RESULTS INTRODUCTION aim METHOD conclusions references Mitochondrial Uncoupler HU6 Reduces Hepatic Oxidative Stress in the DIAMOND™ Mouse Model Thus Abrogating NASH Development. Francisco Portell 3, Stephen Eaton 3, Jameel Dennis 3, Rebecca Caffrey 1, Jonathon Marioneaux 1, Isaac Onyango 3, Omer Khan 3, Shaharyar Khan 3 and Arun Sanyal 2 1. Sanyal biotechnology LLC, Norfolk, VA, United States. 2. Virginia Commonwealth University, Richmond,VA, United States. 3. Gencia INTRODUCTION The Gencia mitochondrial uncoupler HU6 was evaluated for utility in treating NASH in a diet-induced animal model of metabolic syndrome and NASH (DIAMOND™ mice); development of NASH in this model is driven by insulin resistance, oxidative stress, inflammation, and leaky gut induced by Western Diet (WDSW). RESULTS Serum ALT, AST and ALP levels in the HD group were significantly lower than in the VC (P=0.001, 0.0004, and 0.0002, respectively) and PC (P=0.03, 0.01, and 0.00008, respectively) groups. In the HD group the significant reduction in ballooning (P=0.0000001), lobular inflammation and liver transaminotransferase levels is accompanied by a 72% reduction in MCP-1, 37% reduction in IP-10, 50% reduction in MCP-3 and a 43% reduction in MIP-2. Additionally, HU6-treated mice have a near 25% reduction in baseline oxidative damage to nucleic acids. Steatosis percentage and grade (P=0.001 and 0.01, respectively) were reduced in the HD group, which correlated with significantly lower body weights and liver weights compared to the VC and PC groups. Composite histology scores (NAS and SAF activity scores) were also significantly improved in the HD group compared to VC and PC groups. While all VC and PC mice progressed to NASH, HU6 at 5 mg/kg prevented NASH development in all but one mouse. Histology Scores Blood chemistry Vehicle Control Sirius Red Mouse ID 495 – 10X High Dose Sirius Red Mouse ID 619 – 10X Low Dose Sirius Red Mouse ID 619 – 10X Positive Control Sirius Red Mouse ID 574 – 10X Negative Control H&E Mouse ID 699 – 10X Negative Control Sirius Red Mouse ID 699 – 10 Vehicle Control H&E Mouse ID 495 – 10X High Dose H&E Mouse ID 619 – 10X Low Dose H&E Mouse ID 619 – 10X Positive Control H&E Mouse ID 574 – 10X MIP-2 MCP-3 MCP-1 IP-10 DNA/RNA Oxidative damage Chemokine/Cytokine aim The aim of this study was to investigate effects of HU6 on ROS formation and correlate these with disease progression. . METHOD Mice were grouped into 5 groups: vehicle control (VC), high dose HU6 (HD), 5 mg/kg), low dose HU6 (LD, 1 mg/kg), WDSW positive (PC) and NCNW negative (NC) natural history controls. Mice were raised for 12 weeks on diet, corresponding to a baseline NASH F0 in the WDSW groups. Treatment groups were then gavaged once daily with aqueous vehicle or drug in vehicle for 8 weeks. Serum and liver tissue were harvested, snap frozen and fixed, and LFTs, blood lipids and histology on FFPE section stained with H&E and Sirius Red was performed. DNA/RNA oxidative damage was measured via ELISA from representative liver samples. Key chemokines were also assayed from mouse livers utilizing Luminex Bead-based multiplex assays. conclusions With only eight week of dosing low levels of HU6, measures of oxidative stress and chemokines, correlating with decreases in ballooning, inflammation, and progression from simple steatosis to NASH, were significantly reduced. HU6 also produced significant reductions in body and liver weight, correlating with decreased hepatic steatosis. HU6 impacted multiple drivers of NASH in this study, thus supporting the rationale for further study of HU6’s therapeutic potential in NASH. Mansouri A, Gattolliat CH, Asselah T. Mitochondrial Dysfunction and Signaling in Chronic Liver Diseases. Gastroenterology Vol.155, pp 629-647. 2018 references CONTACT INFORMATION Rebecca Caffrey rebecca@sanyalbio.com Francisco Portell frportell@genciabiotech.com