Objective: Dysregulated fecal and epithelial proteolytic activity (PA) has been detected in patients with inflammatory bowel disease (IBD) versus healthy controls. Recently, increased fecal PA that preceded diagnosis of UC was found to have a microbial component that caused inflammation in mice, suggesting it could be used as a luminal diagnostic biomarker in IBD. The aim of this study was to investigate whether PA in IBD patients correlates with disease activity. Methods: 23 adult CD patients were recruited at McMaster Children's Hospital and biopsies were collected from non-inflamed and proximal (within 5 cm) inflamed sites from the same patients. Luminal to serosal flux rate and tissue conductance were measured in biopsies with Ussing chambers. Total proteolytic (trypsin), elastase-like (FITC-elastin and N-Suc-Ala3-pNA), and mucolytic (bioassay) activities were also measured at both sites. In a separate cohort of 16 CD and 19 UC patients recruited by the IMAGINE study (imaginespor.com), overall, elastase-like, and mucolytic fecal PA were measured. IMAGINE patients completed the Symptom IBD Short Index (SIBDSI) survey to classify patients as asymptomatic (UC <13, CD <14) or symptomatic (UC >13, CD >14) at the time of fecal donation. Fecal calprotectin (fCal) levels were measured to determine remission (fCal<50μg/g) or flare (fCal>100μg/g) status of patients. Results: There was higher paracellular permeability and tissue conductance in biopsies from inflamed areas compared with non-inflamed areas from the same patient. Elastase-like and mucolytic activities were higher in inflamed tissue compared with matched non-inflamed (p<0.05 and p<0.04, respectively). In IMAGINE patients, total fecal PA was higher in symptomatic compared with asymptomatic CD patients (p=0.04). There was a trend for higher overall proteolytic activity in IBD patients categorized in flare, or fCal>100μg/g, compared with patients in remission, or fCal<50μg/g. Conclusions: There is growing evidence for the role of host and microbial PA in the progression and exacerbation of IBD. Tissue permeability and elastase-like activity correlate with inflammation and may be causally linked. The results raise the hypothesis that microbial elastase-like activity may cause mucosal inflammation through barrier disruption and constitute a novel non-invasive biomarker for monitoring CD activity.

Postoperative Ileus (POI) is an iatrogenic complication characterized by a transient paralysis of gastrointestinal motility. This transient paralysis leads to food intolerance, nausea, vomiting in patients, and thus prolongs hospitalization time. The underlying mechanisms are described in two phases: a neurogenic phase followed by an inflammatory phase. It has been shown that immune cells such as macrophages and mastocytes are able to produce numerous proteases and releasing them into their environment upon activation. Proteases, in turn, could activate Protease-Activated Receptors (PARs) involved in numerous inflammatory pathways. Therefore, we developed a mouse surgical model of Postoperative Ileus to explore the relationship between this iatrogenic complication and proteolytic activation of PARs. To address this question, PAR-1, PAR-2, or PAR-3 knockout mice were subjected to POI protocol. Additionally, a pre-treatment with PAR antagonists was performed two hours before surgery on wild type (WT) mice. Gastric emptying and small intestine motility were assessed using a charcoal solution. Pro-inflammatory cytokines levels were evaluated by qPCR and ELISA. Immune cells recruitment was measured by flow cytometry. Mice with POI showed no gastric empty and slower gastrointestinal transit compared to mice subjected to laparotomy (sham condition). POI mice exhibited a significant increase mRNA levels of pro-inflammatory markers (CCL2, IL6, TNFa, ICAM-1) in the ileum muscularis externa 2.5 hours after intestinal manipulation compared to sham mice. Additionally, 24 hours post-surgery, mice showed an increase in the chemokine CCL2 in the ileum muscularis externa, as well a recruitment of immune cells including neutrophils, monocytes, and monocyte-derived macrophages. Based on this model of POI, PAR-1 and PAR-3 receptor-deficient mice were not protected from Postoperative Ileus. In contrast, PAR-2 receptor-deficient mice subjected to POI model exhibited a gastric emptying and a normal GI transit. Furthermore, the pre-treatment with PAR-2 antagonist (GB88 at 10uM) protected WT mice from POI. These results highlight a new player in development of Postoperative Ileus: PAR 2, which could be considered as a new pharmacological target.

