MRP2, BSEP, and BCRP Transporter Polymorphisms and Chemotherapy Disposition

描述（由申请人提供）：该提案旨在为主要研究者（PI）提供机会，使其在两名高资质申办者的直接监督下获得分子生物学和临床药理学方面的知识和技能，以增强其发展成为成功的独立研究者的潜力。该提案的范围将包括必要的课程，研讨会和新技术的实践经验，使PI能够在提案的整个五年内完成密集的研究提案。本提案的总体科学目标是确定MRP2、BSEP和BCRP药物外排转运蛋白的功能多态性与化疗处置调节之间的相关性。由于转运蛋白是药物处置过程的关键决定因素，因此将利用模型细胞系统中的功能研究来评价转运活性并进行药代动力学分析。转运蛋白变体对蛋白活性具有显著的功能性影响以及对化疗处置和毒性具有重要意义的假设将通过2个特定目的进行评价：首先，将在HeLa细胞中利用体外重组牛痘介导的转染试验以及免疫印迹和共聚焦免疫荧光分析对MRP 2、BCRP和BSEP变体进行功能性表征，以评价总蛋白和细胞表面蛋白表达。其次，将在模型细胞系统中利用精确的功能测定（包括诱导型基因表达极化稳定细胞系和由内而外的膜囊泡）研究特定目标1中鉴定的功能相关变体，以提供功能改变机制、动力学分析、底物特异性和化疗介导的抑制的综合评价。药物分布和反应的个体间差异是常用处方药的主要问题，但与化疗药物特别相关，因为疗效受到狭窄治疗指数的限制。显著的患者间差异在许多化疗方案中是常见的，并且很明显遗传因素在决定个体对药物治疗的反应中起着至关重要的作用。定义与抗癌药物处置改变相关的药物转运蛋白基因型，通过优化治疗指数和降低与不良反应相关的死亡率和发病率，可能对肿瘤患者使用化疗药物具有显著的治疗效果。

DESCRIPTION (provided by applicant): The proposal seeks the opportunity for the Principal Investigator (PI) to gain knowledge and skills in molecular biology and clinical pharmacology under the direct supervision of two highly qualified sponsors to enhance his potential to develop into a successful independent investigator. The scope of this proposal will incorporate the necessary coursework, seminars, and hands-on experience in new techniques to enable the PI to complete an intensive research proposal over the full five years of the proposal. The overall scientific goal of this proposal is to establish the associations between functional polymorphisms in MRP2, BSEP, and BCRP drug efflux transporters and modulation of chemotherapeutic disposition. Because transporter proteins are critical determinants of the drug disposition process, functional studies in model cell systems will be utilized to evaluate transport activity and perform pharmacokinetic analyses. The hypothesis that transporter variants have significant functional consequences to protein activity and important implications for chemotherapy disposition and toxicity will be evaluated by 2 specific aims: First, MRP2, BCRP, and BSEP variants will be functionally characterized utilizing in vitro recombinant vaccinia-mediated transfection assays in HeLa cells and immunoblot and confocal immunofluoresecent analyses to evaluate total and cell surface protein expression. Second, functionally relevant variants identified in specific aim 1 will be studied in model cell systems utilizing precise functional assays, including inducible gene expression polarized stable cell lines and inside-out membrane vesicles, to provide a comprehensive evaluation of altered mechanisms of function, kinetic analysis, substrate specificity, and chemotherapy-mediated inhibition. Interindividual variation in drug disposition and response is a major concern for commonly prescribed drugs, but has particular relevance with regards to chemotherapeutic agents because efficacy is limited by narrow therapeutic indices. Significant interpatient differences are common with many chemotherapy regimens and it is apparent that genetic factors play vital roles in determining an individual's response to drug therapy. Defining drug transporter genotypes associated with altered disposition of anticancer agents may have significant therapeutic consequences for the use of chemotherapeutic agents in oncology patients by optimizing therapeutic indices and reducing mortality and morbidity associated with adverse effects.