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Non-alcoholic fatty liver disease and steatohepatitis are highly associated with obesity and type 2 diabetes mellitus. Cotadutide, a glucagon-like protein-1 receptor (GLP-1R) and glucagon receptor (GCGR) agonist, was shown to reduce blood glycaemia, body weight and hepatic steatosis in people with type 2 diabetes mellitus. Here, we demonstrate that the effects of cotadutide in reducing body weight and food intake and improving glucose control are predominantly mediated through Glp-1 signalling, whereas its action on the liver to reduce lipid content, drive glycogen flux and improve mitochondrial turnover and function are directly mediated through Gcg signalling. This was confirmed by the identification of phosphorylation sites on key lipogenic and glucose metabolism enzymes in liver of mice treated with cotadutide. Complementary metabolomic and transcriptomic analyses implicated lipogenic, fibrotic and inflammatory pathways, consistent with a unique therapeutic contribution of GCGR agonism by cotadutide in vivo. Notably, cotadutide also alleviated fibrosis to a greater extent than liraglutide or obeticholic acid, despite dose adjustment to achieve similar weight loss in two preclinical mouse models of NASH. Thus, cotadutide, via direct hepatic (GcgR) and extrahepatic (Glp-1R) effects, exerts multifactorial improvement in liver function and is a promising therapeutic option for the treatment of steatohepatitis.

Mice xenotransplanted with human cells and/or expressing human gene products (also known as “humanized mice”) recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. These models can provide a relevant in vivo context for understanding of human‐specific physiology and pathologies. Humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. Here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. As the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. Therefore, an international community‐driven resource called “Minimal Information for Standardization of Humanized Mice” (MISHUM) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. Within MISHUM, we propose comprehensive guidelines for reporting critical information generated using humanized mice., Humanized mice are at the forefront of biomedical research and becoming more mainstream preclinical models. This comprehensive review talks about innovations and challenges regarding the reconstitution of human immunity and introduces “Minimal Information for Standardization of Humanized Mice” (MISHUM).

AIM To comprehensively evaluate mitochondrial (dys) function in preclinical models of nonalcoholic steatohepatitis (NASH). METHODS We utilized two readily available mouse models of nonalcoholic fatty liver disease (NAFLD) with or without progressive fibrosis: Lepob/Lepob (ob/ob) and FATZO mice on high trans-fat, high fructose and high cholesterol (AMLN) diet. Presence of NASH was assessed using immunohistochemical and pathological techniques, and gene expression profiling. Morphological features of mitochondria were assessed via transmission electron microscopy and immunofluorescence, and function was assessed by measuring oxidative capacity in primary hepatocytes, and respiratory control and proton leak in isolated mitochondria. Oxidative stress was measured by assessing activity and/or expression levels of Nrf1, Sod1, Sod2, catalase and 8-OHdG. RESULTS When challenged with AMLN diet for 12 wk, ob/ob and FATZO mice developed steatohepatitis in the presence of obesity and hyperinsulinemia. NASH development was associated with hepatic mitochondrial abnormalities, similar to those previously observed in humans, including mitochondrial accumulation and increased proton leak. AMLN diet also resulted in increased numbers of fragmented mitochondria in both strains of mice. Despite similar mitochondrial phenotypes, we found that ob/ob mice developed more advanced hepatic fibrosis. Activity of superoxide dismutase (SOD) was increased in ob/ob AMLN mice, whereas FATZO mice displayed increased catalase activity, irrespective of diet. Furthermore, 8-OHdG, a marker of oxidative DNA damage, was significantly increased in ob/ob AMLN mice compared to FATZO AMLN mice. Therefore, antioxidant capacity reflected as the ratio of catalase:SOD activity was similar between FATZO and C57BL6J control mice, but significantly perturbed in ob/ob mice. CONCLUSION Oxidative stress, and/or the capacity to compensate for increased oxidative stress, in the setting of mitochondrial dysfunction, is a key factor for development of hepatic injury and fibrosis in these mouse models.

