P068 Anti-fibrotic therapy for Crohn’s disease: in-vitro proof of concept with a selective ROCK2 inhibitor, LYC-53976, in the treatment of human intestinal myofibroblasts in stiffness and transforming growth factor β models
E. Rodansky1, X. Liu2, L. A. Johnson1, K. Demock2, A. J. Celeste2, L. L. Carter2, P. D. R. Higgins*1
1University of Michigan, Internal Medicine - Gastroenterology, Ann Arbor, Michigan, United States, 2Lycera, Inc., Ann Arbor, Michigan, United States
Intestinal fibrosis is the critical final pathway of intestinal failure in Crohn’s disease (CD). Many medications are available to treat inflammation in CD, but no medical therapies exist to treat intestinal fibrosis. The Rho pathway has been implicated in fibrosis in several organ systems. Blocking fibrogenic signals at the convergence point of Rho kinase (ROCK) offers an attractive anti-fibrotic target. However, previous inhibitors of ROCK1 and ROCK2 kinases are limited by known off-target effects and are not clinically used for anti-fibrotic indications. We aimed to develop and test selective ROCK inhibitors with potent anti-fibrotic effects in 2 in-vitro models of CD.
Enzymatic and cell-based assays were used to determine the ROCK selectivity of newly developed compounds, and then candidates were screened for drug-like properties. Promising compounds were tested for their ability to reverse the transforming growth factor beta (TGF-β)-driven induction of a profibrotic phenotype in CCD18co human intestinal myofibroblasts. Successful compounds were then tested for their ability to reverse the pathological stiffness-driven induction of a profibrotic phenotype in CCD18co human intestinal myofibroblasts. Efficacy in each in-vitro model was evaluated with RT-polymerase chain reaction (PCR) expression of profibrotic genes collagen 1 (COL1A1), alpha smooth muscle actin (ACTA2), fibronectin (FN1), and MYLK. Promising compounds were also evaluated on an extended human fibrotic gene PCR array.
A representative compound, LYC-53976, was identified and found to be ROCK2 selective, with potency against ROCK2 at 385 nM and no activity against ROCK1 at the highest concentration tested (10 μM) in the cell-based assay. In the TGF-β-driven profibrotic model, 3 μM LYC-53976 abrogated induction of COL1A1, FN1, and ACTA2 gene expression by RT-PCR, as well as αSMA protein expression. In the pathological stiffness-driven fibrosis induction model, 3 μM LYC-53976 also abrogated induction of COL1A1 and MYLK genes. LYC-53976 was also effective in modulating multiple fibrotic genes in the TGF-β-stimulated LL-29 human fibroblast superarray by 2 to 4 fold.
A ROCK2 selective small molecule inhibitor can repress the expression of multiple profibrotic genes in 2 in-vitro models of intestinal fibrogenesis, suggesting the utility of ROCK2 inhibition as an anti-fibrotic therapy for CD strictures.