Long-enduring primary hepatocyte-based co-cultures improve prediction of hepatotoxicity

摘要

Abstract The failure of drug candidates during clinical trials and post-marketing withdrawal due to Drug Induced Liver Injury (DILI), results in significant late-stage attrition in the pharmaceutical industry. Animal studies have proven insufficient to definitively predict DILI in the clinic, therefore a variety of in vitro models are being tested in an effort to improve prediction of human hepatotoxicity. The model system described here consists of cryopreserved primary rat, dog or human hepatocytes co-cultured together with a fibroblast cell line, which aids in the hepatocytes' maintenance of more in vivo -like characteristics compared to traditional hepatic mono-cultures, including long term viability and retention of activity of cytochrome P450 isozymes. Cell viability was assessed by measurement of ATP following treatment with 29 compounds having known hepatotoxic liabilities. Hμrelrat?, Hμreldog?, and Hμrelhuman? hepatic co-cultures were treated for 24h, or under repeat-dosing for 7 or 13days, and compared to rat and human hepatic mono-cultures following single-dose exposure for 24h. The results allowed for a comparison of cytotoxicity, species-specific responses and the effect of repeat compound exposure on the prediction of hepatotoxic potential in each model. Results show that the co-culture model had greater sensitivity compared to that of the hepatic mono-cultures. In addition, “time-based ratios” were determined by dividing the compounds' 24-hour TC 50 /C max values by TC 50 /C max values measured after dosing for either 7 or 13days. The results suggest that this approach may serve as a useful adjunct to traditional measurements of hepatotoxicity, improving the predictive value of early screening studies. Highlights ? An in vitro hepatocyte model with 14day survival is characterized. ? The model uses rat, human and dog hepatocyte/fibroblast co-cultures. ? Sensitivity and selectivity of the model are compared to 24h hepatic monocultures. ? Results show increased sensitivity for hepatotoxicants using the co-culture model.