Objective Nonalcoholic steatohepatitis (NASH) is an unmet need associated with metabolic syndrome. There are no approved therapies for NASH; however, glucagon-like peptide-1 receptor (GLP-1R) and farnesoid-X receptor (FXR) agonists are promising drug targets. We investigated the therapeutic effects of co-administration of a GLP-1R agonist, IP118, with FXR agonist obeticholic acid (OCA) in mice. Methods OCA and IP118 alone and in combination were sub-chronically administered to Lepob/Lepob mice with diet-induced NASH or diet-induced obese (DIO) mice. Metabolic (body weight and glucose) and liver (biochemical and histological) endpoints were assessed. NASH severity in Lepob/Lepob mice was graded using a customized integrated scoring system. Results OCA reduced liver weight and lipid in NASH mice (both by −17%) but had no effect on plasma ALT or AST levels. In contrast, IP118 significantly reduced liver weight (−21%), liver lipid (−15%), ALT (−29%), and AST (−27%). The combination of OCA + IP118 further reduced liver weight (−29%), liver lipid (−22%), ALT (−39%), and AST (−36%). Combination therapy was superior to monotherapies in reducing hepatic steatosis, inflammation, and fibrosis. Hepatic improvements with IP118 and OCA + IP118 were associated with reduced body weight (−4.3% and −3.5% respectively) and improved glycemic control in OCA + IP118-treated mice. In DIO mice, OCA + IP118 co-administration reduced body weight (−25.3%) to a greater degree than IP118 alone (−12.5%) and further improved glucose tolerance and reduced hepatic lipid. Conclusion Our data suggest a complementary or synergistic therapeutic effect of GLP-1R and FXR agonism in mouse models of metabolic disease and NASH., Highlights • Agonists for FXR (e.g., OCA) and GLP-1R are leading clinical candidates for NASH. • First preclinical evidence for OCA alone or added to GLP-1R agonist IP118 on NASH and metabolism. • OCA + IP118 exerted greater reductions in key morphologic features of NASH. • OCA + IP118 synergistically reduced body weight in DIO mice. • FXR and GLP-1 co-activation may be therapeutically beneficial.

Nonalcoholic steatohepatitis (NASH), characterized by hepatic steatosis, inflammation and fibrosis is an unmet medical need. MEDI0382, a balanced GLP-1/glucagon dual receptor agonist, is under development for the treatment of T2DM. Here we examined the effects of MEDI0382 on NASH compared to liraglutide, a GLP-1 analog. Leptin-deficient ob/ob mice were maintained on high trans-fat, fructose and cholesterol diet for 8 weeks to induce NASH then randomized to four treatment groups: vehicle, MEDI0382 (30 nmol/kg), liraglutide (30 nmol/kg) or vehicle-treated and switched to low-fat diet (LFD). Treatment with MEDI0382 and liraglutide reduced body weight and improved glucose tolerance. Hepatic lipid was reduced by 40% with MEDI0382 treatment (p In conclusion, MEDI0382 exerted similar metabolic control relative to liraglutide, but exhibited superior effects on primary NASH endpoints. Disclosure M. Beaton: Employee; Self; MedImmune. Employee; Spouse/Partner; MedImmune. S. Guionaud: None. J.P. Conway: None. J. Grimsby: Employee; Self; AstraZeneca. C.J. Rhodes: Stock/Shareholder; Self; AstraZeneca. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. J. Trevaskis: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca.

Substances historically thought to cause direct vascular injury in laboratory animals are a heterogeneous group of toxic agents with varied mechanisms of action. Morphologically, the reviewed agents can be broadly categorized into those targeting endothelial cell (ECs) and those targeting smooth muscle cells (SMCs). Anticancer drugs, immunosuppressants, and heavy metals are targeting primarily ECs while allylamine, β-aminopropionitrile, and mitogen-activated protein kinase kinase inhibitors affect mainly SMCs. It is now recognized that the pathogenicity of some of these agents is often mediated through intermediary events, particularly vasoconstriction. There are clear similarities in the clinical and microscopic findings associated with many of these agents in animals and man, allowing the use of animal models to investigate mechanisms and pathogenesis. The molecular pathogenic mechanisms and comparative morphology in animals and humans will be reviewed.

This chapter gives an overview of the embryology of the heart and blood vasculature, a description of the commonly encountered spontaneous lesions seen in Wistar, Sprague-Dawley, and other rat strains observed in routine safety assessment studies (including carcinogenicity studies) and concludes with concise descriptions of classic examples of cardiotoxic compounds. There is a summary of useful biomarkers of cardiac toxicity in general use.

Better biomarkers are needed to identify, characterize, and/or monitor drug-induced vascular injury (DIVI) in nonclinical species and patients. The Predictive Safety Testing Consortium (PSTC), a precompetitive collaboration of pharmaceutical companies and the U.S. Food and Drug Administration (FDA), formed the Vascular Injury Working Group (VIWG) to develop and qualify translatable biomarkers of DIVI. The VIWG focused its research on acute DIVI because early detection for clinical and nonclinical safety monitoring is desirable. The VIWG developed a strategy based on the premise that biomarkers of DIVI in rat would be translatable to humans due to the morphologic similarity of vascular injury between species regardless of mechanism. The histomorphologic lexicon for DIVI in rat defines degenerative and adaptive findings of the vascular endothelium and smooth muscles, and characterizes inflammatory components. We describe the mechanisms of these changes and their associations with candidate biomarkers for which advanced analytical method validation was completed. Further development is recommended for circulating microRNAs, endothelial microparticles, and imaging techniques. Recommendations for sample collection and processing, analytical methods, and confirmation of target localization using immunohistochemistry and in situ hybridization are described. The methods described are anticipated to aid in the identification and qualification of translational biomarkers for DIVI.

