Ada Silos-Santiago

Mo1849 Mechanism of Action for Linaclotide Induced Abdominal Pain Relief

Introduction Cyclic GMP (cGMP) is a 2nd messenger produced in intestinal epithelial cells in response to guanylate cyclase C receptor (GCC) activation. Linaclotide (LIN), an investigational GCC agonist (GCCA), improved constipation and reduced abdominal pain in patients with irritable bowel syndrome with constipation (IBS-C) in clinical trials. We have shown that exogenous extracellular cGMP has contrasting effects on colorectal (CR) afferent mechanosensitivity. 1 Here we assessed the effects of GCCAs on CR afferent mechanosensitivity in healthy and chronic visceral hypersensitivity (CVH) mouse models Methods We investigated CVH in healthy mice and CVH 28 days post-TNBS administration, when inflammation had resolved and nociceptors were mechanically hypersensitive. Mechanosensory responses of CR splanchnic nociceptors and pelvic mucosal afferents were compared in vitro ± GCCAs STc (1, 50, 250, 1000nM) and LIN (1, 30, 100, 300, 1000nM), which were applied individually to the CR mucosal surface. GCC expression in the CR mucosa was determined via qRT-PCR Results In healthy mice, STc dose-dependently (50, 250, 1000nM) reduced nociceptor mechanosensitivity (max. effect at 1000nM [n = 10], –38%; p 0.05). qRT-PCR analysis revealed abundant GCC expression in CR mucosa of both healthy and CVH mice Conclusion STc and LIN significantly reduced colonic nociceptor mechanosensitivity, with greatest effect in CVH. Although these overall effects mirror those of exogenously applied cGMP, GCCAs are more potent at inhibiting nociceptors. Overall, LIN induced the greatest inhibitory effects on nociceptors, particularly in CVH. Reducing colonic nociceptive input would help to reduce pain, which supports LIN clinical data showing a significant reduction in abdominal pain in humans with IBS-C. Increased mucosal afferent sensitivity may help coordinate defecation. Disclosure of Interest S. Brierley Grant/Research Support from: Ironwood Pharmaceuticals, J. Castro: None Declared, A. Harrington: None Declared, P. Hughes: None Declared, C. Martin: None Declared, A. Silos-Santiago Employee of: Ironwood Pharmaceuticals, C. Kurtz Employee of: Ironwood Pharmaceuticals, A. Blackshaw Grant/Research Support from: Ironwood Pharmaceuticals. Reference Gastro. 2011. Supported by Ironwood Pharmaceuticals Inc and by Forest Laboratories Inc. Medical writing assistance was provided by Complete Medical Communications, funded by Almirall.

Gene expression profiling and endothelin in acute experimental pancreatitis.

AIM To analyze gene expression profiles in an experimental pancreatitis and provide functional reversal of hypersensitivity with candidate gene endothelin-1 antagonists. METHODS Dibutyltin dichloride (DBTC) is a chemical used as a polyvinyl carbonate stabilizer/catalyzer, biocide in agriculture, antifouling agent in paint and fabric. DBTC induces an acute pancreatitis flare through generation of reactive oxygen species. Lewis-inbred rats received a single i.v. injection with either DBTC or vehicle. Spinal cord and dorsal root ganglia (DRG) were taken at the peak of inflammation and processed for transcriptional profiling with a cDNA microarray biased for rat brain-specific genes. In a second study, groups of animals with DBTC-induced pancreatitis were treated with endothelin (ET) receptor antagonists [ET-A (BQ123) and ET-B BQ788)]. Spontaneous pain related mechanical and thermal hypersensitivity were measured. Immunohistochemical analysis was performed using anti-ET-A and ET-B antibodies on sections from pancreatic tissues and DRG of the T10-12 spinal segments. RESULTS Animals developed acute pancreatic inflammation persisting 7-10 d as confirmed by pathological studies (edema in parenchyma, loss of pancreatic architecture and islets, infiltration of inflammatory cells, neutrophil and mononuclear cells, degeneration, vacuolization and necrosis of acinar cells) and the pain-related behaviors (cutaneous secondary mechanical and thermal hypersensitivity). Gene expression profile was different in the spinal cord from animals with pancreatitis compared to the vehicle control group. Over 260 up-regulated and 60 down-regulated unique genes could be classified into 8 functional gene families: circulatory/acute phase/immunomodulatory; extracellular matrix; structural; channel/receptor/transporter; signaling transduction; transcription/translation-related; antioxidants/chaperones/heat shock; pancreatic and other enzymes. ET-1 was among the 52 candidate genes up-regulated greater than 2-fold in animals with pancreatic inflammation and visceral pain-related behavior. Treatments with the ET-A (BQ123) and ET-B (BQ-788) antagonists revealed significant protection against inflammatory pain related mechanical and thermal hypersensitivity behaviors in animals with pancreatitis (P < 0.05). Open field spontaneous behavioral activity (at baseline, day 6 and 30 min after drug treatments (BQ123, BQ788) showed overall stable activity levels indicating that the drugs produced no undesirable effects on normal exploratory behaviors, except for a trend toward reduction of the active time and increase in resting time at the highest dose (300 μmol/L). Immunocytochemical localization revealed that expression of ET-A and ET-B receptors increased in DRG from animals with pancreatitis. Endothelin receptor localization was combined in dual staining with neuronal marker NeuN, and glia marker, glial fibrillary acidic protein. ET-A was expressed in the cell bodies and occasional nuclei of DRG neurons in naïve animals. However, phenotypic expression of ET-A receptor was greatly increased in neurons of all sizes in animals with pancreatitis. Similarly, ET-B receptor was localized in neurons and in the satellite glia, as well as in the Schwann cell glial myelin sheaths surrounding the axons passing through the DRG. CONCLUSION Endothelin-receptor antagonists protect against inflammatory pain responses without interfering with normal exploratory behaviors. Candidate genes can serve as future biomarkers for diagnosis and/or targeted gene therapy.

