Feng, Y.; Johnson, L. A.; Clasby, M.; Higgins, P.

Background: Intestinal fibrosis leads to intestinal failure in Crohns disease (CD), but we have no effective antifibrotic medical therapies. Axl inhibitors have been studied in the treatment of fibrosis in liver and lung, but not in intestine. The objective of this study was to test whether CCG264341, a novel gut selective Axl inhibitor, can treat intestinal fibrosis in both in vitro and in vivo IBD fibrosis models. Methods: CCD-18Co human intestinal myofibroblasts were co-treated with 100 ng/mL Fas ligand (FASL) and a dose range of CCG264341 for 5 hours, or treated with TGFb for 48 hours, followed by protein isolation and western blot analysis. Tissue PK experiments in mice were performed to select the CCG264341 dose for the in vivo model. For the in vivo model, intestinal fibrosis was induced in CBA/J mice by S. typhimurium infection, Mice were treated with CCG264341 daily during days 4-21 after S. typhimurium infection, followed by sac on day 22. Cecum and proximal colon (affected tissue) was analyzed by gross pathology, histological fibrosis scoring, qRT-PCR, western blot, and immunofluorescence staining. Results: CCG264341 at doses of 0.1/0.3/1.0 M sensitizes CCD-18Co myofibroblasts to FasL-mediated apoptosis over FasL alone, and this effect was dose dependent, per cleaved Parp protein quantitation. With TGF{beta} treatment, Axl protein expression was increased, along with Col1a1, MYLK, SMA and FN1 protein upregulation. CCG264341 could reduce AXL protein and downstream signal p-Akt expression with dose dependence, along with reduced Col1a1, MYLK, SMA and FN1 protein expression after treatment with CCG264341 treatment. The highest CCG264341 concentrations were detected in the terminal ileum after 5 hours oral feeding in PK experiments, and much lower compound concentrations could still be detected in other organs. A dose of 25mg/kg daily was selected for the mouse intestinal fibrosis treatment model. In the murine S. typhimurium model, AXL protein and downstream signals p-Akt, p-Erk and P-Stat3 expression were all increased in colon and cecum, with the expression of the fibrosis related proteins upregulated. CCG264341 treatment significantly inhibited AXL expression, with downstream signals and fibrosis related protein downregulation. By blinded histopathology with H&E and trichrome staining, as well as immunofluorescence for fibrosis related proteins, colon fibrosis scores significantly decreased. Conclusions: CCG264341is a partially gut selective Axl inhibitor. This compound could play a therapeutic role in IBD fibrosis models. Further work to improve gut selectivity and anti-fibrotic efficacy are needed, but this is a promising pathway for future therapeutic intervention in Crohns disease.