Guanylyl Cyclase C Research Articles

Characterization of immunological cross-reactivity between enterotoxigenic Escherichia coli heat-stable toxin and human guanylin and uroguanylin.

Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable toxin (ST) (human-type [STh] and porcine-type [STp] variants) is among the five most important enteric pathogens in young children living in low- and middle-income countries. ST mediates diarrheal disease through activation of the guanylate cyclase C (GC-C) receptor and is an attractive vaccine target with the potential to confer protection against a wide range of ETEC strains. However, immunological cross-reactivity to the endogenous GC-C ligands guanylin and uroguanylin is a major concern because of the similarities to ST in amino acid sequence, structure, and function. We have investigated the presence of similar epitopes on STh, STp, guanylin, and uroguanylin by analyzing these peptides in eight distinct competitive enzyme-linked immunosorbent assays (ELISAs). A fraction (27%) of a polyclonal anti-STh antibody and an anti-STh monoclonal antibody (MAb) cross-reacted with uroguanylin, the latter with a 73-fold-lower affinity. In contrast, none of the antibodies raised against STp, one polyclonal antibody and three MAbs, cross-reacted with the endogenous peptides. Antibodies raised against guanylin and uroguanylin showed partial cross-reactivity with the ST peptides. Our results demonstrate, for the first time, that immunological cross-reactions between ST and the endogenous peptides can occur. However, the partial nature and low affinity of the observed cross-reactions suggest that the risk of adverse effects from a future ST vaccine may be low. Furthermore, our results suggest that this risk may be reduced or eliminated by basing an ST immunogen on STp or a selectively mutated variant of STh.

Silibinin modulates caudal-type homeobox transcription factor (CDX2), an intestine specific tumor suppressor to abrogate colon cancer in experimental rats.

To authenticate the colon cancer preventive potential of silibinin, the efficacy of silibinin needs to be tested by evaluating an organ-specific biomarker. The aim of this study was to evaluate the impact of silibinin on the colonic expression of the caudal-type homeobox transcription factor (CDX2) an intestine specific tumor suppressor gene and its downstream targets in the colon of rats challenged with 1,2 dimethyl hydrazine (DMH). Rats of groups 1 and 2 were treated as control and silibinin control. Rats under groups 3 and 4 were given DMH (20 mg/kg body weight (b.w.) subcutaneously) once a week for 15 consecutive weeks from the 4th week of the experimental period. In addition, group 4 rats alone were treated with silibinin (50 mg/kg b.w. per os) everyday throughout the study period of 32 weeks. Histological investigation and messenger RNA and protein expression studies were performed in the colonic tissues of experimental rats. Findings of the study revealed that DMH administration significantly decreased the expression of CDX2 and Guanylyl cyclase C (GCC) in the colon of experimental rats. Further the decreased levels of CDX2 protein, colonic mucin content, and increased number of mast cells in the colon of DMH alone-administered rats reflects the onset of carcinogenesis. The pathological changes caused due to CDX2 suppression were attenuated by silibinin supplementation.

Guanylate cyclase-C/cGMP: an emerging pathway in the regulation of visceral pain

Activation of guanylate cyclase-C (GC-C) expressed predominantly on intestinal epithelial cells by guanylin, uroguanylin or the closely related GC-C agonist peptide, linaclotide, stimulates generation, and release of cyclic guanosine-3′,5′-monophosphate (cGMP). Evidence that the visceral analgesic effects of linaclotide are mediated by a novel, GC-C-dependent peripheral sensory mechanism was first demonstrated in animal models of visceral pain. Subsequent studies with uroguanylin or linaclotide have confirmed the activation of a GC-C/cGMP pathway leading to increased submucosal cGMP mediated by cGMP efflux pumps, which modulates intestinal nociceptor function resulting in peripheral analgesia. These effects can be reproduced by the addition of exogenous cGMP and support a role for GC-C/cGMP signaling in the regulation of visceral sensation, a physiological function that has not previously been linked to the GC-C/cGMP pathway. Notably, targeting the GC-C/cGMP pathway for treatment of gastrointestinal pain and abdominal sensory symptoms has now been validated in the clinic. In 2012, linaclotide was approved in the United States and European Union for the treatment of adult patients with irritable bowel syndrome with constipation.

