Antagonism of Protease-Activated Receptor 2 Protects against Experimental Colitis

Abstract

Many trypsin-like serine proteases such as ␤-tryptase are involved in the pathogenesis of colitis and inflammatory bowel diseases. Inhibitors of individual proteases show limited efficacy in treating such conditions, but also probably disrupt digestive and defensive functions of proteases. Here, we investigate whether masking their common target, protease-activated receptor 2 (PAR2), is an effective therapeutic strategy for treating acute and chronic experimental colitis in rats. A novel PAR2 antagonist (5-isoxazoyl-Cha-Ile-spiro[indene-1,4Ј-piperidine]; GB88) was evaluated for the blockade of intracellular calcium release in colonocytes and anti-inflammatory activity in acute (PAR2 agonist-induced) versus chronic [2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced] models of colitis in Wistar rats. Disease progression (disease activity index, weight loss, and mortality) and postmortem colonic histopathology (inflammation, bowel wall thickness, and myeloperoxidase) were measured. PAR2 and tryptase colocalization were investigated by using immunohistochemistry. GB88 was a more potent antagonist of PAR2 activation in colonocytes than another reported compound, N 1 -3-methylbutyryl-N 4 -6-aminohexanoyl-piperazine (ENMD-1068) (IC 50 8 M versus 5 mM). Acute colonic inflammation induced in rats by the PAR2 agonist SLIGRL-NH 2 was inhibited by oral administration of GB88 (10 mg/kg) with markedly reduced edema, mucin depletion, PAR2 receptor internalization, and mastocytosis. Chronic TNBS-induced colitis in rats was ameliorated by GB88 (10 mg/kg/day p.o.), which reduced mortality and pathology (including colon obstruction, ulceration, wall thickness, and myeloperoxidase release) more effectively than the clinically used drug sulfasalazine (100 mg/ kg/day p.o.). These disease-modifying properties for the PAR2 antagonist in both acute and chronic experimental colitis strongly support a pathogenic role for PAR2 and PAR2-activating proteases and therapeutic potential for PAR2 antagonism in inflammatory diseases of the colon.

Tumour progression and cancer-induced pain: A role for protease-activated receptor-2?

“…In vitro, ENMD-1068 has been shown to reduce PAR 2 mediated cell proliferation and to increase apoptosis in human oesophageal adenocarcinoma cells , whilst other studies have reported antiangiogenic and analgesic effects in mouse models of endometriosis and postoperative pain (Oliveira et al, 2013;Wang et al, 2014). Studies utilising GB88 are more limited, however this compound has been shown to antagonise intracellular calcium mobilisation in trypsin treated HT29 cells in vitro and to reduce inflammation in models of arthritis and colitis in vivo (Lohman et al, 2012a;Lohman et al, 2012b;Suen et al, 2012).…”

Pathway‐selective antagonism of proteinase activated receptor 2

“…Furthermore, PAR2 can signal independently of G-proteins via ß-arrestin1/2 (Nichols et al, 2012). PAR2 is an important mediator in many animal models of inflammatory diseases, including asthma (Cocks et al, 1999), pancreatitis (Kawabata et al, 2006), irritable bowel syndrome (Cenac et al, 2007), colitis (Lohman et al, 2012b), arthritis (Lohman et al, 2012a), glomerulonephritis (Moussa et al, 2007), obesity and diabetes (Badeanlou et al, 2011;Lim et al, 2013).…”

Trypsin-induced biphasic regulation of tone in the porcine lower esophageal sphincter

“…However, the observations with a nonpeptide antagonist, ENMD-1068, might cause some controversy for the role of PAR 2 in the trypsin-induced effects. There is a concern regarding the potency of ENMD-1068 because its IC 50 is approximately 3 mM (Lohman et al, 2012), and 5 mM ENMD-1068 was used in the present study. Taken together, the observations obtained with agonists and antagonists appear to provide a suggestive indication but not fully convincing support for the involvement of PAR 2 in the trypsin-induced contraction and relaxation of porcine LES circular smooth muscle.…”