Diabetic nephropathy (DN) remains an unmet medical challenge as its prevalence is projected to continue to increase and specific medicines for treatment remain undeveloped. Activation of the immune system, in particular T-cells, is emerging as a possible mechanism underlying DN disease progression in humans and animal models. We hypothesized that inhibition of T-cell activation will ameliorate DN. Interaction of B7-1 (CD80) on the surface of antigen presenting cells with its binding partners, CTLA4 (CD152) and CD28 on T-cells, is essential for T-cell activation. In this study we used the soluble CTLA4-Fc fusion protein Abatacept to block cell surface B7-1, preventing the cellular interaction and inhibiting T-cell activation. When Abatacept was dosed in an animal model of diabetes-induced albuminuria, it reduced albuminuria in both prevention and intervention modes. The number of T-cells infiltrating the kidneys of DN animals correlated with the degree of albuminuria, and treatment with Abatacept reduced the number of renal T-cells. As B7-1 induction has been recently proposed to underlie podocyte damage in DN, Abatacept could be efficacious in DN by protecting podocytes. However, this does not appear to be the case as B7-1 was not expressed in 1) kidneys of DN animals; 2) stimulated human podocytes in culture; or 3) glomeruli of DN patients. We conclude that Abatacept ameliorates DN by blocking systemic T-cell activation and not by interacting with podocytes.

Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. In recent years, DIVI has been occasionally observed in nonhuman primates given RNA-targeting therapeutics such as antisense oligonucleotide therapies (ASOs) during chronic toxicity studies. While DIVI in laboratory animal species has been well characterized for vasoactive small molecules, and immune-mediated responses against large molecule biotherapeutics have been well described, there is little published information regarding DIVI induced by ASOs to date. Preclinical DIVI findings in monkeys have caused considerable delays in development of promising new ASO therapies, because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans, and the lack of robust biomarkers of DIVI. This review of DIVI discusses clinical and microscopic features of vasculitis in monkeys, their pathogenic mechanisms, and points to consider for the toxicologist and pathologist when confronted with ASO-related DIVI. Relevant examples of regulatory feedback are included to provide insight into risk assessment of ASO therapies.

Drug-induced vascular injury (DIVI) is a common preclinical toxicity usually characterized by hemorrhage, vascular endothelial and smooth muscle damage, and inflammation. DIVI findings can cause delays or termination of drug candidates due to low safety margins. The situation is complicated by the absence of sensitive, noninvasive biomarkers for monitoring vascular injury and the uncertain relevance to humans. The Safer And Faster Evidence-based Translation (SAFE-T) consortium is a public–private partnership funded within the European Commission’s Innovative Medicines Initiative (IMI) aiming to accelerate drug development by qualifying biomarkers for drug-induced organ injuries, including DIVI. The group is using patients with vascular diseases that have key histomorphologic features (endothelial damage, smooth muscle damage, and inflammation) in common with those observed in DIVI, and has selected candidate biomarkers associated with these features. Studied populations include healthy volunteers, patients with spontaneous vasculitides and other vascular disorders. Initial results from studies with healthy volunteers and patients with vasculitides show that a panel of biomarkers can successfully discriminate the population groups. The SAFE-T group plans to seek endorsement from health authorities (European Medicines Agency and Food and Drug Administration) to qualify the biomarkers for use in regulatory decision-making processes.

BACKGROUND Scavenger receptor class B type-1 (SR-B1) is one of the many receptors used by HCV to infect hepatocytes. It is used by the virus not only to directly infect cells but also to facilitate cell-to-cell transmission of the virus. Agents such as anti-human SR-B1 (anti-hSR-B1) antibodies represent potential therapeutics for the treatment of HCV infections. The purpose of this study was to evaluate the safety and pharmacokinetics of an anti-hSR-B1 antibody (Seq2) in cynomolgus monkeys. METHODS The antibody was administered intravenously at 1, 10 and 100 mg/kg body weight; blood samples were taken pre- and post-dose to evaluate the pharmacokinetic profile and blood chemistry. Safety was assessed during treatment and the animals were sacrificed post-treatment for pathological assessment and liver antibody-receptor occupancy determination. RESULTS Following administration of Seq2 antibody to cynomolgus monkeys a non-linear pharmacokinetic profile was observed. The clearance of the antibody decreased from 2 to 0.3 ml/h/kg with increasing doses from 1 to 100 mg/kg, respectively, and the antibody half-life varied from 62 to 218 h for the same doses. An increase in total cholesterol and high-density lipoprotein cholesterol levels after antibody administration was observed, with a good correlation between liver receptor occupancy and dose. CONCLUSIONS The pharmacokinetics and toxicology results were in accordance with the pharmacology of the antibody mechanism. The elevation of total cholesterol was dose-dependent and did not exceed a twofold increase. The safety study indicated no adverse effects during the treatment or in the pathology analysis at any of the doses tested.