Emerging Pharmacologic Therapies for Constipation-predominant Irritable Bowel Syndrome and Chronic Constipation.

Irritable bowel syndrome with constipation and chronic functional constipation are common digestive disorders that negatively impact quality of life and account for billions of dollars in health care costs. Related to the heterogeneity of pathogenesis that underlie these disorders and the failure of symptoms to reliably predict underlying pathophysiology, traditional therapies provide relief to only a subset of affected individuals. The evidence surrounding new and emerging pharmacologic treatments, which include both luminally and systemically acting drugs, is discussed here. These include agents such as lubiprostone, bile acid modulations, guanylate cyclase-C receptor agonists, serotonin receptor modulators and herbal therapies.

Clarification of Linaclotide Pharmacology Presented in a Recent Clinical Study of Plecanatide

Dear Editor: We read with great interest the study of plecanatide, a guanylate cyclase C (GC-C) agonist, reported by Shailubhai et al. [1] in the recent issue of Digestive Diseases and Sciences. We have noticed some factually inaccurate statements with respect to the GC-C agonist linaclotide and the comparisons among GC-C agonists. On behalf of the companies that market linaclotide, we felt compelled to write to address these inaccuracies. In the discussion section, the authors address varying degrees of pH-dependent activity among GC-C agonists. The GC-C binding affinities of guanylin and uroguanylin, and the GC-C activated production of cGMP by these endogenous hormones, have been shown to be affected by pH [2–4]. The authors correctly note that the activity of linaclotide is not pH dependent and that the binding affinities of linaclotide to GC-C were similar across a broad, physiologically relevant pH range (pH 5–8). This pH-independence results in linaclotide being active at GC-C along the entire longitudinal axis of the intestinal tract [2]. However, the authors’ assertion that this pH-independence leads to “uncontrolled activation” of GC-C is not substantiated by the reference cited [2]. In fact, the preclinical study cited demonstrates that linaclotide binds to and activates GC-C in a concentration-dependent manner [2]; in other words, the activity of linaclotide is controlled by virtue of being delivered as a discrete oral dose. In addition, Shailubhai et al. also put forth a theory that the pH-independence of E. coli enterotoxin ST peptide (ST) is the reason for extreme cases of diarrhea that can result from enterotoxigenic E. coli (ETEC) infections. This suggestion is not supported by previous or current research into the causes and treatment of ETEC [5–7]. In an ETEC infection, E. coli infects the entire intestinal tract and is continuously replicating and secreting multiple toxins. It is important to remember that persistent exposure to these toxins throughout the course of an ETEC infection is not in any way analogous to the more transient exposure to a therapeutic GC-C agonist following a discrete oral dose [6–8]. Furthermore, the position of Shailubhai et al. does not take into account the proteolytic degradation that any orally delivered GC-C agonist will undergo [8]. This is an exciting time for the field of GC-C pharmacology. We look forward to contributing to and reading about future research on GC-C agonists and GC-C pharmacology.

From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.

