P1711 Plecanatide and SP-333, Novel Agonists of Guanylate Cyclase-C,

Attenuate Visceral Hypersensitivity in Rat Models Kunwar Shailubhai,1,3 Illona-Marie Boulete,2 John Foss,1 Priya Eddy,1 Anusha Thadi,3 Apoorva Joshi,3 Viren Patwa,3

Vasila Theodorou,2 Vaseem Palejwala,1 and Lionel Bueno2 1Synergy Pharmaceuticals Inc., New York, NY and Doylestown, PA; 2UMR 1331 Toxalim INRA/INPT, Toulouse, France;

3Baruch S. Blumberg Institute, Doylestown, PA

Models of Visceral Hypersensitivity Animals: Male Wistar rats (n=8/group, 220-250g) were used. Colorectal Distention (CRD) Procedure: Rats were placed in a plastic tunnel 1 h after dosage. A balloon (4 cm long) fixed on a rigid catheter was inserted into the rectum to perform colorectal distension (CRD). The balloon, connected to a barostat was inflated progressively in steps of 15 mmHg, from 0 to 60 mmHg, each step of inflation lasting 5 minutes. Colonic volume was also monitored during the procedure. Electromyographic (EMG) Recording: Pairs of nichrome wire electrodes were implanted in the abdominal striated muscle of anesthetized animals at least 8 days prior to testing. The free ends of electrodes were exteriorized on the back of the neck. Electrical activity corresponding to abdominal contractions was recorded with an electroencephalograph machine during each 5-minute segment of the CRD procedure. 1. TNBS-induced Colorectal Hypersensitivity Rectal Inflammation: Colitis was induced in lightly anesthetized rats by intra-rectal instillation of TNBS (80 mg/kg in 50% ethanol). CRD Procedure: This evaluation was conducted 1-2 days before and 4 days following rectal instillation of TNBS. 2. Stress-induced Colorectal Hypersensitivity Partial Restraint Stress (PRS) Procedure: While under light anesthesia, the fore shoulders, upper forelimbs and thoracic trunk were wrapped in a confining harness of paper tape to restrict, but not prevent body movements. The animals were then placed in their home cage for 2 h. CRD Procedure: This evaluation was conducted 30 min after the stress sessions.

Cell Culture: Human colon carcinoma cells T84 and Caco-2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) and Ham's F-12 medium (1:1) containing 10% fetal bovine serum, 1% Glutamax, 1% penicillin and 1% streptomycin at 37 0C in a 5% CO2 controlled incubator. NF-kB Activation: T84 cells were stimulated with E. coli LPS and then treated with 8-Br-cGMP (positive control) or SP-333 for 16 hrs. Nuclear and cytosolic extracts were prepared. Nuclear extract was used for immunoblotting with antibodies for p-NF-kB-p65. Cytosolic extract was used for immunoblotting for IkB, p-IkB-a and IKK-b. Cytokine ELISAs: T84 cells were stimulated with LPS and then treated with indicated concentrations of SP-333 for 16 hr. After the incubation, cell culture media was used for measurement of TNF and IL-8 cytokines by ELISA. Results are expressed as an average of three determinations Permeability Studies: T84 and Caco-2 cells at a seeding density of 150,000 and 8,000 cells/insert, respectively were cultured in 12mm permeable polyester membrane (pore size 0.4 µm) Snapwell inserts. Transepithelial resistance (TER) of cells was monitored with an EVOM epithelial volt ohm meter. T84 cells were used when the TER of the monolayer was > 1000 Ω.cm2 (15-20 days post seeding), Caco-2 cells were used when TER > 400 Ω.cm2 (21-22 days post seeding). To induce tight junction damage, T84 and Caco-2 monolayers were incubated overnight with 100µg/ml LPS in the presence or absence of 1µM SP333. At the end of the incubation, 0.1mg/ml 70KDa rhodamine-dextran or 1mg/ml 3KDa FITC-dextran in Krebs Ringer buffer solution (115mM NaCl, 25mM NaHCO3, 3.3mM KH2PO4, 1.12mM MgCl2, 1.2mM CaCl2, and 10mM Hepes) was added to the apical compartment and incubated for 1h at 37°C. Fluorescence in 100 µl of basolateral buffer solution was measured in Tecan M-1000 plate reader. The excitation and emission wavelengths for FITC were 494 nm and 518 nm while that for rhodamine were 570 nm and 590 nm.

• Plecanatide and SP-333, structural analogs of the endogenous natriuretic peptide uroguanylin, activate intestinal GC-C resulting in increased cGMP production1

• SP-333 treatment suppresses production of pro-inflammatory cytokines, possibly via a cGMP-mediated inhibition of NF-kB activation in T84 cells

• SP-333 treatment attenuates LPS-induced disruption in cellular permeability in T84 and Caco-2 cells, thereby improving barrier function

• In rat models of visceral hypersensitivity, treatment with plecanatide or SP-333 reduces abdominal contractions (possibly due to suppression of inflammation)

• Plecanatide and SP-333 treatment reduced abdominal contractions across all distension pressures in the inflammatory (TNBS) and non-inflammatory (partial restraint stress) models

1. Shailubhai K, Jacob GS, Inventors. Synergy Pharmaceuticals Inc. (New York, NY, US), Assignee. Agonists of guanylate cyclase useful for the treatment of gastrointestinal disorders, inflammation, cancer and other disorders. United States patent US 7,879,802 B2. 2011.

Irritable bowel syndrome (IBS) is a gastrointestinal (GI) disorder with symptoms which include abdominal pain and altered bowel habits. The key mechanism of its pathophysiology is thought to be visceral hypersensitivity, which could be due to increased gut permeability associated with the loss of barrier function. GI inflammation may also be a contributory factor. The balance between the intracellular levels of cyclic AMP and cyclic GMP may be fundamental in the control of visceral hypersensitivity in response to inflammatory mediators. Plecanatide and SP-333 are structural analogs of uroguanylin (UG), an endogenous natriuretic peptide that activates guanylate cyclase-C (GC-C) expressed on the epithelial cells lining the GI mucosa. Activation of GC‐C stimulates cyclic GMP synthesis, a second messenger essential for regulation of ion and fluid transport, homeostasis in epithelial cells, maintenance of barrier function, and amelioration of GI inflammation. The objectives of the present study were to determine the effects of plecanatide or SP-333 on the following: • Activation of NF-κB signaling and expression of inflammatory

cytokines in LPS-treated T84 cells. • Cellular permeability in LPS-treated monolayers of T84 and

Caco-2 cells. • Colonic hypersensitivity in inflammatory (TNBS) and

non‐inflammatory (partial restraint stress, PRS) models in rats.