Introduction: Adverse drug reactions are a major cause for failures of drug development programs, drug withdrawals and use restrictions. Early hazard identification and diligent risk avoidance strategies are therefore essential. For drug-induced liver injury (DILI), this is difficult using conventional safety testing. To reduce the risk for DILI, drug candidates with a high risk need to be identified and deselected. And, to produce drug candidates without that risk associated, risk factors need to be assessed early during drug discovery, such that lead series can be optimized on safety parameters. This requires methods that allow for medium-to-high throughput compound profiling and that generate quantitative results suitable to establish structure-activity-relationships during lead optimization programs.
Methods: We present the validation of such a method, a novel high content screening assay based on six parameters (nuclei counts, nuclear area, plasma membrane integrity, lysosomal activity, mitochondrial membrane potential (MMP), and mitochondrial area) using ~100 drugs of which the clinical hepatotoxicity profile is known.
Results discussion: We find that a 100-fold TI between the lowest toxic concentration and the therapeutic Cmax is optimal to classify compounds as hepatotoxic or non-hepatotoxic, based on the individual parameters. Most parameters have ~50% sensitivity and ~90% specificity. Drugs hitting ≥2 parameters at a concentration below 100-fold their Cmax are typically hepatotoxic, whereas non-hepatotoxic drugs typically hit <2 parameters within that 100-fold TI. In a zone classification model, based on nuclei count, MMP and human Cmax, we identified an area without a single false positive, while maintaining 45% sensitivity. Hierarchical clustering using the multi-parametric dataset roughly separates toxic from non-toxic compounds. We employ the assay in discovery projects to prioritize novel compound series during hit-to-lead, to steer away from a DILI risk during lead optimization, for risk assessment towards candidate selection and to provide guidance of safe human exposure levels.
Keywords: AC50; ALT; CCCP; Cmax; Cytotoxicity; DILI; DMSO; Drug-induced liver injury; FBS; HCS; Hepatotoxicity; High content screening; IC50; LEC; MMP; Methods; Mitochondrial toxicity; PBS; Predictive toxicology; Risk assessment; SD; SEM; TI; activity concentration 50%; alanine transferase; carbonyl cyanide m-chlorophenyl hydrazine; dimethyl sulfoxide; drug-induced liver injury; fetal bovine serum; high content screening; inhibitory concentration 50%; lowest efficacious concentration; maximum total concentration in plasma; mitochondrial membrane potential; phosphate buffered saline; standard deviation; standard error of the mean; therapeutic interval.
© 2013.