The multiple roles of guanylate cyclase C, a heat stable enterotoxin receptor

Guanylate cyclase C (GC-C) is a transmembrane receptor that is expressed primarily on intestinal epithelial cells. Activation of this receptor by its endogenous peptide ligands initiates cyclic guanosine monophosphate-dependent (cGMP) salt and water movement in the intestine. GC-C is targeted by the enterotoxigenic Escherichia coli heat-stable enterotoxin STa, which deregulates this pathway and causes secretory diarrhea. This review discusses current work on the physiological function of GC-C in the intestine. Familial GC-C mutations demonstrate that epithelial cGMP signaling is critical to electrolyte and fluid balance in the neonatal intestine. Chronic deregulation of GC-C activity in early life increases susceptibility to a number of disorders, including obstruction and inflammatory bowel disease. Murine models indicate that GC-C regulates the composition of intestinal commensal microflora and that it suppresses bacterial infection and modulates colonic injury and inflammation. Therapeutic GC-C ligands are used to successfully treat constipation-predominant irritable bowel syndrome and recent studies show that extracellular cGMP is an important mechanism of reducing abdominal pain associated with this disorder. Originally identified as a target of E. coli enterotoxin STa, GC-C is an important regulator of physiological salt and water homeostasis and may directly impact a wide range of intestinal disorders.

Correlation of preoperative guanylate cyclase C mRNA in peripheral blood with postoperative gastric cancer recurrence

Objective To explore the correlation of preoperative guanylate cyclase C(GC-C)mRNA in peripheral blood with postoperative gastric cancer recurrence.Methods The preoperative blood samples were collected from 60 patients with gastric cancer(GC)before operation,also from 21 patients with dysplasia,15 with intestinal metaplasia,and 20 healthy volunteers during the same period.GC-C mRNA in the peripheral blood samples was assessed by real-time fluorescent quantitative PCR(RFQ-PCR).The association of peripheral blood GC-C mRNA expression with pathological parameters and gastric cancer recurrence was analyzed.Results The positive rate of preoperative GC-C mRNA in blood samples of gastric cancer patients(48.3% [29/60])was significantly higher than that of patients with dysplasia(20.0%[3/21],P0.05)and those with intestinal metaplasia(9.5%[2/15],P0.05);GC-C mRNA was not detected in healthy controls.The preoperative blood GC-C mRNA in gastric cancer patients were significantly correlated with Lauren type,clinical TNM stage,depth of invasion,lymph metastasis,and differentiation degree(P0.05 or 0.01).The 1-year,2-year,3-year,and 4-year cumulative recurrence rates of GC-C positive patients were 41.4%,79.3%,96.6%,and 100.0%,respectively;and those of GC-C negative patients were 16.1%,41.9%,67.7%,and 80.6%,respectively.The median recurrence time in GC-C positive patients was significantly shorter than that in GC-C negative patients(P0.01).The 1-4years of cumulative recurrence ratesand overall survival time were significantly different between the two groups after surgery(P0.05 or 0.01).Conclusion The preoperative presence of GC-C mRNA in the peripheral blood of gastric cancer patients is related to poor prognosis and early recurrence after surgery.

Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c(-/-), mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence.

Novel Pharmacological Therapies for Management of Chronic Constipation

Chronic constipation is a common health problem that significantly affects the quality of life of patients and impacts in terms of costs; current treatments based on fiber and laxatives cause dissatisfaction to doctors and patients in more than half of the cases. New drugs are now available or in very advanced stages of research, with different and innovative mechanisms of action as prucalopride, lubiprostone, and linaclotide. Prucalopride an enterokinetic, is a selective high-affinity 5-hydroxytryptamine (5-HT)4 receptor agonist of serotonin that increases the peristaltic reflex and the colonic contractions; lubiprostone, a type 2 chlorine channel activator, or linaclotide, a guanylate cyclase-C agonist of enterocytes, both prosecretory agents, stimulate the secretion of fluid within the intestinal lumen. In general, these promising drugs have proven efficacy and safety as a specific therapeutic option in patients with chronic constipation. Yet the solution might not be sufficient for everybody and still without the ideal drug that might be useful in all cases, the pharmacological revolution for colonic motility disorders has arrived.

Abstract PR12: MLN0264, an investigational, first-in-class antibody-drug conjugate (ADC) targeting guanylyl cyclase C (GCC), demonstrates antitumor activity alone and in combination with gemcitabine in human pancreatic cancer xenograft models expressing GCC.

