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1 Decrease in abdominal pain or discomfort ≥1.0 (5-pt scale) for ≥9 wks 2 CSBMs ≥3and increased ≥1 and straining/BSFS ≥1.0 (5-pt/7-pt scale) for ≥9 wks 3 'Yes' on binaryscale for ≥9 wks 4 Completely/considerably relieved ≥6 wks or at least somewhat relievedall 12 wks 5 Completely relieved for ≥2 wks or at least considerably relieved for all 4 wksfor ≥2 mos 6 Improvement ≥1.0 (5-pt scale) for ≥9 wks 7 Decrease ≥50% in 500-pt IBS-SSS scale at Week 12

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LX1031: A New Approach for Managing Irritable Bowel Syndrome (IBS)Philip Brown, Jessica Jackson, Zhi-Cai Shi, Kenneth Frazier, Qi M. Yang, Qingyun Liu,Stephen C. Pappas

PURPOSE: Gut serotonin (5-HT) plays an important role in regulating GI motility and theperception of visceral pain. Modulating gut 5-HT may be important in the pathophysiologyof IBS. 5-HT synthesis occurs in enterochromaffin cells and is rate limited by tryptophanhydroxylase (TPH). TPH inhibition represents a novel mechanism for reducing gut levelsof serotonin and influencing symptoms associated with GI disorders such as IBS. LX1031is a novel TPH inhibitor that provides local GI exposure with low systemic exposurefollowing oral administration. Preclinical studies demonstrated that LX1031 could reducegastrointestinal 5-HT in a dose-dependent fashion without affecting brain 5-HT. METHODS:LX1031 was evaluated in single and multiple dose studies in normal volunteers; dose levelsevaluated ranged from 250 mg to 4 g for up to 14 days. Subjects were sequestered overthe study period and diet was controlled. 24-hour urine samples were obtained at baselineand on days 5, 10 and 14 for evaluation of urinary 5-HIAA, a biomarker of 5-HT metabolism.RESULTS: Daily doses from 1 g to 4 g (250 mg to 1000 mg QID) over 14 days demonstratedsignificant reduction in 24-hour urinary 5-HIAA relative to placebo (Figure). Observationsat day 5 of therapy revealed reductions in urinary 5-HIAA ranging from 28% (250 mg QID)to 49% (1000 mg QID), suggesting a rapid onset of action which was maintained over theduration of therapeutic exposure. All dose levels were well tolerated, with no evidence of dose-limiting tolerability; adverse events were mild to moderate and resolved without sequelae; noserious adverse events occurred. CONCLUSIONS: Modulating enteric 5-HT production viainhibition of TPH represents a potential new approach to the treatment of GI disorders. Anovel oral TPH inhibitor, LX1031, was well tolerated in total daily doses up to 4 g over 14days. Significant reductions in urinary 5-HIAA indicate successful pharmacological inhibitionof the target enzyme in the GI tract. The reduction in 5-HT production, coupled with theobserved safety profile suggests a potential therapeutic rationale for LX1031 in patients withIBS and serotonin-mediated dysmotility.

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Effects of Rimonabant On Gastric and Small Intestinal Motility in DogsYan Sun, Geng-Qing Song, Jiande Chen

Background and Aims: Rimonabant (SR 141716A), a highly selective cannabinoid receptor1 (CB1) blocker, has been used for treating obesity. The aims of this study were to determinethe effects of rimonabant on gastrointestinal motility in dogs. Methods: 12 female dogs (18-22 kg) were involved in the study. Six dogs were equipped with a duodenal cannula forthe measurement of gastric emptying and small bowel contractions. The other six dogs wereequipped with a gastric cannula for the measurement of gastric motility. The study wascomposed of four experiments as follows: gastric emptying; gastric tone, accommodationand compliance; antral contractions; and small-bowel contractions. Each experiment includedthree randomized sessions (control and rimonabant at doses of 1mg/kg and 0.5mg/kg).Results: 1) Rimonabant (1mg/kg) accelerated gastric emptying of liquids from 62.3 ± 7.7%at 75 min and 67.2 ± 7.7% at 90 min in the control session to 72.1 ± 5.1% at 75 min (p =

