US 2016O235807A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0235807 A1 SHALUBHA (43) Pub. Date: Aug. 18, 2016

(54) AGONSTS OF GUANYLATE CYCLASE Publication Classification USEFUL FOR DOWNREGULATION OF PRO-NFLAMMLATORY CYTOKINES (51) Int. Cl. A638/10 (2006.01) (71) Applicant: SYNERGY PHARMACEUTICALS, A619/00 (2006.01) INC., New York, NY (US) A6II 45/06 (2006.01) A613 L/40 (2006.01) (72) Inventor: Kunwar SHAILUBHAI, Audubon, PA A 6LX3/5377 (2006.01) (US) (52) U.S. Cl. CPC ...... A61K 38/10 (2013.01); A61K 31/40 (21) Appl. No.: 15/026,560 (2013.01); A61 K3I/5377 (2013.01); A61 K (22) PCT Fled: Oct. 9, 2014 45/06 (2013.01); A61 K9/0053 (2013.01) (57) ABSTRACT (86) PCT NO.: PCT/US1.4/S9914 This invention provides a method to prevent, control, and/or S371 (c)(1), treat an inflammatory disease or disorder by administering at (2) Date: Mar. 31, 2016 least one agonist of guanalyte cyclase receptor, or pharma ceutical compositions thereof, either alone or either concur Related U.S. Application Data rently or sequentially with another compound or an active (60) Provisional application No. 61/888,744, filed on Oct. agent used to treat the disease or disorder, and/or with an 9, 2013. inhibitor of c(GMP-dependent phosphodieasterases. Patent Application Publication Aug. 18, 2016 Sheet 1 of 31 US 2016/0235807 A1

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AGONSTS OF GUANYLATE CYCLASE dithiocarbamate (PTDC). In a further embodiment, the c-Src USEFUL FOR DOWNREGULATION OF tyrosine kinase inhibitor is KX2-391. PRO-NFLAMMLATORY CYTOKINES 0005. The invention provides a method for preventing or treating a condition by administering to a subject in need of a CROSS REFERENCE TO RELATED therapeutically effective amount of the composition of the APPLICATIONS invention. For example, the condition is colitis, ulcerative 0001. This application claims the benefit of U.S. Provi colitis, Crohn's disease, irritable bowel syndrome (IBS), non sional Application No. 61/888,744, filed Oct. 9, 2013, which ulcer dyspepsia, chronic intestinal pseudo-obstruction, func is herein incorporated by reference in its entirety for all pur tional dyspepsia, colonic pseudo-obstruction, duodenogas poses. tric reflux, constipation, constipation associated with use of opiate pain killers, post-Surgical constipation, IBS-associated FIELD OF THE INVENTION constipation, constipation associated with neuropathic disor ders, gastroesophageal reflux disease (GERD), Celiac dis 0002 The present invention relates to the therapeutic use ease, gastroparesis, heartburn, poor gastrointestinal motility, of guanylate cyclase C (GC-C) agonists for downregulation congestive heart failure, hypertension, benign prostatic of pro-inflammatory cytokines. The agonists may be used hyperplasia (BPH), gastrointestinal cancer, lung cancer, blad either alone or either concurrently or sequentially with addi der cancer, liver cancer, salivary gland cancer, skin cancer, tional active agents to prevent or downregulate NF-KB acti colon cancer, bronchitis, tissue inflammation, organ inflam Vation and pro-inflammatory cytokines in the human body. mation, respiratory inflammation, asthma, COPD, lipid The GC-C agonists may be used to prevent or treat colitis, metabolism disorder, biliary disorder, cardiovascular disease, including dextran sulphate sodium (DSS) induced colitis, obesity or an endocrine disorder. ulcerative colitis, Crohn's disease, colon cancer, and/or any 0006. The invention provides a method of treating or alle Swelling or inflammation of the large intestine. viating a symptom of a NF-kB mediated inflammation by BACKGROUND OF THE INVENTION administering to a subject in need thereofan effective amount of a GCRA peptide or pharmaceutical composition thereof. 0003. The human chronic inflammatory bowel diseases The invention provides that the amount is effective to inhibit (IBD), Crohn's disease (CD) and ulcerative colitis (UC), NF-KB activation, thus treating or alleviating a symptom of affect over one million Americans. While the etiology of an inflammatory disorder or a NF-kB mediated inflamma these disorders remains unknown, contributing factors tion. For example, the inflammatory disorder or a NF-KB include a poorly regulated immune response against the mediated inflammation is colitis, ulcerative colitis, Crohn's enteric microbiota in a genetically predisposed individual. disease, irritable bowel syndrome (IBS), tissue inflammation, There is currently no cure for IBD and existing therapies such organ inflammation, kidney inflammation, gastrointestinal as corticosteroids, 5-aminosalicylates, and immunomodula system inflammation, necrotizing enterocolitis, pancreatic tory agents (6-mercaptopurine, methotrexate) are of limited inflammation, lung inflammation, respiratory inflammation, effectiveness, have the potential for side effects, and/or are asthma, COPD or skin inflammation. designed to non-specifically reduce intestinal inflammation. 0007. The invention further provides a method of modu As a result of the burden of their clinical symptoms and side lating NF-KB induction in a cell by contacting the cell with an effects of medications, most patients with IBD have a signifi effective amount of a GCRA peptide orpharmaceutical com cantly impaired quality of life. Due to complications of the position thereof, where the GCRA peptide inhibits NF-KB disease, Surgical intervention occurs in a significant propor activation. tion of patients over their lifetime. Increased understanding of specific immune pathways that modulate inflammation in 0008. The invention also provides a method of modulating IBD has led to the FDA approval of anti-TNF antibodies in NF-KB-dependent target gene expression in a cell by contact CD and UC. Although a significant therapeutic advance, these ing the cell with an effective amount of a GCRA peptide or agents are effective in less than 40% of patients long term, pharmaceutical composition thereof. For example, the NF there is the potential for short- and long-term systemic side KB-dependent target gene is IL-1, IL-2, TNF, IL-12p40, effects, high costs, and the need to deliver the drug on a IL-17, IL-23, IL-8, RANTES, MIP-1C. or IL-10. repeated maintenance basis by injection. Therefore, there are 0009. In some embodiments, an effective amount of a urgent needs to identify targets that provide safer and effec GCRA peptide is sufficient to inhibit NF-kB activation. tive means of therapeutic intervention. 0010. In some embodiments, an effective dose of a cQMP dependent phosphodiesterase inhibitor is also administered SUMMARY OF THE INVENTION to a subject in need thereof. In some embodiments of the 0004. The invention provides a composition that includes current invention, the coMP-dependent phosphodiesterase a guanylate cyclase receptor agonist (GCRA). In some inhibitor may be administered either concurrently or sequen embodiments, the invention provides a composition that tially with a GCRA peptide, or pharmaceutical composition includes a guanylate cyclase receptor agonist (GCRA) and thereof. In some embodiments, the coMP-dependent phos another therapeutic compound. In one embodiment, the addi phodiesterase inhibitoris Sulindac Sulfone, Zaprinast, motapi tional therapeutic compound is a NF-KB inhibitor, a c-Src Zone, Vardenafil, or sildenafil. inhibitor, or 5-ASA. In another embodiment, the guanylate 0011. In some embodiments, the invention provides con cyclase receptor agonist is a CGRA peptide. In another current or sequential administration of an anti-inflammatory embodiment, the guanylate is plecanatide (SP-304) or agent with a GCRA peptide or a pharmaceutical composition SP-333. The composition of the invention may further thereof, to a subject in need thereof. The anti-inflammatory include a pharmaceutical carrier, excipient or diluent. In a agent is a steroid or nonsteroid anti-inflammatory drug further embodiment, the NF-KB inhibitor is pyrrolidine (NSAIDs). US 2016/0235807 A1 Aug. 18, 2016

