In vivo Transgenic Reporter for Hepatobiliary Toxicity

描述（由申请人提供）： 组合化学和高通量筛选实践的最新进展已经显著增加了用于药物发现的新化学实体的数量。制药行业现在面临着与通过研发管道进行的主要候选人选择和临床前开发相关的特定挑战。为了满足药物安全性评价的需求，人们越来越需要开发用于评价先导候选药物毒性的新型高通量体外和体内模型。然而，到目前为止，很少有验证，预测，快速通量的体内毒性测定已经建立。在此，我们建议开发一种新的转基因荧光报告基因预测肝胆毒性在临床前开发的化疗药物。提出了两个具体目标：（1）构建细胞特异性转基因透明青鳉，通过诱导γ-谷氨酰转肽酶驱动的荧光报告基因，扩展急性和慢性胆毒性/损伤的无创检测。(2)通过暴露于已知可诱导小管膜损伤、胆管上皮细胞损伤和胆汁淤积并导致γ-谷氨酰转肽酶表达的参考肝毒素，在成年透明青鳉中产生经验证的肝损伤。一旦产生毒性，我们将通过对透明鱼肝脏进行系列非侵入性成像，然后进行临床化学和靶向肝组织学分析，验证报告基因表达与胆管细胞损伤之间的联系。计划用于本研究的透明青鳉是一种独特的动物模型，我们相信它在药物筛选和毒性评估方面具有巨大的潜力。这种鱼是透明的，在所有生命阶段的主要内部器官都可以通过体壁看到。利用透明青鳉，可以非侵入性地成像分子、细胞、组织和器官/系统水平的结构和功能变化，从而大大增强了药物暴露后体内毒性的识别和分析。青鳉的压缩生命周期，再加上其透明的特点，使透明青鳉特别适合研究急性和慢性毒性，诱变和致癌作用。在拟议的研究工作将集中在开发一种非侵入性的报告基因检测方法，用于在临床前评估主要化疗药物时筛选肝毒性。我们设想，该模型可用作先导分子系列选择（即急性毒性研究）优化的快速筛选以及更长期的慢性评价。

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.