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0.003, vs. control) and 76.8 ± 5.3% at 90 min (p=0.005, vs. control) in the session withrimonabant; 2) Rimonabant increased gastric tone, and reduced gastric compliance andaccommodation. The gastric volume was 122.6 ± 10.9 ml in the control session, 93.8 ± 8.8ml with rimonabant 1mg/kg (p=0.028, vs. control) and 106.7 ± 7.0 ml with rimonabant0.5mg/kg (p=0.056, vs. control); Rimonabant with 0.5mg/kg and 1mg/kg decreased gastricaccommodation from 406.8±15.59 ml in the control session to 371.3± 10.3 ml and 298.7±11.4 ml, respectively (P=0.015 vs. control); Gastric compliance was significantly decreasedfrom 9.59 ± 0.72 ml/mmHg in the control session to 7.97 ± 1.15 ml/mmHg with rimonabant1mg/kg and 8.01±0.58 ml/mmHg with rimonabant 0.5mg/kg (P=0.0002 vs. control). 3)Rimonabant inhibited antral contractions. The antral contraction index was 10.06 ± 1.44in the control session and decreased to 7.30 ± 1.46 with rimonabant 0.5mg/kg (P=0.069vs. control) and 5.76±1.15 with rimonabant 1mg/kg (p=0.001,vs. control). 4) Rimonabantincreased small intestinal contractions. The small intestinal contraction index was 9.60 ±2.44 in the control session and increased to 12.35±1.45 with rimonabant 0.5mg/kg (p=0.018vs. control) and 14.75 ± 2.46 with rimonabant 1mg/kg (p=0.008 vs. control). Conclusions:Rimonabant reduces gastric compliance and accommodation by increasing gastric tone; italso inhibits antral contractions but increases intestinal motility. These findings suggest theperipheral mechanisms of rimonabant in reducing food intake and causing weight loss inobesity patients. The CB1 receptor antagonist has different effects on motility of the gastricfundus, gastric antrum and small intestine.

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A Double-Blind, Randomized, Placebo-Controlled Study of Mirtazapine inFunctional Dyspepsia with Weight Loss.Lukas Van Oudenhove, Lieselot Holvoet, Raf Bisschops, Philip Caenepeel, Joris Arts,Dominiek De Wulf, Rita Vos, Daniel Sifrim, Jan F. Tack

Introduction: Therapeutic options in functional dyspepsia (FD) are limited, and this isespecially challenging when symptoms are severe and associated with weight loss. Anecdotalobservations suggest a therapeutic potential for mirtazapine, a dual action (noradrenergic-serotonergic) antidepressant with H1-, α2-, 5-HT2c- and 5-HT3-receptor antagonistic proper-ties. Our aim was to conduct a placebo-controlled trial of mirtazapine in FD with weightloss. Methods: FD patients who lost >10% of original body weight and without depressionor anxiety disorder were recruited for the study. After baseline assessment of solid andliquid gastric emptying, gastric barostat study and a nutrient challenge test, patients wererandomized to placebo or mirtazapine 15 mg in the evening, in a double-blind randomizedfashion. Evaluations during 2-week baseline and 8-week treatment period included FDsymptom severity, the short form Nepean Dyspepsia Index (SF-NDI) quality of life inFD, the hospital anxiety and depression scale (HADS), patient health questionnaire (PHQ:somatization, panic & depression modules) and gastrointestinal-specific anxiety (visceralsensitivity index, VSI). Gastric emptying and nutrient challenge tests were repeated after 8weeks. Data (mean±SEM) were compared by t-test and by two-way ANOVA. Results: Weenrolled 19 FD patients (18 women, mean age 35±3) with a weight loss of 13.2±2.8 kg.Treatment with mirtazapine, but not placebo, was associated with a significant improvementof overall FD symptom severity scores (respectively 14.7±2.4 vs. 6.0±2.3, p<0.05 and13.2±1.6 vs. 10.5±2.1, NS). Early satiety was significantly improved by mirtazapine (2.1±0.1vs. 1.0±0.3, p<0.05), but not by placebo (1.6±0.3 vs. 1.7±0.3, NS). Significantly improvednutrient tolerance (increase of maximum tolerated nutrient volume 75±15 vs. -50±22 ml,p<0.05) and higher weight recovery (6.5±1.6 vs. 0.4±0.5 kg, p=0.001) occurred withmirtazapine compared to placebo. No significant changes occurred in gastric emptyingrate, HADS or PHQ score. Mirtazapine, and not placebo, was associated with significantimprovement in VSI score (respectively 62.7±5.8 vs. 71.6±5.6, p<0.05 and 58.8±5.2 vs.60.7±5.8, NS) and in the NDI domains of interference with daily life and food/drink (bothp<0.05 for mirtazapine and NS for placebo). Conclusion: Mirtazapine significantly improvesoverall FD symptoms, quality of life and gastrointestinal-specific anxiety in FD with weightloss. The favorable effect of mirtazapine is associated with improvement of early satiety andnutrient tolerance, and better recovery of weight loss.