0012. The GCRA peptide may be any one of Tables 1-7. cells incubated with SP-333 subsequent to LPS treatment Preferably, the GCRA peptide is Plecanatide (SP304), SP333 Versus corresponding secretion observed in cells treated with or SP373. LPS alone. 0013. Other features and advantages of the invention will 0018 FIG. 5A-B is a series of graphs showing that Ple be apparent from and are encompassed by the following canatide treatment ameliorates GI inflammation in chemi detailed description and claims. cally induced colitis in BDF1 mice. Efficacy of plecanatide in DSS (A) and TNBS (B) induced colitis was examined as BRIEF DESCRIPTION OF DRAWINGS described. Plecanatide formulated in 0.1M phosphate buffer (pH 7) was administered by oral gavage, once a day at indi 0014 FIG. 1 is a graph showing amino acid sequences of cated dosage. Sulfasalazine (80 mg/kg) was used as reference human uroguanylin and its synthetic analogs Plecanatide and compound. At the end of the study, mice were sacrificed and SP-333. Single letter abbreviations for amino acids are distal section of the large intestine was fixed and embedded in depicted. In designing synthetic hexadecapeptides, aspartic paraffin. Colitis severity Scores were assigned after visualiza acid (D) at position 3 from the NH2-terminus of UG is sub tion of H&E stained sections. All slides were scored in a stituted with glutamic acid (E). This substitution stabilizes the blinded manner. Mean severity score-SEM plotted for indi peptides in an active conformation in aqueous media. In addi cated treatment groups. A significantly lower DSS induced tion, SP-333 has D-stereoisomers of asparagine (N) and leu colitis score was observed in animals that were administered cine (L) instead of the L-forms at position 1 and 16, respec 0.005 mg/kg of plecanatide compared to untreated controls tively. It is designed to be particularly stable against (p=0.02) (A). In TNBS induced colitis model, animals that proteolytic degradation that normally occurs in intestinal received plecanatide at 0.05 mg/kg and higher, exhibited sig fluid as part of the normal digestive process. Uroguanylin as nificantly lower colitis scores as compared to their respective well as its analogs have four cysteine (C) residues enabling untreated controls. In both models, plecanatide treatment was the formation of two intramolecular disulfide bonds. Substi superior to treatment with reference compound. Statistical tuted amino acids in plecanatide and SP-333 are shown in significance calculated by comparing severity score observed bold type. for plecanatide or SulfaSalazine treated group versus corre sponding score for vehicle treated group. All slides were 0015 FIG. 2 is a graph showing stimulation of c(3MP scored in a blinded manner. Mean histological severity of accumulation in T84 cells by plecanatide and SP-333. Syn colitis score-SEM plotted for the indicated treatment groups. thetically generated peptides are able to activate GC-C recep Statistical significance was calculated by comparing severity tors and stimulate accumulation of c(GMP in T84 cells. The score observed for plecanatide or Sulfasalazine treated group activity was assayed as described earlier''. Results are Versus corresponding values in the vehicle treated group. expressed as an average of three determinations-tSD. 0019 FIG. 6A-B is a series of graphs showing Effects of 0016 FIG.3A-D is a series of panels showing that SP-333 plecanatide treatment on GI Inflammation (A) and secretion inhibits LPS induced activation of NF-kB in T84 cells by a of the pro-Inflammatory cytokines IL-12p40, IL-23 and TNF cGMP mediated mechanism. T84 cells were stimulated with in intestinal explants derived from TNBS-induced colitis in LPS (10 ug/ml) for 4 h and then treated either with 8-Br BALB/C mice (B). Colitis was induced in Balb/c mice via cGMP (A) or SP-333 (B) in the presence of 500 uMZaprinast rectal instillation of TNBS. Mice were administered an oral for 16 h. Subsequent to the treatment, nuclear and cytosolic gavage of vehicle or plecanatide (0.5 and 2.5 mg/kg) on day 0 extracts were prepared. Nuclear extract was used to measure and animals were euthanized on day 7 and colitis scores were phosphorylated p65 levels and cytosolic extract was used to determined (A). Colon tissues from the study were harvested examine levels of IKB, phosphorylated IKB and IKK-f3 by for explant culture. Cells from explants were cultured for 24 Western blot. IKK-C/B phosphorylation was examined by hin the presence and absence of plecanatide. At the end of the stimulating T84 cells by LPS (10 g/ml. 4 h) followed by 2h incubation, culture media was Snap frozen until cytokine treatment with SP-333 in the presence of 500 uM Zaprinast analysis was performed. Average levels: SD of IL-12 p40, (C). Cytosolic extracts were prepared and levels of activated IL-23 and TNF in treated explant culture are depicted in B. IKK-C/B, total IKK-B and actin were detected by Western Mice administered plecanatide exhibited significantly lower using appropriate antibodies. Representative immunoblot colitis scores (p<0.05) and correspondingly lower secretion depicting the levels of phosphorylated p65, IKB, p-IKB and of pro-inflammatory cytokines IL-12 p40, IL-23 and TNF as total IKK-B, phosphorylated IKK-C/B and actin are shown. compared to untreated controls. Plotted values represent Relative transcript levels normalized to GAPDH levels in the mean-SEM. sample from three independent determinations--SD are 0020 FIG. 7A-C is a series of graphs showing that ple depicted in panel (D). canatide abrogates colitis in TCRO. mice. Oral adminis 0017 FIG. 4 is a series of graphs showing that SP-333 tration of plecanatide (0.5 and 2.5 mg/kg) for 14 days reduced inhibits LPS-induced secretion of pro-inflammatory cytok the colitis score (A). At the end of the study, colon tissues ines by T84 cells. IL-8 (A) and TNF (B) levels were estimated were harvested and a portion was used for histopathological by ELISA in supernatants of LPS stimulated T84 cells treated colitis scoring, and the remainder was immediately processed with 0.1, 1 and 10 uM of SP-333 as described. Each ELISA for explant culture for 24 hours. The culture media was snap was performed in triplicate with cell-free supernatants from frozen until analysis of IL-17, RANTES, MIP-1C, and IL-10. two independent experiments. The protein concentration of Intestinal explant cultures derived from plecanatide treated each well was assessed by Bio-Rad protein dye detection kit. and untreated mice exhibit reduced secretion of pro-inflam The cytokine/protein ratio was employed to express the matory cytokine (IL-17) and chemokines (RANTES and cytokine production and each result is expressed as the mean MIP-1a) and increased production of anti-inflammatory value of independent experiments+SD. Statistical signifi cytokine IL-10 as compared to vehicle treated Samples (Fig cance calculated by comparing cytokine secretion from T84 ure B and C). Plotted values represent meant-SD. US 2016/0235807 A1 Aug. 18, 2016

0021 FIG. 8 is a series of graphs showing stability and BDF1 mice. Samples derived from mice administered 0.005 biological activity of plecanatide and SP-333 in SIF. HPLC mg/kg SP-333 exhibit the greatest percentage of crypts with chromatographic analyses of plecanatide and SP-333 after normal Ki-67 labeling (A). Myeloperoxidase activity is digestion with heat inactivated SIF for 300 min (B and E) or shown as average increase in absorbance. Values are normal SIF for 120 min. (Cand F) respectively. SIF incubation com ized to a sample protein concentration of 10 mg/ml. DSS/ pletely converts plecanatide into a shorter peptide eluting at vehicle group demonstrated the largest increase in absor 9.4 min (indicated by * in C). Arrows indicate the position of bance. With the exception of mice administered 0.5 mg/kg, plecanatide. As expected, SP-333 is resistant to digestion by absorbance values in samples from SP-333 treatment groups SIF (F). Cyclic GMP synthesis by T84 cells in response to are significantly lower than DSS vehicle group (p<0.05) (B). treatment with control or SIF digested plecanatide and Statistical significance calculated by comparing % crypt SP-333 is shown in A and D. cGMP stimulation at 0 min is labeling or MPO activity observed for SP-333 or 5ASA taken as 100%. The activities in samples at indicated incuba treated group versus corresponding score for vehicle treated tion time is calculated as percent of that observed at 0 min. group. MPO activity is