Abstract Background: MLN0264 consists of a fully human anti-GCC monoclonal antibody linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable linker (MMAE and linker technology licensed from Seattle Genetics). The transmembrane cell surface receptor GCC is expressed by ∼95% of primary and metastatic colorectal cancer (mCRC) tumors. MLN0264 has shown selective binding and antitumor activity in mouse xenograft models of mCRC expressing GCC, and is currently being investigated in a phase 1 study in patients with advanced gastrointestinal malignancies. GCC is also expressed in subsets of pancreatic cancers. Further to the findings in mCRC, we investigated GCC expression in human pancreatic tumors and evaluated MLN0264 activity in mouse xenograft models of GCC-expressing human pancreatic cancer. Methods: GCC expression in multiple human pancreatic cancer samples including tissue microarrays (TMAs) was evaluated by immunohistochemistry (IHC). For in vivo studies, 7 mouse xenograft primary human tumor explant (PHTX) models of pancreatic cancer were developed, including tumor tissue from patients with wild-type and mutant KRAS. Animals were treated when the tumor reached ∼230 mm3. In single-agent studies, animals were administered vehicle, MMAE 0.135 mg/kg once weekly (QW), or MLN0264 3.75 or 7.5 mg/kg QW. In combination studies, animals received vehicle, or MLN0264 7.5 mg/kg QW alone or in combination with gemcitabine 15 or 20 mg/kg twice weekly (BIW), or gemcitabine 15 mg/kg on days 1, 3 each week. Average tumor volume was determined at multiple time points following the start of treatment using vernier callipers. Results: In the GCC-expressing PHTX-249 mouse xenograft model (KRAS mutant G12), single-agent MLN0264 showed significant tumor growth inhibition (TGI) versus vehicle or free MMAE by day 21, with the 7.5 mg/kg dose significantly better than 3.75 mg/kg by day 20-22. Similarly, in the GCC-expressing PHTX-215 model (KRAS wild-type), MLN0264 7.5 mg/kg resulted in significantly greater TGI (79%) versus free MMAE or MLN0264 3.75 mg/kg by day 22, including some tumor regression. Across the 7 models (variable apical GCC expression; KRAS wild-type and mutant), TGI ranged from 24% (p=0.17) to 79% (p<0.001) with single-agent MLN0264. In the PHTX-249 model, MLN0264 7.5 mg/kg plus gemcitabine 15/20 mg/kg BIW or 15 mg/kg, days 1, 3, showed significantly greater TGI than either agent alone. At day 20-21, TGI was 46-47% for single-agent MLN0264, 66-79% for gemcitabine 15 mg/kg BIW and gemcitabine days 1, 3, and 84-88% for the respective combinations; 3 of 7 animals in the latter combination group had a smaller tumor volume at day 20 versus day 0 (TGI, 93%). Conclusions: These findings indicate that MLN0264 has antitumor activity as a single agent and in combination with gemcitabine in GCC-expressing pancreatic cancer xenograft models, and support clinical evaluation of MLN0264 in patients with pancreatic cancer. Data from additional models will be presented. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR12. Citation Format: Julie Zhang, Melissa Gallery, Tim Wyant, Brad Stringer, Mark Manfredi, Hadi Danaee, Petter Veiby. MLN0264, an investigational, first-in-class antibody-drug conjugate (ADC) targeting guanylyl cyclase C (GCC), demonstrates antitumor activity alone and in combination with gemcitabine in human pancreatic cancer xenograft models expressing GCC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR12.

▼Linaclotide for constipation-predominant IBS

Irritable bowel syndrome is a chronic relapsing disorder characterised by abdominal pain or discomfort associated with defaecation, abdominal bloating and a change in bowel habit.1,2 IBS may be classified by...

