Novel Rho associated coiled kinase 2 (ROCK2) inhibitor reduces steatosis and fibrosis in mice model of liver disease

Abstract

Background and AimsRho‐associated coiled kinases 1 (ROCK1) and 2 (ROCK2) are involved in variety of cellular processes including tissue fibrosis. Recently, we demonstrated that hepatic ROCK2 and not ROCK1 is upregulated in hepatic stellate cells in mice model of non‐alcoholic fatty liver disease. The present study was done to investigate if selective blocking of ROCK2 reduces hepatic steatosis, fibrosis and improves liver function.MethodUsing core hopping and structure based drug design approach, we synthesized highly selective (>200 fold selective for ROCK2 vs ROCK1), orally bioavailable ROCK2 inhibitor (ANG4201). We evaluated the antisteatotic and antifibrotic effects of ANG4201 in mice model (C57Bl/6) of NASH. Adult male mice were subjected to high fatty diet (FFD) for three months and CCl4 (0.2 μl (0.3μg/g)) of body wt (once a week, ip)) was administered to accelerate the progression of liver fibrosis. After three months, mice were randomized to vehicle or ANG4201 (10, 25 & 50 mg/kg body wt, PO, BID) for two and half months. Liver injury was analysed by measuring serum transaminases (AST/ALT). RNA was isolated from livers and panel of fibrotic and inflammatory markers were assessed by RT‐PCR. H&E staining was done to analyse NASH and scoring was done to determine NAFLD activity.ResultsCompared to control, ANG4201 treatment dose dependently reduced TAA+FFD induced liver fibrosis in mice, demonstrated by significant reduction in hepatic hydroxyproline as well as alpha‐SMA, collagen I mRNA expression. The reduction in fibrosis was associated with significant improvement in the ratio of liver wt and body wt and reduction in serum transaminases (AST/ALT) and cholesterol. ANG4201 treatment overall reduced NAFLD activity score. ANG4201 also reduced TGF‐β induced gel contraction in hepatic stellate cells which was also associated with significant decrease in connective tissue growth factor (CTGF) mRNA, a marker elevated in fibrotic condition.ConclusionThis study demonstrates that ROCK2 contributes to hepatic fibrosis and hepatic dysfunction in FFD+ CCl4 mice model. ANG4201 is a highly promising orally bioavailable therapeutic ROCK2 inhibitor with potential use in liver fibrosis.Support or Funding Information 1 R43 DK121642 01