Selective disruption of NRF2-KEAP1 interaction leads to NASH resolution and reduction of liver fibrosis in mice.

Background & Aims: Oxidative stress is recognized as a major driver of non-alcoholic steatohepatitis (NASH) progression. The transcription factor NRF2 and its negative regulator KEAP1 are master regulators of redox, metabolic and protein homeostasis, as well as detoxification, and thus appear to be attractive targets for the treatment of NASH. Methods: Molecular modeling and X-ray crystallography were used to design S217879 - a small molecule that could disrupt the KEAP1-NRF2 interaction. S217879 was highly characterized using various molecular and cellular assays. It was then evaluated in two different NASH-relevant preclinical models, namely the methionine and choline-deficient diet (MCDD) and diet-induced obesity NASH (DIO NASH) models. Results: Molecular and cell-based assays confirmed that S217879 is a highly potent and selective NRF2 activator with marked anti-inflammatory properties, as shown in primary human peripheral blood mononuclear cells. In MCDD mice, S217879 treatment for 2 weeks led to a dose-dependent reduction in NAFLD activity score while significantly increasing liver Nqo1 mRNA levels, a specific NRF2 target engagement biomarker. In DIO NASH mice, S217879 treatment resulted in a significant improvement of established liver injury, with a clear reduction in both NAS and liver fibrosis. αSMA and Col1A1 staining, as well as quantification of liver hydroxyproline levels, confirmed the reduction in liver fibrosis in response to S217879. RNA-sequencing analyses revealed major alterations in the liver transcriptome in response to S217879, with activation of NRF2-dependent gene transcription and marked inhibition of key signaling pathways that drive disease progression. Conclusions: These results highlight the potential of selective disruption of the NRF2-KEAP1 interaction for the treatment of NASH and liver fibrosis. Impact and implications: We report the discovery of S217879 - a potent and selective NRF2 activator with good pharmacokinetic properties. By disrupting the KEAP1-NRF2 interaction, S217879 triggers the upregulation of the antioxidant response and the coordinated regulation of a wide spectrum of genes involved in NASH disease progression, leading ultimately to the reduction of both NASH and liver fibrosis progression in mice.

Epigenetic Silencing of RIPK3 in Hepatocytes Prevents MLKL-mediated Necroptosis From Contributing to Liver Pathologies.

BACKGROUND & AIMS: Necroptosis is a highly inflammatory mode of cell death that has been implicated in causing hepatic injury including steatohepatitis/ nonalcoholic steatohepatitis (NASH); however, the evidence supporting these claims has been controversial. A comprehensive, fundamental understanding of cell death pathways involved in liver disease critically underpins rational strategies for therapeutic intervention. We sought to define the role and relevance of necroptosis in liver pathology. METHODS: Several animal models of human liver pathology, including diet-induced steatohepatitis in male mice and diverse infections in both male and female mice, were used to dissect the relevance of necroptosis in liver pathobiology. We applied necroptotic stimuli to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analyzed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes. RESULTS: Identical infection kinetics and pathologic outcomes were observed in mice deficient in an essential necroptotic effector protein, MLKL, compared with control animals. Mice lacking MLKL were indistinguishable from wild-type mice when fed a high-fat diet to induce NASH. Under all conditions tested, we were unable to induce necroptosis in hepatocytes. We confirmed that a critical activator of necroptosis, RIPK3, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis. CONCLUSIONS: We have provided compelling evidence that necroptosis is disabled in hepatocytes during homeostasis and in the pathologic conditions tested in this study.

Characterization and Pharmacological Validation of a Preclinical Model of NASH in Göttingen Minipigs.

Background: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, which is associated with features of metabolic syndrome. NAFLD may progress in a subset of patients into nonalcoholic steatohepatitis (NASH) with liver injury resulting ultimately in cirrhosis and potentially hepatocellular carcinoma. Today, there is no approved treatment for NASH due to, at least in part, the lack of preclinical models recapitulating features of human disease. Here, we report the development of a dietary model of NASH in the Göttingen minipig. Methods: First, we performed a longitudinal characterization of diet-induced NASH and fibrosis using biochemical, histological, and transcriptional analyses. We then evaluated the pharmacological response to Obeticholic acid (OCA) treatment for 8 weeks at 2.5mg/kg/d, a dose matching its active clinical exposure. Results: Serial histological examinations revealed a rapid installation of NASH driven by massive steatosis and inflammation, including evidence of ballooning. Furthermore, we found the progressive development of both perisinusoidal and portal fibrosis reaching fibrotic septa after 6 months of diet. Histological changes were mechanistically supported by well-defined gene signatures identified by RNA Seq analysis. While treatment with OCA was well tolerated throughout the study, it did not improve liver dysfunction nor NASH progression. By contrast, OCA treatment resulted in a significant reduction in diet-induced fibrosis in this model. Conclusions: These results, taken together, indicate that the diet-induced NASH in the Göttingen minipig recapitulates most of the features of human NASH and may be a model with improved translational value to prioritize drug candidates toward clinical development.