significantly lower in colon tissues The data is average of triplicates-SD. After 6 h of incubation from animals dosed with 0.05 mg/kg of SP-333. in SIF, SP-333 retains most of its biological activity while 0025 FIG. 12 is a schematic illustration of the proposed plecanatide retains ~70% of its activity after 2.5-5 h incuba mechanism for the anti-inflammatory effect of synthetic UG tion in SIF. analogs. Binding of plecanatide and/or SP-333 to GC-C 0022 FIG. 9A-B is a series of graphs showing that oral receptor located on the apical surface of intestinal epithelial administration of SP-333 abrogates DSS-induced colitis in cells results in receptor activation and increased intracellular BDF-1 mice. The figure depicts results for colitis severity cGMP production, leading to the activation of PKGII (A), and disease activity index (B). BDF1 mice (n=12) were Enhanced ccjMP levels down regulate NF-KB signaling by administered with 5% DSS in drinking water to induce colitis blocking activation of IKK kinases necessary for phosphory on day 1. Oral gavage with 5-ASA and vehicle (phosphate lation and Subsequent degradation of IKB inhibitor. Increased buffer) served as positive and negative control, respectively. level of unphosphorylated cytosolic IKB binds p05 and p50 SP-333 was administered by oral gavage from day 1 through Subunits and prevents their activation and Subsequent trans day 7. DAI calculated (A) as per the described criteria. location into the nucleus to initiate pro-inflammatory cas Scatter plot depicting DAI values for individual mice, cade. together with mean values (horizontal bar) for each group. 0026 FIG. 13 is a graph showing that activation of Src by Oral administration of SP-333 at 0.05 mg/kg exhibits signifi PV and HgCl2 inhibited GCRA-mediated coMP production cant reduction (p=0.041) in DAI as compared to DSS vehicle by T84 cells. control. Colitis severity (B) was calculated according to out 0027 FIG. 14 is a graph showing that SP-333 is a biologi lined criteria. Data shown are Mean scorestSEM for each cally active agonist of GC-C Receptor. SP-333 treatment group on day 7, determined from the observation of up to 5 stimulated ccjMP synthesis in a dose-dependent manner in mid-colon cross-sections. Oral dose of SP-333 (0.005 mg/kg) T84 cells, and approached a plateau at a concentration of 1 was as effective as 5-ASA (100 mg/kg) in ameliorating colitis uM. in mice. Statistical significance calculated by comparing DAI 0028 FIG. 15A-C is a series of graphs showing treatment or colitis severity score observed for SP-333 or 5ASA treated with SP-333 enhanced coMP production and Expression of group versus corresponding score for vehicle treated group. Protein Kinase GI and II Transcripts. 0023 FIG. 10A-F is a series of tissue staining showing 0029 FIG. 16A-B is a series of graphs demonstrating efficacy of SP-333 in DSS-induced colitis mouse model. compared to vehicle, treatment with SP-333 downregulated Colitis was induced by providing 5% DSS in the drinking NF-kB subunits, IKK-B, c-Src, and p65 as judged by reduc water. Control group received normal drinking water. SP-333 tion in their transcript and protein levels. After treatment with was administered by oral gavage, once a day at 0.005, 0.05, SP-333, a 59% decrease in IKKB expression, a 55% decrease 0.5 and 5 mg/kg, from study day -1 (i.e. prior to initiation of in p65 expression, and a 52% decrease in c-Src expression DSS treatment) until study day 6. Reference compound, compared to untreated cells. 5-ASA (100 mg/kg) was administered in a similar manner. 0030 FIG. 17A-D is series of graphs demonstrating All mice were euthanized on day 7, large bowel processed for SP-333 downregulates c-Myc and transcripts of genes related histopathological analyses. Representative images of the his to cell-cycle in T84 cells. Treatment with SP-333 results in a topathological evaluation of the large bowels from the DSS 92% decrease in c-Myc expression a 58% decrease in Cyclin induced colitis study (described in FIG. 9) are shown. A) D1 expression, and a 50% decrease in Survivin expression. untreated naive mice, histopathology score=0; B) DSS Treatment with SP-333 appears to have no effect on the treated, histopathology score=4; C) DSS+SP-333 (0.005 expression of B-Catenin. mg/kg), histopathology score=1; D) DSS+SP-333 (0.05 0031 FIG. 18A-B is a series of graphs showing SP-333 mg/kg), histopathology score=2; E) DSS+SP-333 (0.5 treatment modulates miRNAs known to be dysregulated in mg/kg, histopathology score=2; and F) DSS+5-ASA (100 inflammation and cancer. In IBD and colon cancer, treatment mg/kg), histopathology score-2. with SP-333 upregulates miR-21 and MiR-155 levels, while 0024 FIG. 11A-B is a series of graphs showing DSS treatment with SP-333 downregulates levels of miR-126 and induced changes in the Ki-67 labeling of crypt epithelial cells miR-101 in colon cancer. (A) and MPO activity in lysates prepared from mid-colon 0032 FIG. 19A-C is a series of graphs demonstrating that samples (B). Samples from 40 mice in the SP-333-adminis SP-333 upregulates expression of miRNAs that are known to tered groups, together with the vehicle control and 5-ASA be expressed following NF-KB activation. NF-kB activation treated groups were randomly selected for analyses. Values down-regulates miR-29 family and let-7i. Treatment with plotted represent Mean-SEM for each group. Administration SP-333 upregulated miRs such as miR-15a (p<0.05), miR-16 of SP-333 improved symptoms of DSS-induced colitis in (p<0.01), let-7i (p<0.005), miR-125b (p<0.001) and the fam US 2016/0235807 A1 Aug. 18, 2016 ily of miR-29 (p<0.05), all of which are negative regulators of lipids, which leads to the activation of cytoplasmic phospho NF-KB signaling, which is known to augment production of lipase A2 (cPLA2), cyclooxygenase-2 (COX-2) and possibly pro-inflammatory cytokines during GI inflammation. 5-lipoxygenase (5-LO) during the process of inflammation, is 0033 FIG. 20 is a schematic representing the mechanism down-regulated by a coMP-dependent mechanism, leading by which SP-333 modulates expression of genes and miR to reduced levels of prostaglandins and leukotrienes, and that NAs implicated in inflammation and cancer. These data will increasing intracellular levels of c(GMP may therefore pro facilitate evaluation of the select miRNAs and corresponding duce an anti-inflammatory response. In addition, a coMP target genes in IBD tissues. dependent mechanism, is thought to be involved in the control of pro-inflammatory processes. Therefore, elevating intracel DETAILED DESCRIPTION lular levels of c(GMP may be used as a means of treating and 0034. It should be understood that singular forms such as controlling lipid metabolism disorders, biliary disorders, gas “a,” “an and “the are used throughout this application for trointestinal disorders, inflammatory disorders, lung disor convenience, however, except where context or an explicit ders, cancer, cardiac disorders including cardiovascular dis statement indicates otherwise, the singular forms are orders, eye disorders, oral disorders, blood disorders, liver intended to include the plural. Further, it should be under disorders, skin disorders, prostate disorders, endocrine disor stood that every journal article, patent, patent application, ders, increasing gastrointestinal motility and obesity. Lipid publication, and the like that is mentioned herein is hereby metabolism disorders include, but not limited to, dyslipi incorporated by reference in its entirety and for all purposes. demia, hyperlipidemia, hypercholesterolemia, hypertriglyc All numerical ranges should be understood to include each eridemia, Sitosterolemia, familial hypercholesterolemia, Xan and every numerical point within the numerical range, and thoma, combined hyperlipidemia, lecithin cholesterol should be interpreted as reciting each and every numerical acyltransferase deficiency, tangier disease, abetalipopro point individually. The endpoints of all ranges directed to the teinemia, erectile dysfunction, fatty liver disease, and hepa same component or property are inclusive, and intended to be titis. Billary disorders include gallbladder disorders such as independently combinable. for example, gallstones, gall bladder