Oral Treatment with SP-333, an Agonist of Guanylate Cyclase-C, Dramatically Ameliorates Morphine-induced Bowel Dysfunction in Rats: Presidential Poster

Purpose: Opioid agonists are commonly used for chronic pain management, but debilitating constipation is a frequent dose-limiting side effect. Guanylate cyclase-C (GC-C) agonists are a new class of drug candidates for the treatment of gastrointestinal (GI) disorders. SP-333, a proteolysis-resistant analog of uroguanylin, activates GC-C to stimulate production of cyclic GMP (cGMP), a second messenger that promotes intestinal secretion and gut motility. Nonclinical toxicology studies have shown that repeated oral doses of SP 333 are well-tolerated in mice and monkeys. SP-333 is currently under clinical development for the treatment of ulcerative colitis. In the current study, the efficacy of SP-333 was assessed in a rat model of opioid-induced bowel dysfunction. Methods: Female CD rats (170-210 g; n=10-12/group) received an intraperitoneal dose of vehicle (water) or 2.5 mg/kg morphine followed by an oral dose of 0 (vehicle), 0.5, 5, or 50 mg/kg of SP-333. A charcoal meal was administered 10 min later, rats were euthanized after an additional 10 min, and GI transit was measured (distance traveled by leading edge of charcoal divided by total length of small intestine). Results: A single dose of morphine significantly reduced GI transit in rats (23.4% in morphine-treated vs 53.3% in vehicle-treated; p≤0.001). SP-333 produced a dose-dependent improvement in gut transit in morphine-treated rats, restoring transit to near normal levels following doses of 5 mg/kg (45.8%) or 50 mg/kg (48.4%) (p≤0.001 for both groups). Conclusion: Treatment with SP-333 accelerated gut transit in morphine-administered rats in a dosedependent manner, possibly via the known cGMP-mediated mode of action. In combination with previously completed toxicology studies, these data support the potential utility of SP 333 and other GC-C agonists as safe oral drug candidates for the treatment of opioid-induced bowel dysfunction. Disclosure - Mr. Kramer, Dr. Palejwala, Dr. Foss and Dr. Shailubhai are employees of Synergy Pharmaceuticals, Inc.Figure

SP-333, a Guanylate Cyclase-C Agonist, Inhibits NF-κB Signaling and Modulates Related Genes and miRNAs Implicated in GI Inflammation and Carcinogenesis

SP-333, a Guanylate Cyclase-C Agonist, Inhibits NF-κB Signaling and Modulates Related Genes and miRNAs Implicated in GI Inflammation and Carcinogenesis

Effect of Linaclotide on Severe Abdominal Symptoms in Patients With Irritable Bowel Syndrome With Constipation

Patients with irritable bowel syndrome with constipation (IBS-C) have abdominal symptoms that vary in severity. Linaclotide, a guanylate cyclase-C agonist, improves abdominal and bowel symptoms in these patients. We examined the prevalence of severe abdominal symptoms in patients with IBS-C and assessed the effects of linaclotide on abdominal symptoms, global measures, and quality of life (QOL). In two phase 3 trials, patients who met modified Rome II criteria for IBS-C were randomly assigned to groups given oral, once-daily linaclotide (290 μg) or placebo for 12 weeks. During the baseline (2 weeks prior to treatment) and treatment periods, patients rated abdominal pain, discomfort, bloating, fullness, and cramping daily (from 0 = none to 10 = very severe). Linaclotide's effects on abdominal symptoms, global measures, and IBS-related QOL were assessed in subpopulations of patients who rated specific individual abdominal symptoms as severe (≥ 7.0) at baseline. In the intent-to-treat population (1602 patients; 797 receiving placebo and 805 receiving linaclotide), baseline prevalence values for severe abdominal symptoms were 44% for bloating, 44% for fullness, 32% for discomfort, 23% for pain, and 22% for cramping, with considerable overlap among symptoms. In patients with severe symptoms, linaclotide reduced all abdominal symptoms; mean changes from baseline severity scores ranged from -2.7 to -3.4 for linaclotide vs -1.4 to -1.9 for placebo (P < .0001). Linaclotide improved global measures (P < .0001) and IBS-QOL scores (P < .01) compared with placebo. Diarrhea was the most common adverse event of linaclotide in patients with severe abdominal symptoms (18.8%-21.0%). Of 5 severe abdominal symptoms assessed, bloating and fullness were most prevalent in patients with IBS-C. Linaclotide significantly improved all abdominal symptoms, global measures, and IBS-QOL in subpopulations of IBS-C patients with severe abdominal symptoms. Clinicaltrials.gov NCT00938717, NCT00948818.