Procollagen C-Proteinase Enhancer-1 (PCPE-1) deficiency in mice reduces liver fibrosis but not NASH progression.

BACKGROUND AND AIMS: Nonalcoholic Steatohepatitis (NASH) is a major cause of end-stage liver diseases such as cirrhosis and hepatocellular carcinoma resulting ultimately in increased liver-related mortality. Fibrosis is the main driver of mortality in NASH. Procollagen C-Proteinase Enhancer-1 (PCPE-1) plays a key role in procollagen maturation and collagen fibril formation. To assess its role in liver fibrosis and NASH progression, knock-out mice were evaluated in a dietary NASH model. METHODS: Global constitutive Pcolce-/- and WT male mice were fed with a Choline Deficient Amino acid defined High Fat Diet (CDA HFD) for 8 weeks. Liver triglycerides, steatosis, inflammation and fibrosis were assessed at histological, biochemical and gene expression levels. In addition, human liver samples from control and NASH patients were used to evaluate the expression of PCPE-1 at both mRNA and protein levels. RESULTS: Pcolce gene deficiency prevented diet-induced liver enlargement but not liver dysfunction. Furthermore, liver triglycerides, steatosis and inflammation were not modified in Pcolce-/- male mice compared to WT under CDA HFD. However, a significant decrease in liver fibrosis was observed in Pcolce-/- mice compared to WT under NASH diet, associated with a decrease in total and insoluble collagen content without any significant modifications in the expression of genes involved in fibrosis and extracellular matrix remodeling. Finally, PCPE-1 protein expression was increased in cirrhotic liver samples from both NASH and Hepatitis C patients. CONCLUSIONS: Pcolce deficiency limits fibrosis but not NASH progression in CDA HFD fed mice.

Beneficial effect of a chronic treatment with rimonabant on pancreatic function and beta-cell morphology in Zucker Fatty rats.

Recent studies suggested the involvement of the endocannabinoid pathway on insulin secretion in RINm5F cells or rat islets. Animal and clinical studies have reported beneficial effects of the selective cannabinoid 1 receptor antagonist rimonabant on glucose homeostasis. The aim of this study was to investigate the in vivo effects of rimonabant on pancreatic function in Zucker Fatty rats. Zucker Fatty rats were treated with rimonabant (10 mg kg(-1) day(-1)) or vehicle for up to 3 months. Pancreatic function was assessed by oral glucose tolerance test and by static incubation of islets in the presence of different glucose concentrations. Islet morphology was assessed by immuno-histochemistry on pancreatic sections. After 3 months, there was no difference in fasting glycaemia or AUC(glucose) during oral glucose tolerance test between rimonabant- and vehicle-treated animals. However, vehicle-treated rats developed a marked hyperinsulinaemia with time in contrast to rimonabant-treated animals, which maintained at 3 months significantly lower fasting insulin levels (7.76+/-0.67 microg l(-1) vs. 5.59+/-0.59 microg l(-1), P<0.01) and lower AUC(insulin) (1380+/-98 microg l(-1)min vs. 926+/-58 microg l(-1)min, respectively, P<0.001). In static incubation, rimonabant significantly decreased insulin secretion in response to low glucose concentration (3 months: 7.68+/-1.29 vs. 12.25+/-2.01 microg l(-1) 5 islets(-1) 45 min(-1) in rimonabant and vehicle respectively, P<0.01), resulting in a trend to increase stimulation index in the presence of 16.7 mM glucose (10.64+/-0.92 vs. 8.52+/-1.70 respectively). Morphological analysis at 3 months showed that rimonabant reduced islet-cell surface (-60%) and the percentage of disorganized islets (-54%).In conclusion, our data suggest that rimonabant has a protective role against the development of hyperinsulinaemia, beta-cell dysfunction and islet modification in Zucker Fatty rats.

A pain management program for chronic cancer-treatment-related pain: a preliminary study.

UNLABELLED: A large proportion of patients may develop chronic pain following cancer treatments such as surgery, radiotherapy, or chemotherapy. These patients can experience significant levels of physical and psychological morbidity. Our aim was to investigate a cognitive-behavioral pain management program (PMP) for cancer patients with chronic treatment-related pain. Thirteen patients (1 man, 12 women; mean age 52 yrs) completed the study, 9 of whom had a history of breast cancer and had received extensive medical treatment, including surgery. A combination of physical and psychological techniques were adapted from previous work in chronic benign pain and implemented by two therapists. Interventions included education, relaxation, exercise training, and goal setting. A variety of outcomes were examined to assess general fitness, psychological distress, coping success, activities of daily living, and pain report. The median number of interventions by each therapist was 10 (4 to 15). Postintervention, there was a significant trend toward improvement in many variables, including anxiety and depression (P < .01), fitness (walking: P < .05), and coping with pain (P < .01). This was a feasibility study and has several limitations. It appears, however, that all patients had a positive outcome. Further research is now required to assess the effectiveness of this approach. PERSPECTIVE: Results of this preliminary study are clinically relevant, as they suggest that a pain management program that uses cognitive-behavioral principles is worthy of further investigation for patients with chronic cancer-treatment-related pain.