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A Double-Blind, Placebo-Controlled Trial to Evaluate the Safety andTolerability of Prucalopride Oral Solution in Constipated Elderly PatientsLiving in a Nursing FacilityMichael Camilleri, Rene Kerstens, Greet Beyens, Patricia Robinson, Lieve Vandeplassche

Background: Prucalopride is a selective 5-HT4 receptor agonist; In Vitro studies show thatselectivity for the 5-HT4 receptor is at least 150 times higher than for any other receptor/channel, including hERG. Objective: To assess the cardiovascular (CV) safety of prucalopride(PRU) in elderly constipated patients. Methods: Phase II, randomized, double-blind, dose-escalation safety study. Patients were treated for 4 weeks with placebo (PLA) or PRU 0.5mg, 1 mg or 2 mg. Vital signs, ECG and laboratory tests (lab) were assessed prior to and3 h after intake of study medication. Centralized ECG analysis was performed for QTcinterval, rhythm, arrhythmia, conduction, morphology, myocardial infarction, ST segment,T- and U waves. Continuous Holter monitoring was performed 24 h before and 24 h afterthe first intake of study medication, and for 24 h on days 7 and 14. Holter data were usedto assess “possible pro-arrythmic effects” (Morganroth J, Am J Cardiol 1987;59:32E-37E).Results: Among 89 patients enrolled, the mean age was 83 yrs and 80% had a history ofCV disease. PRU, at all doses tested, was safe and well tolerated. Of all adverse events (AEs),only diarrhea (PLA: 0%, PRU: 5-15%) and nausea (PLA: 0%, PRU: 8-10%) were morefrequent on PRU. The frequency of CV AEs, SAEs and AEs leading to discontinuation waslow, with no differences between treatment groups. Evaluation of vital signs, lab results,and ECG parameters revealed no clinically important findings; in particular, no differencesbetween treatment groups were observed in QTcF (Table 1). Holter monitoring revealed noevidence of a “pro-arrythmic” effect, with no significant differences supra-ventricular rhythmdisturbances and no events of sustained ventricular tachycardia, ventricular fibrillation ortorsades de pointes. Five patients experienced tachycardia during PRU treatment; all hadmultiple CV disorders at entry and used concomitant medications labelled for tachycardia;in three patients these events were considered not related or doubtfully related to PRU.

Conclusions: PRU up to 2mg o.d. for 4 weeks was safe and well tolerated by constipatedelderly patients. There were no significant differences vs. PLA in ECG parameters, or indica-tions of pro-arrhythmic effects on Holter monitoring.Number of patients with normal QTcF at baseline and borderline, prolonged and > 500 msQTcF intervals during treatment

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Interleukin (IL)-25 Inhibits IL-12 Production and TH1 Cell- DrivenInflammation in the GutRoberta Caruso, Massimiliano Sarra, Carmine Stolfi, Angelamaria Rizzo, Massimo C.Fantini, Francesco Pallone, Thomas T. MacDonald, Giovanni Monteleone