cancer cholangitis, or 0035 About includes all values having substantially the primary Sclerosing cholangitis; or bile duct disorders such as same effect, or providing Substantially the same result, as the for example, cholecystitis, bile duct cancer or fascioliasis. reference value. Thus, the range encompassed by the term Gastrointestinal disorders include for example, irritable “about will vary depending on context in which the term is bowel syndrome (IBS), non-ulcer dyspepsia, chronic intesti used, for instance the parameter that the reference value is nal pseudo-obstruction, functional dyspepsia, colonic associated with. Thus, depending on context, "about can pseudo-obstruction, duodenogastric reflux, gastroesophageal mean, for example, +15%, +10%, +5%, +4%, +3%, +2%, reflux disease (GERD), ileus inflammation (e.g., post-opera +1%, ortless than 1%. Importantly, all recitations of a refer tive ileus), gastroparesis, heartburn (high acidity in the GI ence value preceded by the term “about are intended to also tract), constipation (e.g., IBS-associated constipation, consti be a recitation of the reference value alone. pation associated with use of medications such as opioids, 0036. The present invention is based upon the develop osteoarthritis drugs, osteoporosis drugs; post Surgical consti ment of agonists of guanylate cyclase-C (GC-C) for the treat pation, constipation associated with neuropathic disorders. ment of inflammatory disorders and cancer. Exemplary GC-C Inflammatory disorders include tissue and organ inflamma agonists are analogs of plecanatide, uroguanylin, guanylin, tion Such as kidney inflammation (e.g., nephritis), gas lymphoguanylin and E. coli ST peptide. The invention relates trointestinal system inflammation (e.g., Crohn's disease and to a composition including at least one GC-C peptide (i.e., ulcerative colitis); necrotizing enterocolitis (NEC); pancre GCRA peptide) atic inflammation (e.g., pancreatis), lung inflammation (e.g., 0037. The invention is based upon the surprising discov bronchitis or asthma) or skin inflammation (e.g., psoriasis, ery that CG-C agonists can inhibit NF-kB signaling, thereby eczema). Lung Disorders include for example chronic exerting anti-inflammatory effects. Plecanatide (SP-304) and obstructive pulmonary disease (COPD), and fibrosis. Cancer SP-333, structural analogs of uroguanylin, an endogenous includes tissue and organ carcinogenesis including natriuretic peptide that activates guanylate cyclase-C(CG-C), metastases such as for example gastrointestinal cancer (e.g., ameliorates DSS- and TNBS-induced acute colitis in murine gastric cancer, esophageal cancer, pancreatic cancer colorec models. Plecanatide treatment also ameliorated spontaneous tal cancer including colorectal metastasis, intestinal cancer, colitis in T-cell receptor alpha knockout mice. Consistent anal cancer, liver cancer, gallbladder cancer, or colon cancer); with its in vivo anti-inflammatory activity, plecanatide treat lung cancer, thyroid cancer, skin cancer (e.g., melanoma); ment Suppressed production of inflammatory cytokines and oral cancer, urinary tract cancer (e.g. bladder cancer or kidney chemokines such as IL-12p40, IL-23, TNF, MIP-1C., IL-17 cancer); blood cancer (e.g. myeloma or leukemia) or prostate and RANTES in colon explants. Similarly, SP-333 also ame cancer. Cardiac disorders include for example, congestive liorated DSS-induced colitis in mice. SP-333 treatment inhib heart failure, trachea cardia hypertension, high cholesterol, or ited lipopolysaccharide-mediated activation of nuclear fac high triglycerides. Cardiovascular disorders include for tor-KB (NF-kB) in T84 cells. example aneurysm, angina, atherosclerosis, cerebrovascular 0038. The present invention is based upon several con accident (stroke), cerebrovasculardisease, congestive heart cepts. The first is that there is a coMP-dependent mechanism failure, coronary artery disease, myocardial infarction (heart which regulates the balance between cellular proliferation attack), or peripheral vascular disease. Liver disorders and apoptosis and that a reduction in coMP levels, due to a include for example cirrhosis and fibrosis. In addition, GC-C deficiency of uroguanylin/guanylin and/or due to the activa agonist may also be useful to facilitate liver regeneration in tion of c(GMP-dependent phosphodiesterases, is an early and liver transplant patients. Eye disorders include for example critical step in neoplastic transformation. A second concept is increased intra-ocular pressure, glaucoma, dry eyes retinal that the release of arachidonic acid from membrane phospho degeneration, disorders of tear glands or eye inflammation. US 2016/0235807 A1 Aug. 18, 2016

Skin disorders include for example xerosis. Oral disorders Chemokines include, but are not limited to, IL-8, RANTES include for example dry mouth (xerostomia), Sjögren's Syn and MIP-1C. Anti-inflammatory cytokines include, but are drome, gum diseases (e.g., periodontal disease), or salivary not limited to, IL-10. gland duct blockage or malfunction. Prostate disorders 0044. In some embodiments, GC-C agonists inhibit the include for example benign prostatic hyperplasia (BPH). nuclear localization of NF-kB. Endocrine disorders include for example diabetes mellitus, 0045. In some embodiments, GC-Cagonists mediate inhi hyperthyroidism, hypothyroidism, and cystic fibrosis. bition of NF-KB activating factors. NF-kB activating factors 0039. Uroguanylin is a circulating peptide hormone with are, without being limited to the examples herein, are cytok natriuretic activity and has been found to stimulate fluid and ines such as tumor necrosis factor (TNF) and interleukin electrolyte transport in a manner similar to another family of (IL)-1, lipopolysaccharides, bacterial and viral infections, heat stable enterotoxins (ST peptides) secreted by pathogenic activators of protein kinase C, and oxidants. The inhibition of strains of E. coli and other enteric bacteria that activate gua NF-kB may result in reduced production of cytokines (such nylate cyclase receptor and cause secretory diarrhea. Unlike as, without being limited to the examples herein, TNF, IL-1, bacterial ST peptides, the binding of uroguanylinto guanylate IL-2, IL-6, IL-8, IL-12p40, IL-17, and IL-23), adhesion mol ecules (such as ICAM-1, VCAM-1, E-selectin, and MAd cyclase receptor is dependent on the physiological pH of the CAM-1), and enzymes that are involved in inflammation, gut. Therefore, uroguanylin is expected to regulate fluid and Such as inducible nitric oxide synthase and cyclooxygenase electrolyte transport in a pH dependent manner and without 2. In some embodiments, the invention may provide inhibi causing severe diarrhea. tion of proteins whose genes are switched on by NF-KB, such 0040. The invention also provides a method for preventing as, without being limited to the examples herein, TNF and or treating a condition by administering to a Subject in need of IL-1. a therapeutically effective amount of the composition of the 0046. Some embodiments provide dysregulation of the present invention. The condition that can be treated by this expression of NF-kB target genes, such as TNF, implicated in composition includes colitis, ulcerative colitis, Crohn's dis the pathogenesis of inflammatory disorders or diseases. Such ease, irritable bowel syndrome (IBS), non-ulcer dyspepsia, as inflammatory bowel diseases. In some embodiments, the chronic intestinal pseudo-obstruction, functional dyspepsia, inhibition of NF-kB activation with GC-C agonists may pre colonic pseudo-obstruction, duodenogastric reflux, constipa vent IkB degradation and may attenuate chronic inflamma tion, IBS-associated constipation, constipation associated tion Such as that associated with Crohn's disease. In another with use of opiate pain killers, post-surgical constipation, embodiment, inhibition of p85 subunit of NF-kB may effec constipation associated with neuropathic disorders, gastroe tively abrogate colonic inflammation Such as that associated sophageal reflux disease (GERD), Celiac disease, gastropare with colitis. sis, heartburn, poor gastrointestinal motility, congestive heart 0047. In another embodiment, inhibition of NF-kB with failure, hypertension, benign