ABSTRACT Pathologic tissue fibrosis, impelled by uncontrolled wound healing responses to acute or chronic injury is a significant problem in many organs including kidney, lung, and liver. Liver fibrosis is a major health problem that causes morbidity and mortality in the affected patient population. Liver fibrosis occurs in various pathologies including chronic alcohol exposure, non-alcoholic fatty liver disease (NAFLD) that can lead to non- alcoholic steatohepatitis (NASH), and viral infection, and can lead to cirrhosis and hepatocellular carcinoma. A number of molecular mechanisms have been scrutinized to identify the root causes of liver fibrosis. Among these, prominent studies point to aberrant Hedgehog (Hh) and transforming growth factor-β (TGF-β) signaling as prime factors underlying the pathogenesis of liver fibrosis, and hence extensively investigated. Accumulating evidence also suggests the role of Toll-Like Receptors (TLRs) in mediating the inflammatory responses of liver cells to endogenous factors and bacterial products present in the liver in NAFLD and NASH. At MAX BioPharma, we have identified semi-synthetic oxysterols that act as Hh pathway modulators, both as agonists and antagonists. We found oxysterol Hh pathway antagonists that also have potent inhibitory effects on TGF-β signaling in fibroblastic cells and in primary human hepatic stellate cells (HSC). With support from an SBIR Phase 1 grant from NIDDK, we recently published findings about the anti-NASH properties of an oxysterol analogue, Oxy210, that has led to the present application. Oxy210 has anti-fibrotic as well as anti- inflammatory properties that are mediated through inhibition of Hh, TGF-β and TLR signaling. Oxy210 induced robust inhibitory effects in a humanized hyperlipidemic mouse model of NASH, ApoE*3-Leiden.CETP mice on a high fat Western Diet, evidenced by inhibition of hepatic lipid deposition, inflammatory cytokine expression and fibrosis, associated with reduced hepatic cell apoptosis and improved circulating ALT levels. Based on these properties of Oxy210, in addition to its favorable pharmacokinetic and safety profiles, oral availability, and scalability, in the present SBIR Phase 2 application we propose to continue the examination of the disease modifying effects of Oxy210 in ApoE*3-Leiden.CETP mice in reversing already established disease in contrast to our published report when Oxy210 was administered at the initiation of the disease. We propose to identify the molecular and cellular mechanisms of anti-NASH properties of Oxy210 by performing metabolic tests as well as RNA-sequencing studies in livers of NASH mice with or without Oxy210 treatment. We propose to perform non-GLP in vitro and in vivo safety studies that will provide essential information for future IND-enabling GLP studies required in our anticipated IND filing for Oxy210 as a first-in-class new drug candidate for targeting NASH. NASH is a huge unmet medical need since there are no FDA approved therapies, leaving a large global population susceptible to severe liver failure and the need for liver transplantation.

RELEVANCE TO PUBLIC HEALTH The proposed studies will facilitate the therapeutic development of a novel class of oxysterol molecules that have been found to inhibit transforming growth factor-β (TGF-β), Hedgehog (Hh) and Toll-Like Receptor (TLR) signaling that are the main drivers of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Our lead compound, Oxy210, inhibits NASH as well as atherosclerosis and inflammation in white fat tissue in a humanized mouse model and has favorable pharmacokinetic and safety profiles. In proposed studies in this application, we will provide further support for the candidacy of Oxy210 for development into potentially potent orally available anti-fibrosis compounds for the treatment of NASH.