In rodent models of ulcerative colitis (UC), NX-13 (an orally active, gut selective NLRX1 agonist) improves disease activity through a novel bimodal mechanism. A pig DSS colitis study was performed to generate pharmacokinetic (PK) and mechanistic data, given pigs can be dosed with tablets and their gut and immune systems are markedly more similar to humans. We describe the efficacy and PK of NX-13 tablets in pigs compared to phase 1 clinical trials. NX-13 immediate release (IR) tablets were used in pig studies and human clinical trials with plasma samples collected at hourly intervals. Stool samples were collected at ~24hrs after last dose. The phase 1 clinical trials were double blind, randomized placebo-controlled studies in healthy subjects (1a, Leber et al UEGW J 9(Supp)) or subjects with active UC (1b, Verstockt et al JCC epub 11NOV2023). NX-13 concentrations were determined by LC-MS/MS. In pigs, oral NX-13 dampened colitis as early as day 2, becoming significant at days 4-6 (Fig. 1A). Consistent results were observed in the Phase 1b trial, including rectal bleeding improvement as early as week 2 (Fig. 1B) and endoscopic improvement at week 4. Despite signs of sufficient target engagement in both species, the NX-13 PK characteristics differed. Firstly, NX-13’s low level of human systemic exposure peaked 1 hr after dosing, whereas Tmax in pigs was 7 hrs at comparable doses. Secondly, the NX-13 retained in the stool is greater in pigs (250-1000x plasma levels), compared to humans (150-400x) although both stool and plasma concentrations were greater in pigs (Fig. 1C). This suggests NX-13 is not gut-restricted in humans, but rather gut-selective with low systemic absorption. Interestingly, the stool:plasma ratio was numerically lower in patients with UC, implicating epithelial barrier integrity. The NX-13 PK hypothesis of a gut-selectivity with low systemic absorption will be analyzed in the ongoing phase 2 trial.

Visceral Pain in Inflammatory Bowel Disease: a role for Protease-Activated Receptor-1 C Rolland1, P Rousset1, E Buscail1,2, L Buscail2, B Bournet2, G Le Cosquer1,2, S Sablayrolles3, B Le Grand3, C Deraison1, N Vergnolle1,4 1INSERM U1220, IRSD, Toulouse, France, 2Department of digestive surgery, colorectal surgery unit, Toulouse University Hospital, France 3CVasThera, Castres, France 4Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada Pain is a cardinal sign of inflammation and is associated with flares in inflammatory bowel disease (IBD) patients, where peripheral mediators released by inflamed tissues are responsible for activation of nociceptors and pain pathways. However, 30 to 50% of IBD patients in remission still report significant pain, despite the absence of infiltrated inflammatory cells and complete repair of tissues. We hypothesized that peripheral (colonic) mediators present in repaired mucosa can signal to sensory neurons contributing to pain symptoms. We have investigated the effects of colon biopsy supernatants from IBD patients in remission or healthy controls on cultured dorsal root ganglia (DRG) neurons harvested from mice or human cadavers and tested the effects of Protease-Activated Receptor-1 (PAR1) antagonist. Further, we have induced colitis in rats, by the intracolonic administration of trinitrobenzene sulfonic acid (TNBS), and recorded nociceptive responses. Colon biopsy supernatants from IBD patients in remission but not from healthy controls caused significant activation (calcium signal) of DRG neurons both in mouse and human neurons. Pre-incubation of neurons with a PAR1 antagonist significantly inhibited IBD supernatants-induced DRG calcium signals. Oral treatment with the PAR1 antagonist CVT120165 significantly inhibited, dose-dependently, referred abdominal pain in rats induced by intracolonic TNBS administration 7-days earlier. PAR1 antagonist also favored tissue repair. Peripheral mediators in the colon of IBD patients in remission are able to activate sensory neurons by a PAR1-dependent mechanism. PAR1 blockade relief from pain symptoms in a rat model of colitis, suggesting that PAR1 could be a good target for the treatment of IBD-associated pain in remission.

Mucosal proteolytic homeostasis plays a crucial role in the pathophysiology of functional GI disorders, including Irritable Bowel Syndrome (IBS), one of whose main symptom is abdominal pain and visceral hypersensitivity. Although the exacerbation of wide-spectrum proteolytic activity has been reported in these patients, the exact mechanism to explain how the proteolytic balance controls visceral pain, as well as the type of proteases involved remain to be elucidated. We focused our attention on elastolytic activity, which is known to be exacerbated in high-grade inflammatory diseases, and investigated whether such activity could also be implicated in low-grade inflammatory conditions such as IBS, contributing there to pain signals. To address this question, we used tissue biopsies from IBS patients, several animal models of visceral pain, and calcium signalling in dorsal root ganglia neuron cultures. Intestinal elastolytic activity was increased in tissues from IBS patients and strongly associated with epithelium. Elastolytic activity was also increased in visceral hypersensitivity or pain models induced respectively by stress sessions or mustard oil i.c. administration. Active elastolytic enzymes induced dose-dependent calcium signals in primary afferent neuron cultures and when administered i.c., could cause visceral hypersensitivity in response to colorectal distension. Because stress contributes to the development of IBS, and because we observed that strong elastolytic activity was associated with the epithelium from IBS patients, we investigated the regulation of elastolytic activity by human epithelium. The expression of Elafin, an endogenous inhibitor of elastolytic enzymes, was significantly decreased after exposure of colon organoid cultures to a cocktail of stress hormones. Finally, inhibition of colonic elastolytic activity by Elafin delivery protected mice from visceral hypersensitivity and pain symptoms. Altogether, these data demonstrated that elastolytic activity is dysregulated in IBS and is associated with activation of sensory neurons and pain signals. The nature and source of elastases still has to be identified, but this study highlights elastolytic activity as a potential new target in the management of low-grade inflammation associated visceral pain.