prostatic hyperplasia (BPH), GC-C agonists may prevent mucosal NF-kB activation. In gastrointestinal cancer, lung cancer, bladder cancer, liver can some embodiments, inhibition of NF-kB with GC-Cagonists cer, Salivary gland cancer, skin cancer, colon cancer, bronchi may prevent mucosal NF-KB activation in ulcerative colitis tis, tissue inflammation, organ inflammation, respiratory patients. In particular the GC-C agonists of the current inven inflammation, asthma, COPD, lipid metabolism disorder, bil tion may inhibit NF-kB activation in macrophages. iary disorder, cardiovascular disease, obesity and an endo 0048. The current invention also provides GC-C agonists crine disorder. Preferably, the condition is a gastrointestinal mediated enhancement or stimulation of c(GMP signaling inflammatory disease (for example IBS-associated constipa pathway results in the inhibition of NF-KB. According to tion, ulcerative colitis and Crohn's disease), a gastrointestinal Some embodiments, the GC-C agonist mediated enhance cancer, or colorectal metastasis. ment or stimulation of c(GMP may result in the activation of 0041. The invention also relates in part to the use of GC-C the cyclic dependent protein kinase (PKG). The cyclic GMP agonists to inhibit activation of NF-kB, to reduce production dependent kinase (PKG) is an important mediator of signal of pro-inflammatory cytokines/chemokines and to increase transduction that may induce gene expression through cAMP secretion of anti-inflammatory cytokines to ameliorate coli response element binding protein (CREB). tis. Thus, GC-C agonists and compositions described herein 0049. In a merely illustrative embodiment, Plecanatide may be used either alone or in combination with other anti (SP304), SP333 or SP373 increases cGMP production, lead inflammatory drugs, for example, SulfaSalazine (AZulfidine) ing to the activation of PKG, which is a key regulator that Mesalamine (Asacol, Lialda), balsalazide (Colazal), olsala turns on downstream signaling to activate ion channels, cyclic Zine (Dipentum), Corticosteroids, immune system Suppres nucleotide gated channels and fluid homeostasis ENREF 37. sors, for example, AZathioprine (AZasan, Imuran) and mer Subsequent downstream signaling leads to inhibition of NF captopurine (Purinethol), Cyclosporine (Gengraf, Neoral, KB activation, resulting in reduced production of pro-inflam Sandimmune), Infliximab (Remicade), and/or immuno matory cytokines TNF, IL-12p40, IL-17, IL-23; chemokines modulatory agents (such as 6-mercaptopurine and methotr IL8, RANTES and MIP-1C. and in increased secretion of exate). anti-inflammatory cytokine IL-10, contributing to ameliora tion of colitis in chemically induced and genetically altered 0042 NF-KB may include one or more transcription factor mouse models of colitis. of the NF-KB family, for example without being limited to the 0050. The present invention provides a method of prevent list herein, NF-kB1 (p50), NF-kB2 (p52), p65 (RelA), c-Rel, ing a subject at risk of treating a subject Suffering from, or and RelB, or any protein that share a commonstructural motif ameliorating a symptom of a NF-KB mediated inflammatory called the Rel homology domain. disorder by administering to the Subject an effective amount 0043 Pro-inflammatory cytokines include, but are not of a GC-C agonist or pharmaceutical composition thereof, or limited to, IL-1, IL-2, TNF, IL-12p40, IL-17, and IL-23. a composition described herein. The invention provides that US 2016/0235807 A1 Aug. 18, 2016

the effective amount is sufficient to inhibit NF-kB activation, 0057 For example, the GCRA peptides of the invention thus preventing, treating a Subject at risk or suffering from or stimulate 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or more ameliorating a symptom of a NF-KB mediated inflammatory intracellular ccjMP compared to naturally occurring GC-C disorder. In some embodiments, the invention provides a agonists. The terms induced and stimulated are used inter method of preventing a Subject at risk of treating a subject changeably throughout the specification. The GCRA pep Suffering from, or ameliorating a symptom of gastrointestinal tides described herein are more stable than naturally occur inflammation comprising administering to the Subject an ring GC-C agonists. effective amount of a GC-C agonist or pharmaceutical com 0058. The GCRA peptides described herein have thera position thereof, or a composition described herein. In some peutic value in the treatment of a wide variety of disorders and embodiments, the invention provides a method of preventing conditions including for example lipid metabolism disorders, a Subject at risk of treating a Subject suffering from, or biliary disorders, gastrointestinal disorders, inflammatory ameliorating a symptom of colitis comprising administering disorders, lung disorders, cancer, cardiac disorders including cardiovascular disorders, eye disorders, oral disorders, blood to the subject an effective amount of a GC-C agonist or disorders, liver disorders, skin disorders, prostate disorders, pharmaceutical composition thereof, or a composition endocrine disorders, increasing gastrointestinal motility and described herein. In some embodiments, the invention pro obesity. Lipid metabolism disorders include, but not limited vides a method of preventing a Subject at risk of treating a to, dyslipidemia, hyperlipidemia, hypercholesterolemia, Subject Suffering from, or ameliorating a symptom of cancer hypertriglyceridemia, sitosterolemia, familial hypercholes comprising administering to the Subject an effective amount terolemia, Xanthoma, combined hyperlipidemia, lecithin cho of a GC-C agonist or pharmaceutical composition thereof, or lesterol acyltransferase deficiency, tangier disease, abetalipo a composition described herein. proteinemia, erectile dysfunction, fatty liver disease, and 0051. In some embodiments, the GC-C agonist is admin hepatitis. Billary disorders include gallbladder disorders such istered to the subject concurrently or sequentially with as for example, gallstones, gallbladder cancer cholangitis, or lipopolysaccharide (LPS). primary Sclerosing cholangitis; or bile duct disorders such as 0052. The invention also relates to a method of modulating for example, cholecystitis, bile duct cancer or fascioliasis. Gastrointestinal disorders include for example, irritable NF-kB induction in a cell by contacting the cell with an bowel syndrome (IBS), non-ulcer dyspepsia, chronic intesti effective amount of a GC-C agonist or pharmaceutical com nal pseudo-obstruction, functional dyspepsia, colonic position thereof or a composition described herein. pseudo-obstruction, duodenogastric reflux, gastroesophageal 0053. The present invention also provides a method for reflux disease (GERD), ileus inflammation (e.g., post-opera modulating NF-KB-dependent target gene expression in a cell tive ileus), gastroparesis, heartburn (high acidity in the GI by contacting the cell with an effective amount of a GC-C tract), constipation (e.g., IBS-associated constipation, consti agonist, where the GC-C agonist inhibits NF-kB activation, pation associated with use of medications such as opioids, thereby modulating NF-KB-dependent target gene expression osteoarthritis drugs, osteoporosis drugs; post Surgical consti in a cell. Exemplary NF-KB-dependent target genes include, pation, constipation associated with neuropathic disorders). but are not limited to, IL-1, IL-2, TNF, IL-12p40, IL-17, Inflammatory disorders include tissue and organ inflamma IL-23, IL-8, RANTES, MIP-1C., and IL-10. tion Such as kidney inflammation (e.g., nephritis), gas 0054 Any methods of the present invention may further trointestinal system inflammation (e.g., chronic inflamma include administering to the Subject or contacting the cell tory bowel disease, Crohn's disease, colitis, and ulcerative with one or more other agents. The one or more other agents colitis); necrotizing enterocolitis (NEC); pancreatic inflam include, for example, inhibitor of a NF-kB, inhibitor of c-Src. mation (e.g., pancreatis), lung inflammation (e.g., bronchitis inhibitor of c(GMP-dependent phosphodiesterase, anti-colitis or asthma) or skin inflammation (e.g., psoriasis, eczema). agent, anti-inflammatory