Inhibition of Liver Fibrosis by Oxysterol Drug Candidates in a Mouse Model of NASH

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 due to various pathologies including non-alcoholic fatty liver disease (NAFLD), non-alcoholic liver steatohepatitis (NASH), viral infection, and liver cancer. A number of molecular mechanisms have been scrutinized in an effort to identify the root causes of liver fibrosis, often overlapping with the study of fibrosis in other organs. Among these, prominent studies point to aberrant Hedgehog (Hh) signaling and inappropriate activation of TGFβ signaling as prime factors underlying the pathogenesis of liver fibrosis, and hence extensively investigated. 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 TGFβ signaling in fibroblastic cells and in primary human liver stellate cells (HLS). Activated HLS are considered to be the main mediators of liver fibrosis and a number of studies have shown that their activation results in abundant extracellular matrix formation and fibrosis. Therefore, interference with HLS activation may halt and reverse fibrotic processes in the liver. Based on preliminary in vitro studies and in vivo safety and pharmacokinetic (PK) studies, we identified orally bioavailable Oxy210 as an oxysterol with potent Hh and TGFβ pathway inhibitory activity when applied to NIH3T3 and HLS cells, and Oxy186 as a potent Hh pathway inhibitor without any effect on TGFβ signaling. Oxy210 inhibits the expression of profibrogenic genes induced by TGFβ in NIH3T3 fibroblasts and their expression in activated HLS. We propose that oxysterols might serve as ideal drug candidates for development into orally bioavailable anti-fibrosis agents. Our goal is to examine the efficacy of Oxy210 and Oxy186 in inhibiting liver fibrosis and to further identify the relative roles of TGFβ and Hh signaling in pathogenesis of the disease. We propose to address the following Specific Aims in the present Phase 1 SBIR application: Aim1. Optimization of oral delivery of Oxy210 and Oxy186 to mice when mixed in food, compared to oral gavage for long term studies of liver fibrosis; Aim2. Examination of the effect of Oxy210 and Oxy186 on liver fibrosis in vivo in “humanized” hyperlipidemic mice; Aim 3. Examination of TGFβ and Hh signaling and the effect of Oxy210 and Oxy186 on these signaling pathways during development of liver fibrosis in “humanized” hyperlipidemic mice.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 TGFβ and/or Hedgehog signaling in fibroblasts and in human liver stellate cells. Two lead compounds, Oxy210 and Oxy186, are promising drug candidates that may be useful for intervention in liver fibrosis. In proposed studies in this application we will provide further support for the candidacy of these oxysterols for development into potentially potent orally available anti-fibrosis compounds.