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.

Early risk assessment of drug-induced liver injury (DILI) potential for drug candidates remains a major challenge for pharmaceutical development. We have previously developed a set of rat liver transcriptional biomarkers in short-term toxicity studies to inform the potential of drug candidates to generate a high burden of chemically reactive metabolites that presents higher risk for human DILI. Here we describe translation of those NRF1/NRF2-mediated liver tissue biomarkers to an in vitro assay using an advanced micropatterned co-culture system (HEPATOPAC®) with primary hepatocytes from male Wistar Han rats. A 9-day, resource-sparing and higher throughput approach designed to identify new chemical entities with lower reactive metabolite-forming potential was qualified for internal decision making using 93 DILI positive and negative drugs. This assay provides 81% sensitivity and 90% specificity in detecting hepatotoxicants when a positive test outcome is defined as the bioactivation signature score of a test drug exceeding the threshold value at an in vitro test concentration that falls within 3-fold of the estimated maximum drug concentration at the human liver inlet following highest recommended clinical dose administrations. Using paired examples of compounds from distinct chemical series and close structural analogs, we demonstrate that this assay can differentiate drugs with lower DILI risk. The utility of this in vitro transcriptomic approach was also examined using human HEPATOPAC from a single donor, yielding 68% sensitivity and 86% specificity when the aforementioned criteria are applied to the same 93-drug test set. Routine use of the rat model has been adopted with deployment of the human model as warranted on a case-by-case basis. This in vitro transcriptomic signature-based strategy can be used early in drug discovery to de-risk DILI potential from chemically reactive metabolites by guiding structure activity relationship hypotheses and candidate selection.

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Objectives To evaluate the safety and efficacy of chidamide to reverse HIV‐1 latency in vivo and to compare the effects of four clinically tested histone deacetylase (HDAC) inhibitors on non‐histone proteins in vitro. Methods Participants received chidamide orally at 10 mg twice weekly for 4 weeks while maintaining baseline antiretroviral therapy. The primary outcome was plasma viral rebound during chidamide dosing and the secondary outcomes were safety, pharmacokinetic and pharmacodynamic profiles, changes in cell‐associated HIV‐1 RNA and HIV‐1 DNA, and immune parameters. Western blotting was used to compare the in vitro effects of the four HDAC inhibitors on HSP90, NF‐κB and AP‐1. Results Seven aviraemic participants completed eight oral doses of chidamide, and only grade 1 adverse events were observed. Cyclic increases in histone acetylation were also detected. All participants showed robust and repeated plasma viral rebound (peak viraemia 147–3850 copies/mL), as well as increased cell‐associated HIV‐1 RNA, during chidamide treatment. Furthermore, we identified an enhanced HIV‐1‐specific cellular immune response and a modest 37.7% (95% CI: 12.7–62.8%, P = 0.028) reduction in cell‐associated HIV‐1 DNA. Compared with the other three HDAC inhibitors, chidamide had minimal cytotoxicity in vitro at clinically relevant concentrations and showed mechanistically superior effects on non‐histone proteins, including HSP90, NF‐κB and AP‐1. Conclusions Chidamide safely and vigorously disrupts HIV‐1 latency in vivo, which makes it a promising latency‐reversing agent.

The role of stromal cell-derived factor-1 (SDF-1) in modulating massive liver damage is not well known. In this study, expression of SDF-1 in bone marrow and liver was investigated in rats with acute liver failure (ALF) when mobilized using granulocyte colony-stimulating factor (G-CSF). ALF was induced in rats by D-galactosamine (D-GalN). Starting after 2 hours following D-GalN induction, the animals were injected with G-CSF 50 microg/kg daily or saline as placebo for 5 days. The percentages of CD34+ cells in peripheral blood and the expression of SDF-1 in bone marrow and liver were then determined. The percentages of peripheral CD34+ cells demonstrated a transient increase in placebo rats following D-GalN induction and a significant increase in rats after G-CSF administration. SDF-1 expression showed a transient decrease in bone marrow and a transient increase in liver tissue from placebo rats. However, a significant decrease of SDF-1 expression in bone marrow and a remarkable increase in liver tissue were observed in animals from the G-CSF group. It was concluded that G-CSF can enhance the reduced expression of SDF-1 in bone marrow and increased expression in liver in ALF rats, forming a greater SDF-1 gradient, and chemoattracting CD34+ cells' migration from bone marrow to an injured liver.