OC-068 Measuring the Effect of Ispaghula on Gut Content and Function Using MRI

Introduction Ispaghula husk (IS) is believed to modulate functional gastrointestinal symptoms by drawing water into the bowel to soften stool and accelerate transit, and by adding bulk. It is not thought to be readily fermented. Magnetic Resonance Imaging (MRI) can assess gastrointestinal content and function. The aim of the study was to assess whether MRI could detect and quantify the effects of IS in patients with chronic constipation. Methods A double-blind crossover study in adults with functional constipation or constipation-predominant irritable bowel syndrome. Intervention: Metamucil Original Coarse Fibre® (P&G, USA) 14 g tds – daily IS dose 21 g. Placebo: Maltodextrin(MD) 14 g tds. On day 5 subjects swallowed 5 gadolinium filled capsules. On day 6 MRI scans were taken fasting and hourly for 7 hours while subjects ingested a rice pudding meal and treatment (IS or MD). Whole gut transit was assessed by the weighted average position score of the capsules 24 hours after ingestion (WAPS). Free water in the small bowel (SBWC) and ascending colon (ACWC) was measured as were T1 and T2 relaxation times in the ascending and descending colon (AC & DC), colonic volume and gas.Abstract OC-068 Figure 1 Results 16 subjects completed both treatments. Transit was faster after IS with a mean decrease in WAPS of 24% (p = 0.05, 1 tailed). Postprandial SBWC was markedly higher on IS (p < 0.001) with smaller increases seen in ACWC (p < 0.05). Fasting T1 was significantly higher after IS in both AC and DC. T2 fasting values were also higher. A postprandial rise was seen in both T1AC and T2AC after IS but not MD. Fasting colonic volume increased on PS by mean 332 mL or 48%. Exploratory analysis of colonic gas found that after IS significantly more was detectable both fasting (p < 0.05) and postprandially (p < 0.05). AC gas did not increase until 240 min after PS while transverse colon gas increased steadily through the day.Abstract OC-068 Table 1 Maltodextrin Ispaghula Mean difference(95% confidence interval) WAPS 3.4 (1.6–4.8) 2.2 (1.5–3.0) 0.8 (-0.2–1.7) Colonic Volume (mL) 690 (±55) 1022 (±60) 332 (213–451) T1AC (ms) 596 (±61) 829 (±98) 234 (15–453) T1DC (ms) 366 (±67) 613 (±94) 247 (82–411) Conclusion MRI parameters demonstrated accelerated in transit, increased intestinal water content and increasedcolonic volume with IS. Fasting T1 appears to discriminate constipation from health and responded to treatment. More colonic gas was detected with IS. This may reflect fermentation of IS or interference with small bowel absorption leading to malabsorption of carbohydrate in the rice meal. These novel findings illustrate the potential for MRI to provide insights into the in vivo effects and mechanisms of action of gut modulators. Disclosure of Interest None Declared

Extracellular cGMP, the downstream effector released in response to linaclotide-induced activation of guanylate cyclase-C, reduces excitability of murine and human dorsal root ganglion neurons

PP001 Nesfatin1 (30-59) injected intracerebroventricularly induces a delayed and long lasting anorexigenic effect by inducing satiation in rats P. TEUFFEL*, V. LEMBKE*, P. KOBELT*, T. HOFMANN*, M. GOEBEL-STENGEL, M. ROSE*, B. KLAPP* and A. STENGEL* *Department of Psychosomatic Medicine, Charit e University Hospital Berlin, Germany and Department of Internal Medicine, Martin-Luther Hospital Berlin, Germany

Demonstration of differences in colonic volumes, transit, chyme consistency and response to psyllium between healthy and constipated subjects using magnetic resonance imaging

In functional gastrointestinal disorders a lack of objective biomarkers limits evaluation of underlying mechanisms. We aimed to demonstrate the utility of magnetic resonance imaging for this task using psyllium, an effective constipation treatment, in patients and controls.

