Linaclotide, a potent guanylate cyclase C agonist, is a therapeutic peptide approved in the United States for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation. We present for the first time the metabolism, degradation, and disposition of linaclotide in animals and humans. We examined the metabolic stability of linaclotide in conditions that mimic the gastrointestinal tract and characterized the metabolite MM-419447 (CCEYCCNPACTGC), which contributes to the pharmacologic effects of linaclotide. Systemic exposure to these active peptides is low in rats and humans, and the low systemic and portal vein concentrations of linaclotide and MM-419447 observed in the rat confirmed both peptides are minimally absorbed after oral administration. Linaclotide is stable in the acidic environment of the stomach and is converted to MM-419447 in the small intestine. The disulfide bonds of both peptides are reduced in the small intestine, where they are subsequently proteolyzed and degraded. After oral administration of linaclotide, <1% of the dose was excreted as active peptide in rat feces and a mean of 3-5% in human feces; in both cases MM-419447 was the predominant peptide recovered. MM-419447 exhibits high-affinity binding in vitro to T84 cells, resulting in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3',5'-monophosphate (cGMP). In rat models of gastrointestinal function, orally dosed MM-419447 significantly increased fluid secretion into small intestinal loops, increased intraluminal cGMP, and caused a dose-dependent acceleration in gastrointestinal transit. These results demonstrate the importance of the active metabolite in contributing to linaclotide's pharmacology.

Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman’s Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

Bladder pain syndrome (BPS) is poorly understood; however, there is a female predominance and comorbidity with irritable bowel syndrome (IBS). Here we test the hypothesis that linaclotide, a guanylate cyclase-C (GC-C) agonist approved for the treatment of IBS with constipation (IBS-C), may represent a novel therapeutic for BPS acting through a mechanism involving an inhibition of visceral organ cross-sensitization. We showed previously that infusion of dilute protamine sulfate (PS) into the bladder increased sensitivity and permeability in the bladder and colon. PS was infused into the bladder of female rats; sensitivity was assessed via application of von Frey filaments applied to the suprapubic area and the frequency of withdrawal responses was recorded. Colonic sensitivity was measured via visceromotor behavioral response to graded pressures of colorectal distension (CRD). Permeability was measured in vitro via transepithelial electrical resistance (TEER) and conductance (G). Linaclotide (3 g/kg, p.o.) or vehicle was administered daily for 7 days prior to experiments. Rats treated with PS bladder infusion exhibited visceral hyperalgesia, as shown by a significantly higher response frequency to individual von Frey filaments and increased behavioral responses to CRD. Linaclotide attenuated bladder and colonic hyperalgesia to control levels. PS infusion into the bladder increased bladder and colon permeability measured as a decrease in TEER and increased G. Linaclotide significantly inhibited PS-induced colonic hyperpermeability while having no effect on bladder hyperpermeability. Our findings suggest a novel treatment paradigm for GC-C agonism in IBS-C and BPS mediated through a mechanism involving visceral organ crosstalk.

Our results demonstrate that neonatal rats exposed to unpredictable ELS develop increased sensitivity and permeability of the colon and bladder in adulthood through a mechanism involving activation of peripheral GC-C signaling.