Prosequence switching: An effective strategy to produce biologically active E. coli heat-stable enterotoxin STh

Enterotoxigenic Escherichia coli (ETEC) infections account for the majority of cases of acute secretory diarrhea. The causative agents are enterotoxins secreted by ETEC, among them is the heat-stable enterotoxin, STh. STh is a 19-amino acid peptide containing three disulfide bonds that stimulates fluid secretion in the bowel by binding to the receptor domain of intestinal guanylyl cyclase C (GC-C). Since GC-C agonists have pharmacologic potential for diagnosis and treatment of disorders such as constipation-predominant irritable bowel syndrome (IBS-C), chronic constipation, and colorectal carcinoma, it is crucial to develop methods for the large-scale production of STh and related peptides. Here, we present a strategy for recombinant expression of STh that relies on the use of the prosequence of human uroguanylin to support proper folding and disulfide bond formation. The chimeric protein CysCys-STh consisting of the propeptide of uroguanylin as N-terminus and the STh peptide as C-terminus was expressed in E. coli, and an efficient purification protocol was developed. Trypsin digestion of this protein released the enterotoxin which could be obtained in high purity. NMR and mass spectrometry confirmed the identity and homogeneity of the toxin, and its biological activity was confirmed by a cell-based in vivo assay. The expression scheme introduced here represents a cost-efficient and scalable way of STh production.

Linaclotide Inhibits Colonic Nociceptors and Relieves Abdominal Pain via Guanylate Cyclase-C and Extracellular Cyclic Guanosine 3′,5′-Monophosphate

Linaclotide is a minimally absorbed agonist of guanylate cyclase-C (GUCY2C or GC-C) that reduces symptoms associated with irritable bowel syndrome with constipation (IBS-C). Little is known about the mechanism by which linaclotide reduces abdominal pain in patients with IBS-C. We determined the effects of linaclotide on colonic sensory afferents in healthy mice and those with chronic visceral hypersensitivity. We assessed pain transmission by measuring activation of dorsal horn neurons in the spinal cord in response to noxious colorectal distention. Levels of Gucy2c messenger RNA were measured in tissues from mice using quantitative reverse transcription polymerase chain reaction and in situ hybridization. We used human intestinal cell lines to measure release of cyclic guanosine-3',5'-monophosphate (cGMP) by linaclotide. We performed a post-hoc analysis of data from a phase III, double-blind, parallel-group study in which 805 patients with IBS-C were randomly assigned to groups given an oral placebo or 290 μg linaclotide once daily for 26 weeks. We quantified changes in IBS-C symptoms, including abdominal pain. In mice, linaclotide inhibited colonic nociceptors with greater efficacy during chronic visceral hypersensitivity. Intra-colonic administration of linaclotide reduced signaling of noxious colorectal distention to the spinal cord. The colonic mucosa, but not neurons, was found to express linaclotide's target, GC-C. The downstream effector of GC-C, cGMP, was released after administration of linaclotide and also inhibited nociceptors. The effects of linaclotide were lost in Gucy2c(-/-) mice and prevented by inhibiting cGMP transporters or removing the mucosa. During 26 weeks of linaclotide administration, a significantly greater percentage of patients (70%) had at least a 30% reduction in abdominal pain compared with patients given placebo (50%). We have identified an analgesic mechanism of linaclotide: it activates GC-C expressed on mucosal epithelial cells, resulting in the production and release of cGMP. This extracellular cGMP acts on and inhibits nociceptors, thereby reducing nociception. We also found that linaclotide reduces chronic abdominal pain in patients with IBS-C.

