RESUMO
BACKGROUND & AIMS: Idiosyncratic drug-induced liver injury (DILI) is a complex and unpredictable event caused by drugs, and herbal or dietary supplements. Early identification of human hepatotoxicity at preclinical stages remains a major challenge, in which the selection of validated in vitro systems and test drugs has a significant impact. In this systematic review, we analyzed the compounds used in hepatotoxicity assays and established a list of DILI-positive and -negative control drugs for validation of in vitro models of DILI, supported by literature and clinical evidence and endorsed by an expert committee from the COST Action ProEuroDILI Network (CA17112). METHODS: Following 2020 PRISMA guidelines, original research articles focusing on DILI which used in vitro human models and performed at least one hepatotoxicity assay with positive and negative control compounds, were included. Bias of the studies was assessed by a modified 'Toxicological Data Reliability Assessment Tool'. RESULTS: A total of 51 studies (out of 2,936) met the inclusion criteria, with 30 categorized as reliable without restrictions. Although there was a broad consensus on positive compounds, the selection of negative compounds lacked clarity. 2D monoculture, short exposure times and cytotoxicity endpoints were the most tested, although there was no consensus on drug concentrations. CONCLUSIONS: Extensive analysis highlighted the lack of agreement on control compounds for in vitro DILI assessment. Following comprehensive in vitro and clinical data analysis together with input from the expert committee, an evidence-based consensus-driven list of 10 positive and negative control drugs for validation of in vitro models of DILI is proposed. IMPACT AND IMPLICATIONS: Prediction of human toxicity early in the drug development process remains a major challenge, necessitating the development of more physiologically relevant liver models and careful selection of drug-induced liver injury (DILI)-positive and -negative control drugs to better predict the risk of DILI associated with new drug candidates. Thus, this systematic study has crucial implications for standardizing the validation of new in vitro models of DILI. By establishing a consensus-driven list of positive and negative control drugs, the study provides a scientifically justified framework for enhancing the consistency of preclinical testing, thereby addressing a significant challenge in early hepatotoxicity identification. Practically, these findings can guide researchers in evaluating safety profiles of new drugs, refining in vitro models, and informing regulatory agencies on potential improvements to regulatory guidelines, ensuring a more systematic and efficient approach to drug safety assessment.
RESUMO
Introduction: The development of reliable hepatic in vitro models may provide insights into disease mechanisms, linking hepatocyte dysmetabolism and related pathologies. However, several of the existing models depend on using high concentrations of hepatocyte differentiation-promoting compounds, namely glucose, insulin, and dexamethasone, which is among the reasons that have hampered their use for modeling metabolism-related diseases. This work focused on modulating glucose homeostasis and glucocorticoid concentration to improve the suitability of a mesenchymal stem-cell (MSC)-derived hepatocyte-like cell (HLC) human model for studying hepatic insulin action and disease modeling. Methods: We have investigated the role of insulin, glucose and dexamethasone on mitochondrial function, insulin signaling and carbohydrate metabolism, namely AKT phosphorylation, glycogen storage ability, glycolysis and gluconeogenesis, as well as fatty acid oxidation and bile acid metabolism gene expression in HLCs. In addition, we evaluated cell morphological features, albumin and urea production, the presence of hepatic-specific markers, biotransformation ability and mitochondrial function. Results: Using glucose, insulin and dexamethasone levels close to physiological concentrations improved insulin responsiveness in HLCs, as demonstrated by AKT phosphorylation, upregulation of glycolysis and downregulation of Irs2 and gluconeogenesis and fatty acid oxidation pathways. Ammonia detoxification, EROD and UGT activities and sensitivity to paracetamol cytotoxicity were also enhanced under more physiologically relevant conditions. Conclusion: HLCs kept under reduced concentrations of glucose, insulin and dexamethasone presented an improved hepatic phenotype and insulin sensitivity demonstrating superior potential as an in vitro platform for modeling energy metabolism-related disorders, namely for the investigation of the insulin signaling pathway.
