Limitations of acetaminophen as a reference hepatotoxin for the evaluation of in vitro liver models.

Acetaminophen is commonly used as a reference hepatotoxin to demonstrate that in vitro human liver platforms can emulate features of clinical drug-induced liver injury. However, the induction of substantial cell death in these models typically requires acetaminophen concentrations (∼10 mM) far higher than blood concentrations of the drug associated with clinical hepatotoxicity (∼1 mM). Using the cytochrome P450 inhibitor 1-aminobenzotriazole, we show that acetaminophen toxicity in cultured human, mouse, and rat hepatocytes is not dependent on N-acetyl-p-benzoquinonimine formation, unlike the in vivo setting.

Biomarkers of NRF2 signalling: Current status and future challenges.

The cytoprotective transcription factor NRF2 regulates the expression of several hundred genes in mammalian cells and is a promising therapeutic target in a number of diseases associated with oxidative stress and inflammation. Hence, an ability to monitor basal and inducible NRF2 signalling is vital for mechanistic understanding in translational studies. Due to some caveats related to the direct measurement of NRF2 levels, the modulation of NRF2 activity is typically determined by measuring changes in the expression of one or more of its target genes and/or the associated protein products.

A systems approach reveals species differences in hepatic stress response capacity.

To minimize the occurrence of unexpected toxicities in early phase preclinical studies of new drugs, it is vital to understand fundamental similarities and differences between preclinical species and humans. Species differences in sensitivity to acetaminophen (APAP) liver injury have been related to differences in the fraction of the drug that is bioactivated to the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI). We have used physiologically based pharmacokinetic modeling to identify oral doses of APAP (300 and 1000 mg/kg in mice and rats, respectively) yielding similar hepatic burdens of NAPQI to enable the comparison of temporal liver tissue responses under conditions of equivalent chemical insult.

Modulating the expression of tumor suppressor genes using activating oligonucleotide technologies as a therapeutic approach in cancer.

Tumor suppressor genes (TSGs) are frequently downregulated in cancer, leading to dysregulation of the pathways that they control. The continuum model of tumor suppression suggests that even subtle changes in TSG expression, for example, driven by epigenetic modifications or copy number alterations, can lead to a loss of gene function and a phenotypic effect. This approach to exploring tumor suppression provides opportunities for alternative therapies that may be able to restore TSG expression toward normal levels, such as oligonucleotide therapies.

Advances and challenges in therapeutic targeting of NRF2.

Activation of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) is emerging as an attractive therapeutic approach to counteract oxidative stress, inflammation, and metabolic imbalances. These processes underpin many chronic pathologies with unmet therapeutic needs, including neurodegenerative disorders and metabolic diseases. As the NRF2 field transitions into the clinical phase of its evolution, the need for an understanding of the factors influencing NRF2 pharmacology has never been greater.

Interruption of bile acid uptake by hepatocytes after acetaminophen overdose ameliorates hepatotoxicity.

Background & Aims: Acetaminophen (APAP) overdose remains a frequent cause of acute liver failure, which is generally accompanied by increased levels of serum bile acids (BAs). However, the pathophysiological role of BAs remains elusive. Herein, we investigated the role of BAs in APAP-induced hepatotoxicity.

Pharmacological Activation of Nrf2 Enhances Functional Liver Regeneration.

Background And Aims: The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates an array of cytoprotective genes, yet studies in transgenic mice have led to conflicting reports on its role in liver regeneration. We aimed to test the hypothesis that pharmacological activation of Nrf2 would enhance liver regeneration.

Approach And Results: Wild-type and Nrf2 null mice were administered bardoxolone methyl (CDDO-Me), a potent activator of Nrf2 that has entered clinical development, and then subjected to two-thirds partial hepatectomy.

Managing the challenge of drug-induced liver injury: a roadmap for the development and deployment of preclinical predictive models.