drugs, for example, SulfaSalazine Lung Disorders include for example chronic obstructive pull (AZulfidine), Mesalamine (Asacol, Lialda), balsalazide (Co monary disease (COPD), and fibrosis. Cancer includes tissue lazal), olSalazine (Dipentum), Corticosteroids, immune sys and organ carcinogenesis including metatases such as for tem. Suppressors, for example, AZathioprine (AZasan, Imu example gastrointestinal cancer (e.g., gastric cancer, esoph ran) and mercaptopurine (Purinethol), Cyclosporine ageal cancer, pancreatic cancer, colorectal cancer including (Gengraf, Neoral, Sandimmune), Infliximab (Remicade) or colorectal metastasis, intestinal cancer, anal cancer, liver can immunomodulatory agents (such as 6-mercaptopurine and cer, gallbladder cancer, or colon cancer); lung cancer, thyroid methotrexate). In some embodiment of the current invention, cancer, skin cancer (e.g., melanoma); oral cancer, urinary the one or more other agents may be administered either tract cancer (e.g. bladder cancer or kidney cancer); blood concurrently or sequentially with a GC-C agonist or pharma cancer (e.g. myeloma or leukemia) or prostate cancer. Car ceutical composition thereof. diac disorders include for example, congestive heart failure, 0055. The GC-C agonists according to the invention trachea cardia hypertension, high cholesterol, or high tryg include amino acid sequences represented by Formulas I-XX lycerides. Cardiovascular disorders include for example as well as those amino acid sequence Summarized below in aneurysm, angina, atherosclerosis, cerebrovascular accident Tables 1-7. The GC-C agonists according to the invention are (stroke), cerebrovasculardisease, congestive heart failure, collectively referred to herein as “GCRA peptides'. In some coronary artery disease, myocardial infarction (heart attack), embodiments, the GC-C agonist has the sequence of SEQID or peripheral vascular disease. Liver disorders include for NO: 1 (SP-304), SEQID NO.:9 (SP-333), or SEQID NO: example cirrhosis and fibrosis. In addition, GC-C agonist 250 (SP-373). may also be useful to facilitate liver regeneration in liver 0056. The GCRA peptides described herein bind the gua transplant patients. Eye disorders include for example nylate cyclase C (GC-C) and stimulate intracellular produc increased intra-ocular pressure, glaucoma, dry eyes retinal tion of cyclic guanosine monophosphate (cGMP). Option degeneration, disorders of tear glands or eye inflammation. ally, the GCRA peptides induce apoptosis. In some aspects, Skin disorders include for example xerosis. Oral disorders the GCRA peptides stimulate intracellular ccjMP production include for example dry mouth (xerostomia), Sjögren's Syn at higher levels than naturally occurring GC-C agonists (e.g., drome, gum diseases (e.g., periodontal disease), or salivary uroguanylin, guanylin, lymphoguanylin and E. coli ST pep gland duct blockage or malfunction. Prostate disorders tides). include for example benign prostatic hyperplasia (BPH). US 2016/0235807 A1 Aug. 18, 2016

Endocrine disorders include for example diabetes mellitus, apoptosis, e.g., programmed cell death or activates the cystic hyperthyroidism, hypothyroidism, and cystic fibrosis. fibrosis transmembrane conductance regulator (CFTR). In 0059. As used herein, the term “guanylate cyclase receptor further embodiments, the GCRA peptide modulates NF-kB (GCR) refers to the class of guanylate cyclase C receptor on expression. In further embodiments, the GCRA peptide any cell type to which the inventive agonist peptides or natural modulates NF-KB signaling. In yet a further embodiment, the agonists described herein bind. As used herein, “intestinal NF-kB expression and/or signaling is inhibited. guanylate cyclase receptor is found exclusively on epithelial 0067. As used herein PEG3, 3 PEG, is meant to denote cells lining the GI mucosa. Uroguanylin, guanylin, and ST polyethylene glycol Such as include aminoethyloxy-ethy peptides are expected to bind to these receptors and may loxy-acetic acid (AeeA). induce apoptosis. The possibility that there may be different 0068. As used herein, the term “AMIDE is meant to receptors for each agonist peptide is not excluded. Hence, the denote that the terminal carboxylic acid is replaced with an term refers to the class of guanylate cyclase receptors on amide group, i.e., the terminal COOH is replaced with epithelial cells. CONH. 0060. As used herein, the term “GCR agonist' is meant to 0069. As used herein, the term "pyGlu' refers to pyro refer to peptides and/or other compounds that bind to an glutamic acid. intestinal guanylate cyclase receptor and stimulate fluid and 0070. As used herein, (e.g., in Formulas I-XX) X is any electrolyte transport. This term also covers fragments and natural, unnatural amino acid or amino acid analogue; M is pro-peptides that bind to GCR and stimulate fluid and water a Cysteine (Cys), Penicillamine (Pen) homocysteine, or secretion. 3-mercaptoproline. Xaa, is meant to denote an amino acid 0061. As used herein, the term “substantially equivalent” sequence of any natural, unnatural amino acid or amino acid is meant to refer to a peptide that has an amino acid sequence analogue that is one, two or three residues in length, Xaa, is equivalent to that of the binding domain where certain resi meant to denote an amino acid sequence of any natural, dues may be deleted or replaced with other amino acids unnatural amino acid or amino acid analogue that is Zero or without impairing the peptide’s ability to bind to an intestinal one residue in length; and Xaa, is meant to denote an amino guanylate cyclase receptor and stimulate fluid and electrolyte acid sequence of any natural, unnatural amino acid or amino transport. acid analogue that is Zero, one, two, three, four, five or six 0062. Addition of carriers (e.g., phosphate-buffered saline residues in length. Additionally, any amino acid represented or PBS) and other components to the composition of the by Xaa, may be an L-amino acid, a D-amino acid, a methy present invention is well within the level of skill in this art. In lated amino acid, a florinated amino acid or any combination addition to the compound, Such compositions may contain of thereof. Preferably the amino acids at the N-terminus, pharmaceutically acceptable carriers and other ingredients C-terminus or both are D-amino acids. Optionally, any known to facilitate administration and/or enhance uptake. GCRA peptide represented by Formulas I-XX may contain Other formulations, such as microspheres, nanoparticles, on or more polyethylene glycol residues at the N-terminus, liposomes, and immunologically-based systems may also be C-terminus or both. An exemplary polyethylene glycol used in accordance with the present invention. Other includes aminoethyloxy-ethyloxy-acetic acid and polymers examples include formulations with polymers (e.g., 20% w/v. thereof. polyethylene glycol) or cellulose, or enteric formulations. 0071 Specific examples of GCC agonist peptides that can 0063 GCRA Peptides be used in the methods and formulations of the invention 0064. The GCRA peptides of the present invention are include a peptide selected from Tables 1-7. analogues of plecanatide, uroguanylin, guanylin, lym 0072. In some embodiments, GCC agonist peptides phoguanylin and ST peptides. No particular length is implied include peptides having the amino acid sequence of Formula by the term "peptide'. In some embodiments, the GCRA I, wherein at least one amino acid of Formula I is a D-amino peptide is less than 25 amino acids in length, e.g., less than or acid or a methylated amino acid and/or the amino acid at equal to 20, 15, 14, 13, 12, 11, 10, or 5 amino acid in length. position 16 is a serine. Preferably, the amino acid at position 0065. The GCRA peptides can be polymers of L-amino 16 of Formula I is a D-amino acid or a methylated amino acid. acids, D-amino acids, or a combination of both. For example, For example, the amino acid at position 16 of Formula I is a in various embodiments, the peptides are D retro-inverso d-leucine or a d-serine. Optionally, one or more of the amino peptides. The term “retro-inverso isomer refers to an isomer acids at positions 1-3 of Formula I are D-amino acids or of a linear peptide in