In vivo Transgenic Reporter for Hepatobiliary Toxicity

肝胆毒性的体内转基因报告基因

基本信息

批准号：
6840566
负责人：
Seth William Kullman
金额：
$ 13.86万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-01 至 2006-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Recent advances in combinatorial chemistry and high throughput screening practices have dramatically increased the number of new chemical entities for pharmaceutical discovery. The pharmaceutical industry is now faced with specific challenges associated with lead candidate selection and preclinical development through the Research and Development pipeline. To meet the demands of drug safety assessment, there is an increasing demand for development of novel high throughput in-vitro and in-vivo models for assessing toxicity of lead drug candidates. To date however, few validated, predictive, rapid throughput in vivo toxicity assays have been established. Herein we propose to develop a novel transgenic fluorescent reporter for prediction of hepatobiliary toxicity during preclinical development of chemotherapeutic drugs. Two specific aims are proposed: (1) Construct a cell specific transgenic see-through medaka to extend noninvasive detection of acute and chronic biliary toxicity/injury, through induction of a gamma-glutamyl transpeptidase driven fluorescent reporter gene. (2) Produce verified hepatic injury in adult see-through medaka by exposure to reference hepatotoxins known to induce canalicular membrane damage; bile duct epithelia cell damage and cholestasis with resultant expression of gamma-glutamyl transpeptidase. Once toxicity is produced, we shall verify linkage between reporter gene expression and biliary cell injury by serial, noninvasive imaging of liver in the transparent fish followed by clinical chemistry and targeted hepatic histological analysis. The see-through medaka planned for use in this study is a unique animal model, which we believe has enormous potential for drug screening and assessment of toxicity. This fish is transparent with principal internal organs visible through the body wall in in all life stages. With the see-through medaka, structural and functional changes at molecular, cellular, tissue and organ/system levels may be imaged noninvasively, leading to greatly enhanced identification and analysis of in vivo toxicity following pharmaceutical exposure. The compressed life cycle of the medaka, when coupled with its transparent features, makes the see-through medaka particularly well suited to study both acute and chronic toxicity, mutagenesis and carcinogenesis. Effort in the proposed research will focus on the development of a non-invasive reporter assay for screening hepatotoxicity during preclinical evaluation of leading chemotherapy drugs. We envision that this model can be employed as both a rapid screen for optimization of the lead molecule series selection (i.e. acute toxicity studies) as well as for more long-term chronic evaluations.

描述（由申请人提供）：联合化学和高吞吐量筛查实践的最新进展显着增加了药物发现的新化学实体的数量。现在，通过研发管道，制药行业面临着与候选人选择和临床前开发相关的具体挑战。为了满足药物安全评估的需求，对新型高通量体内和体内模型的发展需求不断增长，以评估铅药物候选者的毒性。然而，迄今为止，很少建立经过验证的，预测的，快速的体内毒性测定法。本文中，我们建议开发一种新型的转基因荧光报告基因，以预测化学治疗药物临床前发育过程中肝胆固醇毒性。提出了两个具体的目的：（1）通过诱导γ-谷氨酰胺转肽酶驱动的荧光报告基因，构建一个细胞特异性转基因透明的Medaka，以扩展急性和慢性胆汁毒性/损伤的无创检测。（2）通过暴露于已知可诱导小管膜损伤的参考肝毒素中，会在成人透明的Medaka中造成经过验证的肝损伤；胆管上皮细胞损伤和胆汁淤积，导致γ-谷氨酰基肽酶的表达。一旦产生毒性，我们将通过透明鱼类中肝脏的连续，无创成像进行肝脏的无创成像验证报告基因表达与胆道细胞损伤之间的联系，然后进行临床化学，并靶向肝组织学分析。计划在这项研究中使用的透明的Medaka是一种独特的动物模型，我们认为这具有巨大的药物筛查和毒性评估潜力。这种鱼是透明的，在所有生命阶段，都可以通过人体壁可见。借助透明的Medaka，可以无创地成像分子，细胞，组织和器官/系统水平的结构和功能变化，从而大大增强药物暴露后体内毒性的鉴定和分析。 Medaka的压缩生命周期与其透明特征相结合，使透明的Medaka特别适合研究急性和慢性毒性，诱变和致癌作用。拟议的研究中的努力将集中于开发非侵入性记者测定法，以筛选肝毒性，期间在临床前化学疗法药物的临床前评估期间。我们设想，该模型既可以用作优化铅分子序列选择（即急性毒性研究）的快速筛选，也可以用作更长期的慢性评估。