Linaclotide Attenuates Visceral Organ Crosstalk: Role of Guanylate Cyclase-C Activation in Reversing Bladder-Colon Cross-Sensitization

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 µg/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.

MP42-05 CHRONIC ORAL ADMINISTRATION OF THE GUANYLATE CYCLASE-C AGONIST LINACLOTIDE ATTENUATES COLITIS INDUCED BLADDER AFFERENT AND DORSAL ROOT GANGLION HYPERACTIVITY

INTRODUCTION AND OBJECTIVES: Nerve growth factor (NGF) has been implicated as an important mediator to induce C-fiber bladder afferent hyperexcitability, which contributes to the emergence of neurogenic detrusor overactivity (NDO) following spinal cord injury (SCI). In this study, we examined whether NGF neutralization using anti-NGF antibody normalizes the SCI-induced changes in electrophysiological properties of capsaicin-sensitive C-fiber bladder afferent neurons in the mouse model. METHODS: In female C57BL/6 mice, the spinal cord was transected at the Th8/9 level. Two weeks later, an osmotic pump was placed subcutaneously to administer anti-NGF antibody at 10 mg/kg/h for 2 weeks. Bladder afferent neurons were labeled with axonal transport of Fast Blue (FB), a fluorescent retrograde tracer, injected into the bladder wall 3 weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurons were prepared. Whole cell patch clamp recordings were then performed in FB-labeled bladder afferent neurons, and the data were compared between SCI and spinal intact (SI) mice. After recording action potentials (AP) or voltage-gated K (Kv) currents, the sensitivity of each neuron to capsaicin was evaluated. RESULTS: In capsaicin-sensitive bladder afferent neurons, the resting membrane potentials and the peak and duration of AP did not changed by SCI. On the other hand, the threshold for eliciting AP was significantly reduced in SCI vs. SI mice. Also, SCI increased the number of AP during 800 ms membrane depolarization. These SCI induced changes were reversed by NGF neutralization. SCI induced significant increases in the diameter and cell input capacitance of capsaicin-sensitive bladder afferent neurons, which were not reversed by NGF neutralization. Densities of slow decaying KA and sustained KDR currents evoked by depolarization to 0 mV were significantly reduced by SCI. NGF neutralization reversed the SCI-induced reduction in the KA current density. CONCLUSIONS: In SCI mice, NGF plays an important role in hyperexcitability of capsaicin sensitive C-fiber bladder afferent neurons due to KA current reduction. Thus, NGF-targeting therapies could be effective for treatment of afferent hyperexcitability and NDO in SCI.

PWE-171 Assessing The Utility Of Key Mri Parameters In Characterising The Mode Of Action Of A Proven Effective Laxative, Ispaghula

Introduction Half of the patients who suffer from chronic constipation are dissatisfied with their treatments, which has initiated a recent surge in novel treatments. Assessing the efficacy of these treatments requires techniques that are patient acceptable and non-invasive and allow characterisation of the underlying mode of action. This study assessed the utility of key parameters likely to give insight into laxatives’ mode of action including: colonic volumes, transit, ascending colon water content (ACWC) and small bowel water content (SBWC) by means of a randomised double-blind, placebo-controlled, crossover study (RCT) of a known effective laxative, ispaghula. Methods 16 healthy volunteers (24 ± 4 years old, BMI 23 ± 4 kg m-2) participated in this three-way RCT. They took either 21 g/day of ispaghula, 10.5 g/day of ispaghula or a placebo daily on days 1–6. On day 5 they swallowed 5 transit marker pills (TMP) filled with a dilute MR contrast agent and on day 6 were scanned serially for 7 h. The TMPs were assigned a weighted average position score (WAPS) based on their location in the bowel. Protocol compliance was assessed and 9 subjects were included in the per protocol analysis. Results (mean AUC±SEM, n = 9) Relative to the placebo, 21 g/day but not 10.5 g/day of ispaghula increased the ACWC (Figure 1) significantly (11 ± 4 L.min vs. 1.0 ± 0.5 L.min, p < 0.001), while SBWC (Figure 2) was increased significantly by both doses (68 ± 15 L.min vs placebo 25 ± 6 L.min, p= < 0.01), and (49 ± 11 L.min, p= < 0.05) respectively. Both doses significantly increased the total colon volume, and there was a significant increase in colon volume at fasted baseline measurement. Ascending colon T1 was also increased by the 21 g/day dose (p < 0.01), but 24 h WAPS for transit were not significantly changed by treatment. Abstract PWE-171 Figure 1 Time course of freely mobile water in the ascending colon (ACWC) throughout the MRI study day Abstract PWE-171 Figure 2 Time course of the freely mobile water in the small bowel (SBWC) throughout the MRI study day Conclusion The volume of water in both the ascending colon and the small bowel are significantly increased by ispaghula and could be useful biomarkers of a laxative effect. Ispaghula also increased colonic volumes and ascending colon T1 without altering transit times, suggesting it or its metabolites does not stimulate motility. These MRI methods could be readily used in assessing the mode of action of a range of novel agents in constipated patients will provide unique information on the mechanisms of action. Disclosure of Interest None Declared.

