摘要
人类基因组中的遗传变异,是人体对药物及其他外源性化合物产生不同反应的重要原因。同时,在一定程度上,人类对大部分疾病的易感性也决定于人类基因中的各种变异。在过去的40年里,随着分子生物学的发展,遗传药理学从一个小学科逐渐发展成为推动临床药理学进步的中坚力量,同时也成为生物医药研究中一个炙手可热的领域。目前,人类基因组拥有超过一千万个单核苷酸突变以及大量的其他类型的遗传变异。在任何一项临床研究中,只要经费允许,我们都可以对超过一百万个遗传多态性或者超过两万五千个基因的表达进行分析。尽管如此,目前这些工作仍没能有效改变传统的临床治疗模式,大部分的临床药物治疗依然遵循经验用药模式,但转变正在悄然进行,相当多的临床医生已经开始在日常医疗工作中考虑基因多态性的影响,如CYP2C9,CYP2C19,CYP2D6,TPMT以及VKORC1等。更明显的是,遗传药理学已经改变了临床药物研发的要求和模式。遗传药理学准则已经成为许多大型药物临床研究的基本要求。美国食品药品管理局(FDA)也鼓励临床试验者提供实验药物的相关遗传药理学资料。
自2002年开始人类基因组单体型图计划(International HapMap Project)正式启动。由这项由美国、日本、英国、加拿大、中国和尼日利亚的科学家们合作的,从人类基因组计划延续而来的项目,旨在开发一个面向公众的,人类全基因组单体型数据库。该数据库的信息将用来指导各种遗传研究以及临床表型相关的研究。HapMap计划所研究的DNA样本为来自不同地域,不同种族的270个个体。到Ⅱ期数据发布时,共分析了三百多万个SNP,分析密度达到了每1kb序列中分析1个SNP。这大概覆盖了九百万到一千万个常见SNP中的25%～35%。目前,其数据也已经可以通过互联网无偿获得。
基于以上研究背景,本课题旨在查明有机阴离子转运体OATP1B1的常见遗传多态性对他汀类降脂药物阿托伐他汀代谢动力学的影响,以及通过竞争性抑制和非竞争性抑制,改变OATP1B1转运能力后,阿托伐他汀以及罗素他汀药代动力学的改变。此外,通过严格设计的临床实验,应用HapMap数据库提供的基因单体型信息,考察多基因,多遗传变异以及多个单体型,对接受阿糖胞苷标准化疗的急性髓细胞样白血病(AML)患者临床预后和药物疗效的影响进行研究。本课题不仅为药物反应个体差异提供分子水平的解释,还为临床遗传药理学开辟新的研究方法和研究方向。
本课题的主要研究结果如下:
1.证实在中国健康人群中,OATP1B1单体型显著影响阿托伐他汀在不同个体中的血药浓度。OATP1B1*5突变携带者与OATP1B1*1a/*1a相比,阿托伐他汀血药浓度显著升高;
2.发现单剂量口服600mg利福平,可显著抑制阿托伐他汀的肝摄取。OATP1B1单体型显著影响利福平对阿托伐他汀肝脏摄取的抑制效应。利福平抑制作用在OATP1B1*1a/*1a个体中较OATP1B1*5突变携带者中更显著。
3.连续14天服用熊脱氧胆酸胶囊,血浆罗素他汀及胆红素水平显著升高。这可能是由于熊脱氧胆酸通过抑制转录因子HNF1α,进而抑制OATP1B1活性所致。这种抑制水平要相对低于直接抑制OATP1B1转运。由于熊脱氧胆酸所抑制的转录因子HNF1α能参与调节多种药物代谢酶的表达,临床应用熊脱氧胆酸时,应注意可能通过此机制产生的多种药物相互作用。
4.研究了参与阿糖胞苷体内代谢的多种代谢酶(CDA,dCK),转运体(hCNT1)的多个单核苷酸变异以及单体型对接受阿糖胞苷标准化疗的急性髓细胞样白血病(AML)患者的总体生存率,无事件生存率,无复发生存率等临床预后指标的影响。结果证实,hCNT1,1561C＞T(rs2242046)遗传变异,与完全缓解组AML患者的总体生存率以及药物毒性反应显著相关。此外,在出现药物治疗失效以及治疗早期死亡患者中,骨髓移植以及加用全反式维甲酸可显著改善患者的总体生存率。
5.首次将人类基因组单体型图(HapMap)数据应用于临床研究,并证实,在候选基因已知的情况下,HapMap数据库能有效指导临床遗传药理学研究,并提供有力的数据分析工具。
总之,本项目从遗传药理学角度研究了阿托伐他汀,罗素他汀的药物反应个体差异以及药物相互作用,从药物基因组学水平综合研究了阿糖胞苷药物疗效的遗传机制对AML患者预后的影响,并首次将HapMap数据应用于临床研究,为临床遗传药理学研究开辟了新的研究方向。
The different response to drugs and other xenobiotics among individuals and the susceptibility to almost all diseases are to some extent due to variations in human genome.In the past 40 years,driven by the advances in molecular biology,pharmacogenetics has evolved from a minor discipline to a major driving force of clinical pharmacology. Nowadays,over 10 million SNPs and other kinds of variants have been detected in human genome.If the budget is enough,we can assess more than 1 million polymorphisms or the expression of more than 25,000 genes in each clinical study.However,this has not yet changed daily therapeutic mode in clinic which is somehow empirical.But some physicians are aware of the importance of some gene polymorphisms, such as those in CYP2C9,CYP2C19,CYP2D6,TPMT and VKORC1.To more practical situation,pharmacogenetics has changed the practices and requirements in preclinical and clinical drug research.Pharmacognetics displines have become a basic requirement of many large clinical trials on new drugs.The US Food and Drug Administration(FDA) also has encouraged the voluntary deposition of supplementary pharmacogenomics data.