Linaclotide induces secretion of cGMP from mouse colonic epithelium

Linaclotide is a novel receptor guanylyl cyclase C (GC-C) agonist approved for treatment of abdominal pain and constipation in patients with irritable bowel syndrome with constipation (IBS-C). Linaclotide effects on bowel movements are mediated by the intracellular cGMP that is produced upon activation of GC-C. It is hypothesized that the effects of linaclotide on abdominal pain are mediated by extracellular cGMP, which was shown to decrease the activity of pain-sensing nerves [1]. Here we used an ex vivo Ussing chamber assay to measure the secretion of cGMP from the mouse colonic mucosa in response to linaclotide treatment. Ion transport and epithelial barrier function were monitored by measuring short-circuit current (Isc) and trans-epithelial electrical resistance (TEER). Stimulation with linaclotide (1 µM) elicited a robust short-circuit current across mouse colonic epithelium. Isc reached a maximum within ten minutes following stimulation with linaclotide and remained steady during the duration of the study (60 min). Treatment of colonic mucosa with linaclotide induced release of cGMP from the apical, as well as, the basolateral side of the epithelium. The time course of cGMP accumulation in the basolateral bath of the Ussing chamber was linear with an estimated cGMP secretion rate equal to 23 fmol/min×cm2. The trans-epithelial electrical resistance of the colonic mucosa remained high over the course of the study indicating that the barrier to diffusion of cGMP between apical and basolateral sides remained intact throughout the study.. In summary, these data demonstrate that linaclotide-stimulated mouse colonic epithelium secretes cGMP from both the apical and basolateral sides and that cGMP is available in the submucosal interstitial space to inhibit colonic nociceptors.

Linaclotide-a Novel Secretagogue in the Treatment of Irritable Bowel Syndrome with Constipation and Chronic Idiopathic Constipation

Irritable bowel syndrome with constipation (IBS-C) and chronic idiopathic constipation (CIC) are highly prevalent gastrointestinal disorders. Traditional symptoms based therapies had somewhat limited success and efficacy in addressing the disorders. Recently, linaclotide emerged as novel peptide capable of improving abdominal symptoms in patients suffering from IBS-C and CIC. Guanylate cyclase C (GC-C) receptor a multi domain protein, found to be molecular target for linaclotide which acts by activating GC-C receptor on the apical surface of intestinal epithelial cells. Binding of linaclotide to GC-C receptor triggers the elevation of second messenger cGMP that elicits fluid secretion into intestinal cells which play a critical role in maintaining homeostasis through cystic fibrosis transmembrane conductance regulator (CFTR). Data from Phase II and III clinical trials demonstrated that linaclotide seems to produce a statistically significant increase in stool frequency, improved straining, decreased abdominal pain and discomfort.

Emerging receptor target in the pharmacotherapy of irritable bowel syndrome with constipation

Preclinical experiments in rodent models have recently provided new information on the mechanisms underlying pain sensation in chronic visceral hypersensitivity, as well as insights into the mechanism of action of new drugs targeting abdominal pain in irritable bowel syndrome (IBS). This article describes the evidence base supporting the role of guanylate cyclase C (GC-C) activation in the modulation of gastrointestinal transit and, in particular, in visceral hypersensitivity. We propose that GC-C activation represents an important emerging target for pharmacotherapy in IBS with constipation (IBS-C), particularly given the recent regulatory approval of the GC-C agonist linaclotide as a treatment for IBS-C. More specifically, we address the following questions: “How is pain transmitted from the colon?”; “How is abdominal pain increased in IBS-C?”; “How can we reduce IBS-related abdominal pain – what drugs have been developed?”; “Does linaclotide reduce abdominal pain in animals and humans?”; and “How does linaclotide reduce abdominal pain?”