Prediction of Drug-Induced Hepatotoxicity Using Long-Term Stable Primary Hepatic 3D Spheroid Cultures in Chemically Defined Conditions

Abstract

High failure rates of drug candidates in the clinics, restricted-use warnings as well as withdrawals of drugs in postmarketing stages are of substantial concern for the pharmaceutical industry and drug-induced liver injury (DILI) constitutes one of the most frequent reasons for such safety failures. Importantly, as DILI cannot be accurately predicted using animal models, animal safety tests are commonly complemented with assessments in human in vitro systems. 3D spheroid cultures of primary human hepatocytes in chemically defined conditions, hereafter termed CD-spheroids, have recently emerged as a microphysiological model system in which hepatocytes retain their molecular phenotypes and hepatic functions for multiple weeks in culture. However, their predictive power for the detection of hepatotoxic liabilities has not been systematically assessed. Therefore, we here evaluated the hepatotoxicity of 123 drugs with or without direct implication in clinical DILI events. Importantly, using ATP quantifications as the single endpoint, the model accurately distinguished between hepatotoxic and nontoxic structural analogues and exceeded both sensitivity and specificity of all previously published in vitro assays at substantially lower exposure levels, successfully detecting 69% of all hepatotoxic compounds without producing any false positive results (100% specificity). Furthermore, the platform supports the culture of spheroids of primary hepatocytes from preclinical animal models, thereby allowing the identification of animal-specific toxicity events. We anticipate that CD-spheroids represent a powerful and versatile tool in drug discovery and preclinical drug development that can reliably flag hepatotoxic drug candidates and provide guidance for the selection of the most suitable animal models.

Studying 3D cell cultures in a microfluidic droplet array under multiple time-resolved conditions

“…While the results discussed here do not address the question of whether spheroids are better predictors of in vivo drug response than other formats (44)(45)(46), they do indicate that the spheroid response to a drug involves collective structural effects that are not present in 2D monolayers. Such effects play a role in the response of solid tumors to chemotherapy and may provide important insights on the sensitivity or survival of micro-niches within a tumor, in particular as cancer cells may be interspersed with fibroblasts or other stromal cell types.…”

3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases, Drug Metabolism, and Toxicity: Emerging Culture Paradigms and Applications

“…Proctor et al used a commercial proprietary model to test the toxicity of 110 drugs (69 DILI‐positive and 41 DILI‐negative) during 14 days of repeated exposures using the InSphero model and reported overall sensitivity and specificity of 59% and 80%, respectively. In contrast, we used chemically defined media and ULA 3D PHH spheroids to expose a panel of 123 drugs (70 DILI‐positive and 53 DILI‐negative), correctly predicting the hepatotoxicity of 48 out of 70 compounds (sensitivity = 69%) without a single false‐positive result (specificity = 100%; Figure A) . Classification of compounds into DILI‐positive and ‐negative for this study was based on the current expert consensus and regulatory classifications.…”

“…Error bars indicate SD ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Reproduced with permission . Copyright 2018, Society of Toxicology.…”

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

“…In addition, human hepatocytes engineered to allow in vitro expansion and cell lines are frequently used in this context (Tolosa et al 2019;Wink et al 2018;O'Brien et al 2006). However, the predictive performance of four so far published in vitro studies with PHH in 2D and 3D spheroid culture is limited, resulting in sensitivities of 51, 66, 59 and 69% and accuracies of 71, 71, 67 and 82%, respectively (Xu et al 2008;Khetani et al 2013;Proctor et al 2017;Vorrink et al 2018). Several reasons may be responsible for the limited predictive performance in vitro.…”

“…Several reasons may be responsible for the limited predictive performance in vitro. One is that some studies only used PHH from one donor per compound (Proctor et al 2017;Vorrink et al 2018), thus not taking interindividual variability into account. A second limitation is that data was analyzed using only the margin of safety (MoS) concept (e.g.…”