Quantitative analysis of aryl hydrocarbon receptor activation using fluorescence-based cell imaging--a high-throughput mechanism-based assay for drug discovery.

Early identification of toxicity associated with new chemical entities is important for reducing compound attrition in late stage drug discovery. Activation of the aryl hydrocarbon receptor (AhR) by xenobiotics is a recognised mechanism of toxicity: the AhR mediates most, if not all, of the serious toxicities caused by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition to compounds such as TCDD, the AhR can be activated by compounds with drug-like properties; consequently there is a desire to eliminate AhR activity in candidate drug programs. Endogenous AhR translocates from the cytoplasm to the nucleus in response to prototypical AhR ligands. This trafficking was monitored in mouse Hepa-1 cells, human HepG2 cells and rat primary hepatocytes using an anti-AhR antibody. A confocal imaging plate reader, the InCell Analyzer 3000, was used to image fixed cells cultured in 96 well plates, and algorithms were used to analyse both population data and individual cell responses. The subsequent induction of the CYP1A1 gene, in the three cell models, was also assessed using quantitative real-time polymerase chain reaction and showed good correlation with the translocation assay. To conclude, we have established robust, automated high throughput assays for the identification of AhR activators in primary hepatocytes and cell lines.

Investigations into the liver effects of ximelagatran using high content screening of primary human hepatocyte cultures.

BACKGROUND: Ximelagatran, the first oral agent in the new class of direct thrombin inhibitors, was withdrawn from the market due to increased rates of liver enzyme elevations in long-term treatments. Despite intensive pre clinical investigations the cellular mechanisms behind the observed hepatic effects remain unknown. OBJECTIVE: The aim of this study was to assess drug-induced cytotoxicity in primary human hepatocyte cultures by ximelagatran and other reference pharmaceutical agents with known in vivo hepatotoxic profiles. METHODS: Drugs cause liver injury by many distinct mechanisms that result in abnormal cellular functioning and different patterns of injury. To address many potential toxic mechanisms in a human-relevant model, freshly isolated human hepatocytes were used in automated imaging assays. Ximelagatran was used as a test compound to study biochemical and morphological changes in human hepatocytes. In addition, 11 control, reference and comparator compounds with known liver-toxic potential in humans were used. The response to these compounds was assessed across five different hepatocyte donor preparations. RESULTS: Cytotoxicity induced by a number of compounds was quantitatively monitored using an automated imaging technique. A variety of morphological changes in hepatocyte cytoskeleton and mitochondrial function could be identified at sublethal doses of test compounds. Doses of ximelagatran up to 500 microM did not cause a cytotoxic response in the majority of preparations and no subcytotoxic response was observed at doses below 125 microM. CONCLUSIONS: The experiments described here demonstrate that primary human hepatocytes may be used in a medium-throughput format for screening using imaging-based assays for the identification of cellular responses. Overall, it is concluded that ximelagatran did not cause a significant decrease in cell viability when incubated for 24 h at considerably higher concentrations than are found in plasma following therapeutic dosing.