs in clinical are metabolismed by CYP1A2、CYP2C9、CYP2C19、CYP2D6、CYP3A4 isozymes. There are many factors influence the activities of drug metabolism enzymes, such as drug, toxins and other environmental factors; age, gender and other physiological factors; liver, kidney or heart disease pathological factors and genetic factor.The most important factor is genetic factor. The gene polymorphisms are the main source for the differences of drug metabolism among the ethnics and individuals. CYP450 gene polymorphisms cause the differences of drug metabolisms among the individuals, the identical dose of the same drug may cause serious adverse effects in poor metabolizers, but in the extensive metabolizers the concentration of drug is so low that does not reach the effective therapeutic concentration. As a result, the activity of drug metabolism enzyme of patient in clinic should be predicted to realize the individual treatment. There are two research methods to predict the enzyme activity. One is the "cocktail" probe drugs method and the other is the gene analysis method. The advantages of the "cocktail" probe drugs method are more than one metabolic pathway information can be obtained in a single test process, reducing the variations of drug metabolizing enzymes among individuals, reducing time and cost. However, the people of liver and kidney dysfunction, and people who have adverse reactions to drugs limited the application of probe drugs. In addition, the problems of ethics and the high analytical methods requirement also should be considered. However, the gene analysis method is cheap and little plasma will meet the need of gene analysis, moreover, the method has small individual variability. Gene analysis method can be more convenient for clinical application.The individual treatment base on the genotype is necessary. It can be reduce the adverse drug reaction, improve the drug treatment and conserve the resource. Each isoenzyme has many mutation alleles, the relationship between the genetic polymorphism and enzyme activity need to be explored to determine the change of enzyme function which caused by the gene mutations. The individual administration can be realized by predicting the enzyme activity base on the individual genotype test. The gene analysis method is also an effective method for screening the healthy volunteer in clinical trial, that the exclusion of the poor metabolizers in clinical trial can improve the safety and efficiency of the trial. The part one developed a LC-MS/MS analytic method to simultaneously determine the five probe parent drugs and their metabolites of cytochrome P450 (CYP450) including tolbutamide/4-hydroxytolbutamide(C YP2C9),omeprazole/5-hydroxyomeprazole(C YP2C19), dextro-methorphan/dextrorphan (CYP2D6) and midazolam/1’-hydroxymidazolam (CYP3A4) in human plasma. The pretreatment of sample is directly protein precipitated with acetonitrile then the analytes are restituted after being dried by nitrogen. An aliquot (10μL) was injected into a CAPCELL PAK C18column (MGⅢ,100 mm×2.0 mm ID,5μm) which were kept at room temperature. The flow rate was 0.3 mL/min. The mobile phase consisted of 0.05% formic acid in acetonitrile (B) and 0.05% formic acid in water (A). During the gradient elution step (0～5 min), the solvent composition of mobile phase B was increased linearly from 10% to 90%, then maintained to 90% for 0.5 min, and reduced from 90%(B) to 10%(B) in 0.5 min, then maintained the 10% (B) until the end of the analytic time. The column was then allowed to re-equilibrate at initial conditions. The complete analytic run was lasted for 11 min. A 3200 QTrap triple-quadrupole mass spectrometer, equipped with an electrospray ionization (ESI) source, was used for the mass analysis and detection. The positive and negative ions were respectively determined by the multiple reaction monitoring (MRM).Omeprazole/5-hydroxyomeprazole, dextromethorphan/dex-trorphan, midazolam/1’-hydoxymidazolam were determined in positive ionization mode with the internal standard phenacetin; whereas, tolbutamide/4-hydroxytolbutamide were determined in negative ionization mode with the internal standard chlorzoxazone. The result indicated that the calibration curves for each analyte were linear (r>0.999) over the range of 0.4～40 ng/mL for omeprazole; 0.2～20 ng/mL for 5-hydroxyomeprazole, dextromethorphan,l’-hydroxy-midazolam and 4-hydroxytolbutamide; 0.1～10 ng/mL for dextrorphan and tolbutamide; 0.3～30 ng/mL for midazolam. The precision of intra and inter batches for each analyte were less than 15%, the accuracy of intra and inter batches for each analyte were in the range of 85%～115%. The method is simple, quick, accurate and sensitive to determine eight parent drugs and metabolites at a time. It can promote the "cocktail" probe drugs widely used in future, and meet the requirements of high-throughput analysis.The part two, we select caffeine, omeprazole, dextromethorphan, tolbutamide and midazolam as the probe drugs for the research activities of CYP1A2, CYP2C9, CYP2C19,C-YP2D6, CYP3A4 enzymes. Fifty healthy volunteers were selected and the plasma samples were drawn at 3,5,24h after administration of five probe drugs simultaneously. The concentration of probe parent drugs and their metabolites in plasma were determined by the LC-MS/MS method developed in the part one. The result indicated that five probe drugs were safe to human and had no drug interactions. All the monitoring indexes for the subjects were in the normal range and no adverse events were happened during the whole research period. Each of analyte was determined precisely except the caffeine and paraxanthine due to the interferences in bank human plasma. The concentration of each analyte in plasma laid the foundation for further research. The "cocktail" probe drugs of caffeine, omeprazole, dextromethorphan, tolbutamide and midazolam determine the activities of five CYP450 enzymes simultaneously, this method is quick and convenient. This cocktail has the potential to become a useful tool to determine the activity of enzyme and research the phenotype, as well as in the detection of clinically important drug-drug interactions during drug development.The part three mainly studied the relations between the gene polymorphisms of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 enzymes and the differences of probe drugs metabolisms. The DNA direct sequencing of single nucleotide polymorphisms (SNPs) of the important mutation alleles of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 enzymes in the fifty Chinese healthy volunteers. The genotype frequencies and allelic frequencies of the important mutation alleles of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 enzymes and the difference of probe drugs metabolisms among the different genotypes were investigated in the the above mentioned volunteers. The frequencies of distribution of mutation alleles of the CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 enzymes are 10% for CYP1A2*1B,31.9% for CYP1A2*1C,16.4% for CYP1A2*1D,59% for CYP1A2*1F, 6% for CYP2C9*3,0% for CYP2C9*13,20.8% for CYP2C19*2,8.2% for CYP2C19*3, 64.1% for CYP2D6*10,0% for CYP3A4*15,26.7% for CYP3A4*1G and 2% for CYP3A4*18. The mutation sites of CYP2C9*3, CYP2C19*2, CYP2D6*10 can reduce the activities of the enzymes. The patients who carry these mutation alleles should reduce the dose of drug in clinical in order to avoiding the drug adverse reactions. There are no relation between the activity of drug enzymes and mutation alleles of CYP1A2*1B, CYP1A2*1C, CYP1A2*1D, CYP1A2*1F, CYP2C19*3, CYP3A4*1G, CYP3A4*18. The influence of CYP2C9*13, CYP3A4*15 on the activity of enzyme was not determined because no mutation alleles were found in the subjects. The result will provide the basis for the individual drug administration base on the genotype, and promote the rational use of drug in clinical to ensure the safety of drug.更多还原