Evaluation of multiple mechanism-based toxicity endpoints in primary cultured human hepatocytes for the identification of drugs with clinical hepatotoxicity: Results from 152 marketed drugs with known liver injury profiles

Highlights

• 152 drugs with known DILI profiles evaluated in primary cultured human hepatocytes.

• Four toxicological endpoints: ATP, ROS, GSH, and caspase.

• AUC of dose–response curves and receiver operating characteristics used in data analysis.

• The ROS/ATP ratio accurately identified drugs associated with severe DILI.

Abstract

We report here the results of a collaborative research program to develop a robust and reliable in vitro system to allow an accurate definition of the drug-induced liver injury (DILI) potential of new drug entities during drug development. The in vitro hepatotoxic potential of 152 drugs with known DILI profiles were evaluated in primary cultured human hepatocytes with four mechanistically-relevant endpoints: cellular ATP depletion, reactive oxygen species (ROS), glutathione (GSH) depletion, and caspase activation for apoptosis. The drugs, 80 in the testing set and 72 in the validation set, were classified based on serious clinical/regulatory outcomes as defined by reported acute liver failure, black-box warning, and/or withdrawal. The drugs were further sub-categorized for dominant types of liver injury. Logistic regression models were performed to calculate the area under the receiver operating characteristics curve (AUROC) and to evaluate the prediction potential of the selected endpoints for serious clinical/regulatory outcomes. The ROS/ATP ratio was found to yield an excellent AUROC in both the testing (0.8989, P < 0.0001) and validation set (0.8545, P < 0.0001), and was found to distinguish drugs associated with severe from non-severe DILI cases (p < 0.0001). The results suggest that evaluation of drugs in primary human hepatocytes using the ROS/ATP ratio endpoint may aid the definition of their potential to cause severe DILI.

Introduction

Drug-induced liver injury (DILI), being one of the most common reasons for early termination of drug development and post-marketing regulatory actions [16], [28], continues to be a major challenge in the drug development and regulatory decision making processes. The occurrence of severe DILI (sDILI) events leading to acute liver failure (ALF) of marketed drugs illustrates the inadequacy of the current testing strategy to accurately identify perpetrators that can cause death or result in the need for liver transplantation. The shortfall of preclinical safety evaluations may be attributed to the significant species difference in drug-metabolizing enzyme pathways (e.g. species differences in metabolic activation and detoxification) and toxicological mechanisms [2], [20]. The failure of clinical trials to identify sDILI events can be explained by their low incidence, which has been estimated to be 1 case per 100,000 patients per year [3], [23].

Therefore, there is an unmet need for effective strategies at an early stage in drug development to avoid the occurrence of sDILI. One strategy is to identify the at-risk population. However, in spite of an apparently promising finding that the major histocompatibility complex (MHC) variant alleles HLA-B*5701 genotype is a major determinant of drug-induced liver injury due to flucloxacillin [7], [8], an international genome-wide association study (GWAS) failed to identify common genetic variants amongst patients with serious drug-induced liver injuries, thus suggesting that either genetic determinants are drug-specific or that the genetic events are rare and are not readily identifiable [25], [31]. It has been postulated that the individual differences in sensitivity to hepatotoxic drugs are a result of the co-occurrence of multiple, transient, environmentally-related phenomena, and therefore cannot be identified by genome characterization alone [19]. One promising ongoing focus is to develop accurate diagnostic early biomarkers such as microRNA-122 to complement serum transaminases in order to allow early therapeutic intervention to avoid full development of DILI events [29], [33], [34].

The study reported here is based on the hypothesis that drugs found to cause idiosyncratic hepatotoxicity may have common chemical and biological properties which can be used for their identification [19]. We have selected primary human hepatocytes as the experimental system to evaluate the hypothesis for the following reasons: 1. While DILI may involve multiple cell types, damage to hepatocytes is a major pathological finding for acute liver failure [11], [32]. 2. Primary human hepatocytes are known to retain key liver functions, especially human-specific drug metabolizing enzymes and cofactors, and are generally considered the “gold standard” for the evaluation of human drug metabolism and hepatotoxicity [12], [21].

We report here results of the evaluation of 152 marketed drugs in primary human hepatocytes with well-defined clinical hepatotoxicity using cellular ATP depletion, reactive oxygen species (ROS), glutathione (GSH) depletion, and caspase activation for apoptosis as endpoints. We observed that the drug-induced elevation of ROS/ATP ratio in human hepatocytes accurately identifies drugs associated with severe DILI events.