Development and validation of an integrated tiered in vitro screening approach for predicting drug induced liver injury (DILI)

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Liver injury is a major cause of drug attrition as a result of preclinical toxicity and usually occurs late on in clinical trials. Currently, liver injury is largely assessed using animal (rodent) models. However, there is an urgent need to develop alternative in vitro and in silico models that once validated would have a number of important potential advantages. Because multiple modes of action are known to be important an integrated tiered screening approach based on a mechanistic understanding is required. The aim of the project is o develop and assess in vitro assays including bile salt export pump and multidrug resistance protein 2 inhibition assay, UDP-glucuronosyl transferase 1A1 and 1A3 enzyme inhibition, cell toxicity (MTT, adenylate kinase), impairment of mitochondrial membrane potential (TMRE), mitochondrial (mitosox) and cellular ROS (DCF-DA) using liver cell lines (e.g. HepG2) and primary hepatocytes in combination with state-of-the-art technologies including the Hi Content Imaging Express, Incucyte Cell Imager for real time analysis and imaging of cells in culture and LC-MS-TOF for reactive metabolite assessment. In addition we will explore use of confocal-imaging in combination with tagged-reporter proteins to develop assays for stress-response mediated by specific transcriptional activation pathways (e.g. Nrf2 nuclear translocation as a marker of the "anti-oxidant response"). These assays will be validated using a "training set" of known toxic (e.g. carbon tetrachloride) and non-toxic compounds to build a database of responses to known hepatoxicants that exert their effects by different mechanisms. Once validated the predictive power of developed assays will be tested using a panel of chemicals (and metabolites, acyl,N-,O-glucuronides for example) where little information exists on their ability to cause liver injury or which are currently under in vivo assessment. A link to potential systemic drug levels will also be assessed,