Background. In Crohn's disease (CD), the tissue-destructive Th1 cell-mediated inflammationis driven by interleukin (IL)-12, a cytokine produced by mucosal CD14+ monocyte-likecells. Factors/mechanisms that control this pathogenic response are not yet fully understood.IL-25 is a recently identified cytokine that promotes Th2 cell responses by targeting MHCclass II-positive non-T/non-B cells. Objective. Since Th1/Th2 cells and the cytokines theyrelease are mutually antagonistic, we examined whether IL-25 controls IL-12 productionand Th1 cell-mediated inflammation in the gut. Methods. IL-25 receptor (IL-25R) wasevaluated in intestinal lamina propria mononuclear cells by flow-cytometry, and IL-25 wasexamined in inflamed and control mucosal samples by real-time PCR and Western blotting.CD mucosal CD14+ cells were treated with IL-25 and/or LPS/PGN. IL-25 was injectedintraperitoneally in mice with PGN- and TNBS-colitis. To assess the role of IL-13 in thenegative regulation of PGN-colitis by IL-25, a blocking IL-13 antibody was administeredbefore IL-25 treatment. Cytokines were evaluated by real-time PCR and/or ELISA. Results.CD mucosal CD14+ cells expressed high levels of IL-25R and responded to IL-25 bydecreasing the synthesis of IL-12 and IL-23. In Vivo IL-25 largely prevented and reversedexperimental Th1 mediated-colitis. IL-25 treatment induced IL-13 in the colon, but thiscytokine was not required for suppression of PGN-colitis. Significant less IL-25 productionwas found in patients with CD and patients with ulcerative colitis in comparison to controls.Discussion. Data show that IL-25 inhibits IL-12 production and IL-12-driven Th1-mediatedinflammation in the gut, thus suggesting that IL-25 may be therapeutically useful in CD.

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Melatonin Prevents Colitis-Induced Dysplasia Through Inhibition of PI3K-Mediated β-Catenin SignalingGoo Lee, Tatiana Goretsky, Ajaypal Singh, Jae Wook Dong, Dong Hoon Kim, James B.Young, Terrence A. Barrett

Background/Aim; Our studies in human (Sun et al. DDW 2008)suggest PI3K-mediated β-catenin signaling in crypt epithelial progenitors increases in colitis and colitis-induced cancer.Melatonin is known as a sleep-inducing drug. Recent data suggest that melatonin reducesoxidative injury and inhibits proliferation as well as ameliorates inflammation in humanlung disease (ARDS). The aim of this study is to examine chemopreventive effect of melatoninin IBD through inhibition of PI3K/Akt pathway. Methods;Piroxicam-induced (D0-D14)IL10-/- mouse model of colitis-induced dysplasia was used. Melatonin was administered i.p.at the doses of 2 mg/kg (M), 2 mg/kg with 100 mg/Kg of 5-ASA p.o. (M-5ASA), and 5-ASAonly (5ASA) for 6 weeks (D14-D56) daily. Colitis scores and dysplasia were examined byH&E. To examine PI3K/Akt pathway, phosphorylated-Akt (P-Akt) and Akt-phosphorylatedactive, nuclear β-catenin (P-β-catenin, He et al. Nat Genet 2007) were assessed by IHC andwestern blot (WB). 2hr BrdU label and nitrotyrosine (NT) were detected by IHC. Colonictissue cytokine levels were measured by real time PCR (RT-PCR). Results; As shown inTable, untreated mice exhibited severe colitis with increased dysplasia, cytokines (TNF, IL-1β,IFN-γ), nuclear P-Akt, P-β-catenin, BrdU incorporation and NT (+) stromal cells. Melatoninsignificantly reduced colitis scores by 50-90% and dysplasia incidence by 60-90%. WBanalyses confirmed that epithelial nuclear P-Akt and P-β-catenin levels were significantlyreduced by melatonin. Combination therapy revealed better outcomes than either 5-ASA ormelatonin alone. Conclusion; Melatonin effectively blocks the PI3K/Akt-mediated β-cateninpathway in crypt epithelial progenitors in chronic colitis and prevents progression of colitis-induced dysplasia. The synergistic effect of melatonin+5-ASA may relate to both agents'reducing PI3K signaling.Results of Colitis score, Dysplasia incidence, IHC (P-Akt, P-β-catenin,BrdU, Nitrotyrosine), and RT-PCR (IFNγ, IL-1β, TNF)

mean±SD, colitis score; total 13 (Seril et als Dig Dis Sci 2002), P-Akt, P-β-catenin, andBrdU; (+)epithelial cells/HPF, nitrotyrosine; (+) stromal cells/HPF, IFNγ , IL-1β, and TNF;

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mRNA fold induction, *p<0.05 compared to control, # p<0.05 compared to D56 untreated,$ p<0.05 comapared to 5ASA or M

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Use of Enterally-Delivered Angiotensin II Type IA Receptor Antagonists toReduce the Severity of ColitisManabu Okawada, Hiroyuki Koga, Scott D. Larsen, Hollis D. Showalter, Peter C. Lucas,Daniel H. Teitelbaum