Following the International Hunam Genomics Project,International HapMap Project is carried out under the cooperation of scientists coming from US,Japan,Britain,Canada,China and Nigeria.It was launched in 2002 with the aim of providing a public resource to accelerate medical genetic research.The information in this database will be used for genetic studies and clinic studies on phenotyps.In HapMap project,DNA samples retrieved were from 270 donors come from four different places and four different species.With the releasing of PhaseⅡdata,over three million SNPs have been analyzed.All the data have been open to public through internet now.
Based on these information,the aim of our study was to clarify the impact of the common genetic polymorphisms occurred on OATP1B1 on the pharmacokinetics of atorvastatin and how does the kinetics of atorvastatin and rosuvastain change when the activity of OATP1B1 is inhibited via a competitive inhibition manner or non-competitive inhibition manner.We also lanched a well designed clinical study which is accomplished in Ulm University,Germany,on the clinical outcome of AML patients to the perspective of Pharmacogenomics with the guide of HapMap knowledge.This project provided new explains for individual differences of drug response and explored a new way to translate HapMap knowledge into the pharmacogenetics and pharmacogenomics study.
In this project we have found that:
1.OATP1B1 haplotypes significantly changes the pharmacokinetics of atorvastatin in vivo.
2.OATP1B transporters represent the major hepatic uptake systems for atorvastatin.Single oral dose of 600mg rifamin can significantly inhibit the hepatic uptake of atorvastain.OATP1B1 haplotypes significantly influence the inhibition effect of rifampin on the activity of OATP1B1. Individuals with OATP1B1~*1a/~*1a wild type were more sensivie to the inhibition of rifampin than OATP1B1~*5 carriers.
3.Confirm the findings from in vitro studies that UDCA inhibits OATP1B1 activity by inhibition of the transcription factor HNF1αin vivo.
4.Frequencies of several SNPs of hCNT1,CDA and dCK are different between AML patients and healthy individuals.In RD and ED group, bone marrow transplantation and the use of ATAR can improved the prognosis of AML patients,hCNT1 genetic variat,1561C＞T(rs2242046), was associated with drug toxicity and overall survival rate in patients who were complete remission.
5.The PhaseⅡdatabase released in HapMap provides aboundant and confidential information for the analysis of dCK genome.Using HapMap database can provide a new method for pharmacogenetics and pharmacogenomics research.
In summary,we studied the inter-individual difference of atorvastatin response to the perspective of pharmacogentics and drug interactions of atorvastatin and rosuvastain.We also explored the impact of genetics variants involved in cytarabine metabolism on the clinical outcomes of AML patiens and for the first time translate the HapMap knowledge into clinical study which provides a new approach to proceed with pharmacogenetics and pharmacogenomics study.
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