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.