Purpose: Renin-angiotensin blockade can reduce intestinal inflammation and apoptosis.Based on the finding of a 4-fold increase in angiotensin II typeIa receptor(AT1aR)expressionin a dextran sodium sulfate (DSS;2.5%water) colitis model, we targeted blockade with AT1aRantagonists (AT1aR-A). Because a major consequence of AT1aR-A use is hypotension, wehypothesized that the use of AT1aR-A compounds which lack mucosal absorption, wouldallow for direct enteral delivery at far higher concentrations than would be tolerated systemic-ally, yet retain efficacy. Methods: Using the anti-hypertensive losartan as a structural AT1aR-A model, the compound deschloro-losartan(DCL)was produced which has extremely poorcell membrane permeability. To determine AT1aR-A efficacy, we capitalized on the fact thatAT1aR signaling leads to NF-κB activation;and used a HepG2A cell line stably transfectedwith AT1aR, and transiently with an IKKB luciferase reporter gene. DSS colitis was inducedin C57BL/6 mice and both AT1aR-A were given at 10-fold higher doses than typically givensystemically(QD,0.25 ml transanally) and compared to placebo (water) for 7 days. Results:DCL showed similar AT1aR blockade vs. losartan (85% vs. 90% inhibition of NF-κB activationat 60μM dosing, respectively). Both compounds significantly decreased clinical and histologiccolitis scores and apoptosis(Table1). Proinflammatory cytokine expression significantlydecreased with both compounds (Table2). To test the compound's systemic effect bloodpressure (Delta BP), and mucosal and colonic blood flow (MBF and CBF via laser Doppler)was determined. Losartan resulted in an increase in Delta BP and decline intestinal bloodflow; DCL did not affect either (Table1). Conclusions: This study demonstrated efficacy forhigh-dose AT1R-A in reducing the severity of DSS colitis. The results suggest that interactionbetween the angiotensinII and AT1aR might be implicated in the pathogenesis of DSS colitis.We postulate that specially designed AT1R-A with poor oral absorption may have greatpotential as a new therapeutic agent for inflammatory bowel disease.Table1

*P<0.05; **P<0.01; †P<0.001. ‡vs. placebo; ‡‡ vs. Naïve using ANOVA.Table2

*P<0.05; **P<0.01; †P<0.001

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Anti-Interleukin (IL)-21 Monoclonal Antibody Reduces Disease Severity andInflammatory Cytokines in Murine ColitisKatherine E. Lewis, Janine Bilsborough, Mark F. Maurer, Kristen Bontadelli, Kim Waggie,Cecile M. Krejsa, Stacey R. Dillon

Interleukin (IL)-21 is a type I cytokine expressed by specialized subsets of activated CD4+T cells and by natural killer T (NK-T) cells. In mice, IL-21 is produced by Th17 and TFH(follicular helper) T cells, while in humans it is expressed by Th1, Th17, TFH and NK-Tcells. Recent evidence suggests that IL-21 plays a role in several autoimmune and inflammat-ory diseases, including inflammatory bowel disease (IBD). IL-21 is overexpressed in inflamedtissues from IBD patients, and has direct effects on tissues and cell types associated withIBD pathogenesis. IL-21 receptor (IL-21R) is expressed on both hematopoietic and non-hematopoietic cells in IBD patients, including fibroblasts and epithelial cells from patientswith Crohn's disease. We are developing a neutralizing human anti-human IL-21 monoclonalantibody (mAb) for clinical use and here describe our evaluation of the efficacy of a neutraliz-ing anti-mouse IL-21 mAb in a CD4+CD25- T-cell transfer SCID mouse model of colitis.In mice treated prophylactically with 0.4 mg/week (20 mg/kg) anti-IL-21 mAb, we observedsignificant (p < 0.05 or better) prevention of body weight loss and significant reductions incolitis symptoms (stool consistency and hemoccult) throughout the study period comparedto mice treated with vehicle (PBS) or an isotype control mAb. By the end of the study (day45), mice treated with anti-IL-21 mAb were at 100% of starting body weight compared toonly ~84% of starting body weight in the control groups. Anti-IL-21 mAb-treated mice had6-7-fold lower average clinical colitis scores and significant reductions in histological indicesof colitis (severity of inflammation and morphologic abnormalities) compared to PBS- andisotype control-treated mice. Mice treated with IL-21 mAb also had significantly lower serumIL-6, RANTES, TNF-alpha, and MIP-1-beta levels compared to PBS-treated mice, further

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