Application of a Rat Liver Drug Bioactivation Transcriptional Response Assay Early in Drug Development That Informs Chemically Reactive Metabolite Formation and Potential for Drug-induced Liver Injury

Abstract

Drug induced liver injury (DILI) is a major reason for drug candidate attrition from development, denied commercialization, market withdrawal, and restricted prescribing of pharmaceuticals. The metabolic bioactivation of drugs to chemically reactive metabolites (CRM) contribute to liver-associated adverse drug reactions (ADR) in humans that often goes undetected in conventional animal toxicology studies. A challenge for pharmaceutical drug discovery has been reliably selecting drug candidates with a low liability of forming CRM and reduced DILI potential, at projected therapeutic doses, without falsely restricting the development of safe drugs. We have developed an in vivo rat liver transcriptional signature biomarker reflecting the cellular response to drug bioactivation. Measurement of transcriptional activation of integrated Nuclear factor erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (Keap1) electrophilic stress, and Nuclear factor erythroid 2-related factor 1 (NRF1) proteasomal endoplasmic reticulum (ER) stress responses, is described for discerning estimated clinical doses of drugs with potential for bioactivation-mediated hepatotoxicity. The approach was established using well benchmarked CRM forming test agents from our company. This was subsequently tested using curated lists of commercial drugs and internal compounds, anchored in the clinical experience with human hepatotoxicity, while agnostic to mechanism. Based on results with 116 compounds in short-term rat studies, with consideration of the maximum recommended daily clinical dose, this CRM mechanism-based approach yielded 32% sensitivity and 92% specificity for discriminating safe from hepatotoxic drugs. The approach adds new information for guiding early candidate selection and informs structure activity relationships (SAR) thus enabling lead optimization and mechanistic problem solving. Additional refinement of the model is ongoing. Case examples are provided describing the strengths and limitations of the approach.

The human hepatocyte TXG-MAPr: WGCNA transcriptomic modules to support mechanism-based risk assessment

“…For instance, how well a certain process is captured in gene expression data from the input experiments, which can influence gene-to-gene correlation patterns, could be a hypothesis to be explored in future studies. Nevertheless, literature reports that kinetics and activation of the non-preserved Nrf2/p53 processes are different across species (MacRae et al, 2015; Martin & Chang, 2018; Monroe et al, 2020), suggesting that preservation of co-expression networks is a promising approach for the evaluation of the applicability domain of new approach methodologies (NAMs) for extrapolating safety assessments across species (Parish et al, 2020).…”

Mechanistic Investigations Support Liver Safety of Ubrogepant

“…For MK-3207 at the highest testable concentration (due to solubility limitations) of 4.4 times the calculated unbound liver inlet concentration, no significant impact was observed on biliary excretion ( Table 1 ) in human HEPATOPAC. Parameters reflecting the in vitro abilities of the 3 molecules to generate reactive metabolites and electrophilic stress were consistent in demonstrating DILI risk for MK-3207 and telcagepant, and are summarized in Table 1 and further described elsewhere ( Kang et al , forthcoming ; Monroe et al , forthcoming ). The calculated reactive metabolite body burden for ubrogepant (4600) was lower than for telcagepant (14 560) or MK-3207 (14 720), indicating that ubrogepant has a lower potential to form reactive metabolites at dosing likely to achieve therapeutic results.…”

“…These initial mechanistic studies concluded that the production of reactive metabolites was a primary causative factor of the clinical DILI observed with telcagepant and MK-3207 and that production of reactive metabolites with ubrogepant in the same test systems was sufficiently reduced to warrant internal approval for nonclinical and clinical development. Those study data and methodologies are published separately, along with the supporting platform development data from over 100 other compounds ( Kang et al , forthcoming ; Monroe et al , forthcoming ; Podtelezhnikov et al , 2020 ), and the telcagepant, MK-3207, and ubrogepant data are also summarized there. For business reasons, ubrogepant was sold by Merck to Allergan during early clinical development.…”

Gene Signatures Reduce the Stress of Preclinical Drug Hepatotoxicity Screening

“…Typically, the hepatic covalent binding burden of a drug is determined using in vitro or in vivo radiolabel and nucleophile trapping methods, but the Merck team found these approaches could not adequately distinguish compounds that are hepatotoxic from those that are safe in the clinic, even after taking into account the maximum human daily dose. ( 1 ) Because the Nrf2/1‐driven stress responses are triggered as part of the cellular adaptation to chemical insult, they have for some time been considered more reflective of the consequence (risk), as opposed to the mere occurrence (hazard), of CRM formation. However, until now, this concept has not been examined in a comprehensive and systematic manner in the context of DILI.…”