which the direction of the sequence is methylated amino acids or a combination of D-amino acids or reversed and the chirality of each amino acid residue is methylated amino acids. For example, Asn', Asp or Glu (or inverted. See, e.g., Jameson et al., Nature, 368, 744-746 a combination thereof) of Formula I is a D-amino acid or a (1994); Brady et al., Nature, 368, 692-693 (1994). The net methylated amino acid. Preferably, the amino acid at position result of combining D-enantiomers and reverse synthesis is Xaa' of Formula I is a leucine, serine or tyrosine. that the positions of carbonyl and amino groups in each amide 0073. In alternative embodiments, GCC agonist peptides bond are exchanged, while the position of the side-chain include peptides having the amino acid sequence of Formula groups at each alpha carbon is preserved. Unless specifically II, wherein at least one amino acid of Formula II is a D-amino stated otherwise, it is presumed that any given L-amino acid acid or a methylated amino acid. Preferably, the amino acid sequence of the invention may be made into a D retro-inverso denoted by Xaa, of Formula II is a D-amino acid or a methy peptide by synthesizing a reverse of the sequence for the lated amino acid. In some embodiments, the amino acid corresponding native L-amino acid sequence. For example a denoted by Xaa, of Formula II is a leucine, a d-leucine, a GCRA peptide includes the sequence defined by Formulas serine, or ad-serine. Preferably, the one or more amino acids I-XX and those listed on Tables 1-7. denoted by Xaa, of Formula II are D-amino acids or methy 0066 By inducing c(3MP production is meant that the lated amino acids. Preferably, the amino acid at positionXaa GCRA peptide induces the production of intracellular cGMP. of Formula II is a leucine, a serine, or a tyrosine. Intracellular ccjMP is measured by methods known in the art. 0074. In some embodiments, GCC agonist peptides For example, the GCRA peptide of the invention stimulate include peptides having the amino acid sequence of Formula 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or more intrac III, wherein at least one amino acid of Formula III is a ellular cGMP compared to naturally occurring GC-C ago D-amino acid or a methylated amino acid and/or Maa is not a nists. In further embodiments, the GCRA peptide stimulates cysteine. Preferably, the amino acid denoted by Xaa, of US 2016/0235807 A1 Aug. 18, 2016

Formula III is a D-amino acid or a methylated amino acid. In of Formula XVIII is a valine or isoleucine. Preferably, the some embodiments the amino acid denoted by Xaa, of For amino acid at position 9 of Formula XVIII is an asparagine. mula III is a leucine, a d-leucine, a serine, or a d-serine. Preferably, the amino acid at position 10 of Formula XVIII is Preferably, the one or more amino acids denoted by Xaa, of a valine or a methionine. Preferably, the amino acid at posi Formula III are D-amino acids or methylated amino acids. tion 11 of Formula XVIII is an alanine. Preferably, the amino Preferably, the amino acid at positionXaa' of Formula III is a acid at position 13 of Formula XVIII is a threonine. Prefer leucine, a serine, or a tyrosine. ably, the amino acid at position 14 of Formula XVIII is a 0075. In other embodiments, GCC agonist peptides glycine. Preferably, the amino acid at position 16 of Formula include peptides having the amino acid sequence of Formula XVIII is a leucine, serine or threonine IV, wherein at least one amino acid of Formula IV is a 0080. In alternative embodiments, GCRA peptides D-amino acid or a methylated amino acid, and/or Maa is not include peptides containing the amino acid sequence of For a cysteine. Preferably, the Xaa, of Formula IV is a D-amino mula XIX. Preferably, the amino acid at position 1 of Formula acid or a methylated amino acid. In some embodiments, the XIX is a serine or asparagine. Preferably, the amino acid at amino acid denoted by Xaa, of Formula IV is a leucine, a position 2 of Formula XIX is a histidine or an aspartic acid. d-leucine, a serine, or ad-serine. Preferably, the one or more Preferably, the amino acid at position 3 of Formula XIX is a of the amino acids denoted by Xaa, of Formula IV are threonine or a glutamic acid. Preferably, the amino acid at D-amino acids or methylated amino acids. Preferably, the position 5 of Formula XIX is a glutamic acid. Preferably, the amino acid denoted Xaa, of Formula IV is a leucine, a serine, amino acid at position 6 of Formula XIX is an isoleucine, or a tyrosine. In further embodiments, GCC agonist peptides leucine, Valine or tyrosine. Preferably, the amino acid at posi include peptides having the amino acid sequence of Formula tion 8, 10, 11, or 13 of Formula XIX is an alanine. Preferably, V, wherein at least one amino acid of Formula V is a D-amino the amino acid at position 9 of Formula XIX is an asparagine acid or a methylated amino acid. Preferably, the amino acid at or a phenylalanine. Preferably, the amino acid at position 14 position 16 of Formula V is a D-amino acid or a methylated of Formula XIX is a glycine. amino acid. For example, the amino acid at position 16 (i.e., I0081. In further embodiments, GCRA peptides include Xaa') of Formula V is a d-leucine or a d-serine. Optionally, peptides containing the amino acid sequence of Formula XX. one or more of the amino acids at position 1-3 of Formula V Preferably, the amino acid at position 1 of Formula XX is a are D-amino acids or methylated amino acids or a combina glutamine. Preferably, the amino acid at position 2 or 3 of tion of D-amino acids or methylated amino acids. For Formula XX is a glutamic acid oranaspartic acid. Preferably, example, Asn', Asp or Glu (or a combination thereof) of the amino acid at position 5 of Formula XX is a glutamic acid. Formula V is a D-amino acids or a methylated amino acid. Preferably, the amino acid at position 6 of Formula XX is Preferably, the amino acid denoted at Xaa' of Formula V is a threonine, glutamine, tyrosine, isoleucine, or leucine. Prefer leucine, a serine, or a tyrosine. ably, the amino acid at position 8 of Formula XX is isoleucine 0076. In additional embodiments, GCRA peptides include or valine. Preferably, the amino acid at position 9 of Formula peptides having the amino acid sequence of Formula VI, XX is asparagine. Preferably, the amino acid at position 10 of VII-a, VII-b, VIII, or IX. Preferably, the amino acid at posi Formula XX is methionine or valine. Preferably, the amino tion 6 of Formula VI, VII-a, VII-b, VIII, or IX is a leucine, a acid at position 11 of Formula XX is alanine. Preferably, the serine or a tyrosine. In some aspects the amino acid at position amino acid at position 13 of Formula XX is a threonine. 16 of Formula VI, VII-a, VII-b, VIII or IX is a leucine or a Preferably, the amino acid at position 1 of Formula XX is a serine. Preferably, the amino acid at position 16 of Formula glycine. Preferably, the amino acid at position 15 of Formula VI, VII-a, VII-b, VIII or IX is a D-amino acid or a methylated XX is a tyrosine. Optionally, the amino acid at position 15 of amino acid. Formula XX is two-amino acid in length and is Cysteine 0077. In additional embodiments, GCRA peptides include (CyS), Penicillamine (Pen) homocysteine, or 3-mercaptopro peptides having the amino acid sequence of Formula X, XI, line and serine, leucine or threonine. XII, XIII, XIV. XV, XVI or XVII. Optionally, one or more 0082 In certain embodiments, one or more amino acids of amino acids of Formulas X, XI, XII, XIII, XIV, XV, XVI or the GCRA peptides can be replaced by a non-naturally occur XVII are D-amino acids or methylated amino acids. Prefer ring amino acid or a naturally or non-naturally occurring ably, the amino acid at the carboxyl terminus of the peptides amino acid analog. There are many amino acids beyond the according to Formulas X, XI, XII, XIII, XIV. XV, XVI or standard 20 (Ala, Arg, ASn, Asp, Cys, Gln, Glu, Gly, His, Ile, XVII is a D-amino acid or a methylated amino acid. For Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val). Some are example the amino acid at the carboxyl terminus of the pep naturally-occurring others are not. (See, for example, Hunt, tides according to Formulas X, XI, XII, XIII, XIV,XV,XVI or The Non-Protein Amino Acids: In Chemistry and Biochem XVII is a D-tyrosine. istry of the Amino Acids, Barrett, Chapman and Hall, 1985). 