Determination of cGMP levels in rodent tissues following oral dosing of a soluble guanylate cyclase stimulator

Background In the vasculature, nitric oxide (NO) binds and activates smooth muscle soluble guanylate cyclase (sGC), leading to increased intracellular cGMP, which triggers smooth muscle relaxation and vasodilation. sGC stimulators are a class of small molecule allosteric modulators, which stimulate cGMP production independently of NO but also act in synergy with NO. Evidence to date suggests that sGC stimulators may be balanced vasodilators, meaning that they elicit vasorelaxation in both the arterial and venous vasculature; however, there have been conflicting reports [1,2]. Our approach to developing a better understanding of the

Sa1487 Gene Expression Profiling and Endothelin in Acute Experimental Pancreatitis

AIM:To analyze gene expression profiles in an experimental pancreatitis and provide functional reversal of hypersensitivity with candidate gene endothelin-1 antagonists.METHODS:Dibutyltin dichloride(DBTC) is a chemical used as a polyvinyl carbonate stabilizer/catalyzer,biocide in agriculture,antifouling agent in paint and fabric.DBTC induces an acute pancreatitis flare through generation of reactive oxygen species.Lewis-inbred rats received a single i.v.injection with either DBTC or vehicle.Spinal cord and dorsal root ganglia(DRG) were taken at the peak of inflammation and processed for transcriptional profiling with a cDNA microarray biased for rat brain-specific genes.In a second study,groups of animals with DBTC-induced pancreatitis were treated with endothelin(ET) receptor antagonists [ET-A(BQ123) and ET-B BQ788)].Spontaneous pain related mechanical and thermal hypersensitivity were measured.Immunohistochemical analysis was performed using anti-ET-A and ET-B antibodies on sections from pancreatic tissues and DRG of the T10-12 spinal segments.RESULTS:Animals developed acute pancreatic inflammation persisting 7-10 d as confirmed by pathological studies(edema in parenchyma,loss of pancreatic architecture and islets,infiltration of inflammatory cells,neutrophil and mononuclear cells,degeneration,vacuolization and necrosis of acinar cells) and the painrelated behaviors(cutaneous secondary mechanical and thermal hypersensitivity).Gene expression profile was different in the spinal cord from animals with pancreatitis compared to the vehicle control group.Over 260 up-regulated and 60 down-regulated unique genes could be classified into 8 functional gene families:circulatory/acute phase/immunomodulatory;extracellular matrix;structural;channel/receptor/transporter;signaling transduction;transcription/translation-related;antioxidants/chaperones/heat shock;pancreatic and other enzymes.ET-1 was among the 52 candidate genes upregulated greater than 2-fold in animals with pancreatic inflammation and visceral pain-related behavior.Treatments with the ET-A(BQ123) and ET-B(BQ-788) antagonists revealed significant protection against inflammatory pain related mechanical and thermal hypersensitivity behaviors in animals with pancreatitis(P 0.05).Open field spontaneous behavioral activity(at baseline,day 6 and 30 min after drug treatments(BQ123,BQ788) showed overall stable activity levels indicating that the drugs produced no undesirable effects on normal exploratory behaviors,except for a trend toward reduction of the active time and increase in resting time at the highest dose(300 μmol/L).Immunocytochemical localization revealed that expression of ET-A and ET-B receptors increased in DRG from animals with pancreatitis.Endothelin receptor localization was combined in dual staining with neuronal marker NeuN,and glia marker,glial fibrillary acidic protein.ET-A was expressed in the cell bodies and occasional nuclei of DRG neurons in na ve animals.However,phenotypic expression of ET-A receptor was greatly increased in neurons of all sizes in animals with pancreatitis.Similarly,ET-B receptor was localized in neurons and in the satellite glia,as well as in the Schwann cell glial myelin sheaths surrounding the axons passing through the DRG.CONCLUSION:Endothelin-receptor antagonists protect against inflammatory pain responses without interfering with normal exploratory behaviors.Candidate genes can serve as future biomarkers for diagnosis and/or targeted gene therapy.