Drug-induced liver injury (DILI) is a patient-specific, temporal, multifactorial pathophysiological process that cannot yet be recapitulated in a single in vitro model. Current preclinical testing regimes for the detection of human DILI thus remain inadequate. A systematic and concerted research effort is required to address the deficiencies in current models and to present a defined approach towards the development of new or adapted model systems for DILI prediction.

SFX-01 reduces residual disability after experimental autoimmune encephalomyelitis.

Background: Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is a master transcriptional regulator of the protective cellular response to oxidative stress. Sulforaphane is a Nrf2 activator but is unstable at ambient temperature. SFX-01 is a novel composition comprised of synthetic sulforaphane stabilised within the pocket of an α-cyclodextrin complex.

Attenuation of doxorubicin-induced cardiotoxicity in a human in vitro cardiac model by the induction of the NRF-2 pathway.

Dose-dependent cardiotoxicity is the leading adverse reaction seen in cancer patients treated with doxorubicin. Currently, dexrazoxane is the only approved drug that can partially protect against this toxicity in patients, however, its administration is restricted to those patients receiving a high cumulative dose of anthracyclines. Investigations into the mechanisms of cardiotoxicity and efforts to improve cardioprotective strategies have been hindered by the limited availability of a phenotypically relevant in vitro adult human cardiac model system.

Correction: The Nrf2 inhibitor brusatol is a potent antitumour agent in an orthotopic mouse model of colorectal cancer.

[This corrects the article DOI: 10.18632/oncotarget.25497.

Characterisation of the NRF2 transcriptional network and its response to chemical insult in primary human hepatocytes: implications for prediction of drug-induced liver injury.

The transcription factor NRF2, governed by its repressor KEAP1, protects cells against oxidative stress. There is interest in modelling the NRF2 response to improve the prediction of clinical toxicities such as drug-induced liver injury (DILI). However, very little is known about the makeup of the NRF2 transcriptional network and its response to chemical perturbation in primary human hepatocytes (PHH), which are often used as a translational model for investigating DILI.

The Nrf2 inhibitor brusatol is a potent antitumour agent in an orthotopic mouse model of colorectal cancer.

Nrf2 is a transcription factor that regulates cellular stress response and irinotecan-metabolising pathways. Its aberrant activity has been reported in a number of cancers, although relatively few studies have explored a role for Nrf2 in colorectal cancer (CRC). This study assessed the expression of Nrf2 in patient CRC tissues and explored the effect of Nrf2 modulation alone, or in combination with irinotecan, in human (HCT116) and murine (CT26) cell lines and in an orthotopic syngeneic mouse model utilising bioluminescent imaging.

NRF2 regulates the glutamine transporter Slc38a3 (SNAT3) in kidney in response to metabolic acidosis.

Expression of the glutamine transporter SNAT3 increases in kidney during metabolic acidosis, suggesting a role during ammoniagenesis. Microarray analysis of Nrf2 knock-out (KO) mouse kidney identified Snat3 as the most significantly down-regulated transcript compared to wild-type (WT). We hypothesized that in the absence of NRF2 the kidney would be unable to induce SNAT3 under conditions of metabolic acidosis and therefore reduce the availability of glutamine for ammoniagenesis.

Real-time in vivo imaging reveals localised Nrf2 stress responses associated with direct and metabolism-dependent drug toxicity.

The transcription factor Nrf2 coordinates an adaptive response to chemical and oxidative stress characterised by the upregulated expression of cytoprotective target genes. In order to understand the mechanistic relevance of Nrf2 as a marker of drug-induced stress it is important to know if this adaptive response is truly localised in the context of organ-specific drug toxicity. Here, we address this knowledge gap through real-time bioluminescence imaging of transgenic Nrf2-luciferase (Nrf2-luc) reporter mice following administration of the metabolism-dependent hepatotoxin acetaminophen (APAP) or the direct nephrotoxin cisplatin.

A tetraoxane-based antimalarial drug candidate that overcomes PfK13-C580Y dependent artemisinin resistance.

