SP-333, a Proteolysis-resistant Agonist of Guanylate Cyclase-C, Inhibits Activation of NF-κB and Suppresses Production of Inflammatory Cytokines to Ameliorate DSS-induced Colitis in Mice

Abstract

Purpose: Uroguanylin (UG) and guanylin (GN) are the physiological natriuretic peptides that activate guanylate cyclase-C (GC-C) to stimulate production of cyclic GMP, a second messenger known to mediate anti-inflammatory effects of cellular molecules such as nitric oxide and hemeoxygenase-1. Earlier we reported that UG expression is suppressed in human colon tumors and that the oral treatment with UG inhibited polyp formation in ApcMin/+ mice via a cGMP-mediated mechanism. Subsequently, we also reported that GC-C agonists (SP-304 and SP-333), highly potent analogs of UG, ameliorated GI inflammation in murine colitis models via downregulation of certain inflammatory cytokines. These results raised the possibility that SP-333 might inhibit NF-κB activation via stimulation of GC-C/cGMP signaling. Methods: Effect of oral treatment with SP-333 was evaluated in DSS-induced colitis in BDF1 mice. H&E stained colonic tissue sections were assessed for colitis by a pathologist blinded to treatment groups. Tissue sections were also scored for expression of Ki-67. Myeloperoxidase activity was measured in colon tissue. Intestinal explants were cultured for 24 hrs and levels of cytokines were measured in culture supernatants by ELISA. Human colon carcinoma T84 cells were treated with lipopolysaccharide (LPS) and SP-333 (0.1 to 10 μM) for 24 hrs and levels of cytokines secreted in the culture media were measured by ELISA. Cytosolic and nuclear fractions were isolated and levels of p-NF-κB-p65, IκB and IKKβ were measured by immunoblotting. Results: Oral treatment with SP-333 ameliorated DSS-induced colitis in mice. In T84 cells, treatment with SP-333 reduced levels of p-NF-κB-p65 in the nuclear fraction and increased levels of cytosolic IκB. Similar observations were made when cells were treated with 8-Br-cGMP, suggesting a cGMP-mediated mechanism to inhibit NF-κB activation and to reduce production of IL-17a, IL-8, IL-23 and TNF-α in T-84 cells. Conclusion: This is the first ever study to show that SP-333 treatment, through enhancement of GC-C/cGMP signaling, inhibits NF-κB activation and suppresses production of inflammatory cytokines to ameliorate DSS-induced colitis in BDF1 mice. SP-333 is currently being developed as an oral drug candidate for treatment of ulcerative colitis in humans. Disclosure: Dr. Kunwar Shailubhai is CSO of Synergy Pharmaceuticals, Inc.Figure: No Caption available.