【作者】 刘端；

【导师】 陈超；

【作者基本信息】 西北大学 ， 微生物学， 2010， 博士

【摘要】 药物的有效性和毒性主要取决于药物在体内的药代动力学过程,包括药物在体内的吸收、分布、代谢和排泄等步骤。这些步骤若发生改变将会影响到药物的安全性。药物基因组学研究表明,当与药物相关的基因(如药物代谢酶、转运体和靶标的基因)发生变化后,将会改变药物的药代动力学过程,进而影响到药物的安全性和疗效。目前,对药物代谢的研究较为清楚。很多药物代谢酶已经被鉴定出来,其中最重要的一类就是细胞色素P450(CYP)家族。CYP家族参与代谢的药物约占市场上销售药物的80%以上。这些酶的基因多态性已经被证明可以影响药物在体内的清除和药物的相互作用,因此会增加药物不良反应的风险。目前在人体中发现的CYP单核苷酸多态性(SNP)已经多达1000多种,其中包括大量的非同义SNP (nsSNP)。这些nsSNP会导致表达氨基酸发生变化的CYP蛋白,因此很有可能会影响到这些CYP酶代谢药物的活性。少数CYP nsSNP的功能已经被证实可以引起药物代谢活性的降低,它们的临床效应也已经较好的阐明。但是,大部分CYP nsSNP造成的影响还不清楚。由于缺乏有效的工具和手段,CYP nsSNP对药物安全性的影响在药物开发过程中还没有能被很好的进行评估。为了弥补这个缺陷,本文建立了一套体外评估CYP nsSNP对药物安全性影响的实验体系。首先利用酵母表达了八种主要的CYP亚型和CYP3A4的17种多态性酶。利用这些重组CYP酶,选择荧光探针底物建立了快速、可靠的生化试验。进而检测这些CYP酶的代谢活性和药物对它们的抑制能力,并预测其对药物安全性的影响。利用这一实验体系,本文对17种CYP3A4多态性酶的代谢活性,以及8种多态性酶对17种药物抑制能力的影响进行了评估。结果发现9种CYP3A4多态性酶会导致其代谢活性的降低或完全丧失。导致活性降低的CYP3A4多态性酶也会影响大部分受测试药物的抑制能力,使它们的抑制能力明显减弱。此外,本文还利用建立的体系对一个新的候选药物——丹参素冰片酯(DBZ)的体外代谢进行了研究。对比了DBZ经过人和大鼠肝微粒体代谢的异同,包括代谢动力学、代谢产物的比较,以及代谢产物的鉴定；还鉴定出了参与DBZ代谢的主要CYP酶,CYP2C8; DBZ主要抑制的CYP亚型,CYP3A4。在此基础上,进一步研究了CYP2C8和CYP3A4的nsSNP对DBZ代谢和抑制能力影响。其结果显示CYP2C8*3会导致DBZ代谢清除率下降54%；CYP3A4*2、CYP3A4*12和CYP3A4*16会导致DBZ抑制能力的减弱。这些结果可以帮助了解DBZ的作用机制及DBZ的有效性和安全性,为DBZ的进一步临床试验提供了指导信息。通过本文的实验结果可知,本文建立的体系可以成功的用来评估CYP nsSNP对药物安全性的影响。这一体系可以用来进行药物基因组学的基础研究,检测CYP基因多态性对其活性的影响；也可以用在药物开发过程中,根据CYP基因型来设计临床试验。更多还原

【Abstract】 CYPs are the most important drug-metabolizing enzymes, being responsible for 70-80% of all phase I-dependent metabolism of clinically useful drugs. More than 1000 single-nucleotide polymorphisms (SNPs) have been discovered in CYP genes, of which the most important ones are the non-synonymous SNPs (nsSNPs). nsSNPs lead to the expression of variant CYP enzymes that may possess altered metabolic activities and increase the risk of ADRs for individuals carrying the nsSNPs. Pharmacogenomic studies also indicate that CYP nsSNPs can have profound effects on the ability and potency of drugs to inhibit CYPs, thus resulting in unexpected drug-drug interactions (DDIs) in individuals carrying polymorphic genotypes.CYP genetic polymorphisms and CYP-mediated DDIs are both important in the assessment of drug safety and efficacy. However, the effects of CYP nsSNPs on DDIs are still underappreciated in drug development because methods are lacking to study the effects of a large number of SNPs.To address this deficiency, we established and experimentally validated a homogeneous yeast assay system consisting of eight human CYP isoforms and 17 CYP3A4 nsSNP variants. We tested the metabolic activities of those enzymes and their abilities to alter the drug’s inhibitory potencies. The results suggested that 9 of the 17 CYP3A4 nsSNP variants can lead to deceased metabolic activities. Those variants also can weaken the inhibitory potencies of most tested drugs to CYP3A4.Furthermore, we tested a new chemical entity, tanshinol borneol ester (DBZ), in this system to systematically evaluate the effects of CYP nsSNPs on both the CYP-inhibition potential of DBZ and on DBZ metabolism. The inhibitory potency of DBZ toward three CYP3A4 allelic variants, CYP3A4.2,3A4.12, and 3A4.16, was reduced by 2-10 folds relative to prototype CYP3A4, which implies that DBZ may have less potential to interact with CYP3A4-metabolised drugs in patients carrying these three alleles. Furthermore, compared with prototypic CYP2C8, the allelic variant, CYP2C8.3, produced a 54% decrease in the intrinsic clearance of DBZ, which suggests a dosage adjustment of DBZ may be needed in patients carrying the CYP2C8*3 allele. This pharmacogenomic information could help to rationalize clinical trials of DBZ, including improving the selection of drug doses, drug combinations and volunteers. Our results suggest applications for this in vitro CYP assay system both for basic research in pharmacogenomics and for drug development.更多还原