K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P.

Characterization of Drug-Specific Signaling Between Primary Human Hepatocytes and Immune Cells.

It is now apparent that antigen-specific T-cells are activated in certain patients with drug-induced liver injury (DILI). Since cross-talk between hepatocytes and immune cells is likely to be critical in determining the outcome of drug exposure, the aim of this study was to profile the signals released by drug-treated hepatocytes and to characterize the impact of these molecules on dendritic cells. Human hepatocytes were exposed to 3 drugs (flucloxacillin, amoxicillin, and isoniazid) associated with DILI potentially mediated by the adaptive immune system as drug-specific T-cells have been isolated from DILI patients, and the metabolite nitroso-sulfamethoxazole (SMX-NO).

Circulating levels of miR-122 increase post-mortem, particularly following lethal dosing with pentobarbital sodium: implications for pre-clinical liver injury studies.

microRNA-122 (miR-122) is increasingly being measured in pre-clinical and clinical settings due to greater sensitivity and hepatic specificity compared to the gold standard liver injury biomarker alanine aminotransferase (ALT). In pre-clinical studies, various culling methods can be employed prior to collection of blood samples, including lethal injection with pentobarbital sodium (Pentoject). However, little is known about whether such an approach could alter the circulating levels of miR-122 and compromise the interpretation of data.

Design and Synthesis of Irreversible Analogues of Bardoxolone Methyl for the Identification of Pharmacologically Relevant Targets and Interaction Sites.

Semisynthetic triterpenoids such as bardoxolone methyl (methyl-2-cyano 3,12-dioxooleano-1,9-dien-28-oate; CDDO-Me) (4) are potent inducers of antioxidant and anti-inflammatory signaling pathways, including those regulated by the transcription factor Nrf2. However, the reversible nature of the interaction between triterpenoids and thiols has hindered attempts to identify pharmacologically relevant targets and characterize the sites of interaction. Here, we report a shortened synthesis and SAR profiling of 4, enabling the design of analogues that react irreversibly with model thiols, as well as the model protein glutathione S-transferase P1, in vitro.

Value of monitoring Nrf2 activity for the detection of chemical and oxidative stress.

Beyond specific limits of exposure, chemical entities can provoke deleterious effects in mammalian cells via direct interaction with critical macromolecules or by stimulating the accumulation of reactive oxygen species (ROS). In particular, these chemical and oxidative stresses can underpin adverse reactions to therapeutic drugs, which pose an unnecessary burden in the clinic and pharmaceutical industry. Novel pre-clinical testing strategies are required to identify, at an earlier stage in the development pathway, chemicals and drugs that are likely to provoke toxicity in humans.

Integrated transcriptomic and proteomic analyses uncover regulatory roles of Nrf2 in the kidney.

The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 h, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared with those of wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared with those of vehicle control.

Brusatol provokes a rapid and transient inhibition of Nrf2 signaling and sensitizes mammalian cells to chemical toxicity-implications for therapeutic targeting of Nrf2.

The transcription factor Nrf2 regulates the basal and inducible expression of a battery of cytoprotective genes. Whereas numerous Nrf2-inducing small molecules have been reported, very few chemical inhibitors of Nrf2 have been identified to date. The quassinoid brusatol has recently been shown to inhibit Nrf2 and ameliorate chemoresistance in vitro and in vivo.

Chemical tuning enhances both potency toward nrf2 and in vitro therapeutic index of triterpenoids.

The transcription factor Nrf2 protects against a number of experimental pathologies, and is a promising therapeutic target. The clinical investigation of a potent Nrf2-inducing agent, the triterpenoid (TP) bardoxolone methyl (BARD), was recently halted due to adverse cardiovascular events in chronic kidney disease patients, although the underlying mechanisms are yet to be resolved. The majority of small molecule Nrf2 inducers are electrophilic and trigger Nrf2 accumulation via the chemical modification of its redox-sensitive repressor Keap1.