0078 Preferably, the amino acid denoted by Xaa' of For For example, an aromatic amino acid can be replaced by mula XIV is a tyrosine, phenylalanine or a serine. Most pref 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodot erably the amino acid denoted by Xaa' of Formula XIV is a hyronine, L-thyroxine, phenylglycine (Phg) or nor-tyrosine phenylalanine or a serine. Preferably, the amino acid denoted (norTyr). Phg and norTyrand otheramino acids including Phe by Xaa' of Formula XV. XVI or XVII is a tyrosine, a pheny and Tyrcan be substituted by, e.g., a halogen, —CH3, —OH, lalanine, or a serine. Most preferably, the amino acid position CH2NH3, —C(O)H, CH2CH3, –CN, Xaa' of Formula XV. XVI or XVII is a phenylalanine or a —CH2CH2CH3, SH, or another group. Any amino acid S1. can be substituted by the D-form of the amino acid. 0079. In some embodiments, GCRA peptides include pep I0083. With regard to non-naturally occurring amino acids tides containing the amino acid sequence of Formula XVIII. or naturally and non-naturally occurring amino acid analogs, Preferably, the amino acid at position 1 of Formula XVIII is a glutamic acid, aspartic acid, glutamine or lysine. Preferably, a number of Substitutions in the polypeptide and agonists the amino acid at position 2 and 3 of Formula XVIII is a described herein are possible alone or in combination. glutamic acid, or an aspartic acid. Preferably, the amino acid I0084. For example, glutamine residues can be substituted at position 5 is a glutamic acid. Preferably, the amino acid at with gamma-Hydroxy-Glu or gamma-Carboxy-Glu. position 6 of Formula XVIII is an isoleucine, valine, serine, Tyrosine residues can be substituted with an alpha substituted threonine or tyrosine. Preferably, the amino acid at position 8 amino acid such as L-alpha-methylphenylalanine or by ana US 2016/0235807 A1 Aug. 18, 2016 logues such as: 3-Amino-Tyr; Tyr(CH3); Tyr(PO3(CH3)2): 3-nitro-tyrosine; norleucine; pyro-glutamic acid; Z (Car Tyr(SO3H); beta-Cyclohexyl-Ala; beta-(1-Cyclopentenyl)- bobenzoxyl); e-Acetyl-Lysine; B-alanine; B-aspartic acid; Ala; beta-Cyclopentyl-Ala; beta-Cyclopropyl-Ala; beta B-cyclohexylalanine; aminobenzoyl derivative; aminobu Quinolyl-Ala; beta-(2-Thiazolyl)-Ala; beta-(Triazole-1-yl)- tyric acid (Abu); citrulline; aminohexanoic acid (Ahk); ami Ala; beta-(2-Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe: noisobutyric acid (AIB); cyclohexylalanine; d-cyclohexyla Fluoro-Phe: Cyclohexyl-Gly; tBul-Gly; beta-(3-benzothie lanine; cyclohexylglycine; hydroxyproline; nitro-arginine; nyl)-Ala; beta-(2-thienyl)-Ala; 5-Methyl-Trp.; and A-Methyl nitro-phenylalanine; nitro-tyrosine; norvaline; octahydroin Trp. Proline residues can be substituted with homopro dole carboxylate; ornithine (Orn); penicillamine (PEN); tet (L-pipecolic acid); hydroxy-Pro; 3,4-Dehydro-Pro; 4-fluoro rahydroisoquinoline; diaminobutyric acid; diaminopimelic Pro; or alpha-methyl-Pro or an N(alpha)-C(alpha) cyclized acid; pyroglutamic acid; homocysteine; homoserine; N-e- amino acid analogues with the structure: n=0, 1, 2, 3 Alanine dinitrophenyl-lysine; N-e-methyl-lysine; N-e-dimethyl residues can be substituted with alpha-substitued or N-me lysine; N.N.N-e-trimethyl-lysine; acetamidomethyl pro thylated amino acid such as alpha-amino isobutyric acid (aib), L/D-alpha-ethylalanine (L/D-isovaline), L/D-meth tected amino acids and pegylated amino acids. Further ylvaline, or L/D-alpha-methyleucine or a non-natural amino examples of unnatural amino acids and amino acid analogs acid such as beta-fluoro-Ala. Alanine can also be substituted can be found in U.S. 20030108885, U.S. 20030082575, with: n=0, 1, 2, 3 Glycine residues can be substituted with US20060019347 (paragraphs 410-418) and the references alpha-amino isobutyric acid (aib) or L/D-alpha-ethylalanine cited therein. The polypeptides of the invention can include (L/D-isovaline). further modifications including those described in US20060019347, paragraph 589. 0085. Further examples of unnatural amino acids include: an unnatural analog of alanine (e.g., L-1-Nal or L-2-Nal); an I0086) “Nal' used herein refers to both L-1-naphthylala unnatural analog of tyrosine; an unnatural analogue of nine (L-1-Nal) and L-2-naphthylalanine (L-2-Nal). glutamine; an unnatural analogue of phenylalanine; an unnatural analogue of serine; an unnatural analogue of threo 0087. In some embodiments, an amino acid can be nine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, replaced by a naturally-occurring, non-essential amino acid, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, Sulfonyl, e.g., taurine. Seleno, ester, thioacid, borate, boronate, phospho, I0088 Alternatively, the GCRA peptides are cyclic pep phosphono, phosphine, heterocyclic, enone, imine, aldehyde, tides. GCRA cyclic peptides are prepared by methods known hydroxylamine, keto, or amino substituted amino acid, or any in the art. For example, macrocyclization is often accom combination thereof, an amino acid with a photoactivatable plished by forming an amide bond between the peptide N- and cross-linker, a spin-labeled amino acid; a fluorescent amino C-termini, between a side chain and the N- or C-terminus acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another e.g., with KFe(CN) at pH 8.5 (Samson et al., Endocrinol molecule; a metal binding amino acid; an amino acid that is ogy, 137:5182-5185 (1996)), or between two amino acid side amidated at a site that is not naturally amidated, a metal chains, such as cysteine. See, e.g., DeGrado, Adv. Protein containing amino acid; a radioactive amino acid; a photo Chem, 39: 51-124 (1988). In some embodiments, the GCRA caged and/or photoisomerizable amino acid; a biotin or peptides of the present invention are bicyclic peptides. In biotin-analogue containing amino acid; a glycosylated or car various aspects the GCRA peptides are 4,12; 7.15 bicycles. bohydrate modified amino acid; a keto containing amino I0089. In some GCRA peptides one or both members of acid; amino acids comprising polyethylene glycol or poly one or both pairs of Cys residues which normally form a ether; a heavy atom Substituted amino acid (e.g., an amino disulfide bond can be replaced by homocysteine, penicil acid containing deuterium, tritium, C, N, or 'O); a lamine, 3-mercaptoproline (Kolodzie et al. 1996 Int J Pept chemically cleavable orphotocleavable amino acid; an amino Protein Res 48:274); B, B dimethylcysteine (Hunt et al. 1993 acid with an elongated side chain; an amino acid containing a toxic group; a Sugar Substituted amino acid, e.g., a Sugar Int JPept Protein Res 42:249) or diaminopropionic acid Substituted serine or the like; a carbon-linked Sugar-contain (Smith et al. 1978 J. Med Chem 21:117) to form alternative ing amino acid; a redox-active amino acid; an O-hydroxy internal cross-links at the positions of the normal disulfide containing acid; an amino thio acid containing amino acid; an bonds. C. C. disubstituted amino acid; a 3-amino acid; a cyclic amino 0090. In addition, one or more disulfide bonds can be acid other than proline; an O-methyl-L-tyrosine; an L-3-(2- replaced by alternative covalent cross-links, e.g., an amide naphthyl)alanine; a 3-methyl-phenylalanine; a p-acetyl-L- linkage ( CH2CH(O)NHCH 2- or -CH2NHCH(O)CH phenylalanine; an O-4-allyl-L-tyrosine; a 4-propyl-L-ty 2-), an ester linkage, a thioester linkage, a lactam bridge, a rosine; a tri-O-acetyl-GlcNAc B-serine; an L-Dopa; a carbamoyl linkage, aurea linkage, a thiourealinkage, a phos fluorinated phenylalanine; an isopropyl-L-phenylalanine; a phonate ester linkage, al alkyl linkage p-azido-L-phenylalanine; a p-acyl-L-phenylalanine; a p-ben (—CH2CH2CH2CH2-), an alkenyl linkage (—CH Zoyl-L-phenylalanine; an L-phosphoserine; a phospho 2CH=CHCH2-), an ether linkage (-CH2CH2OCH2- or noSerine; a phosphonotyrosine; a p-iodo-phenylalanine; a —CH2OCH2CH2-), a thioether linkage (-CH2CH2SCH2 4-fluorophenylglycine; a p-bromophenylalanine; a p-amino O CH2SCH2CH2-), al amine linkage L-phenylalanine; an isopropyl-L-phenylalanine; L-3-(2- ( CH2CH2NHCH2- or CH2NHCH2CH2-) or a thioam naphthyl)alanine: D-3-(2-naphthyl)alanine (dNal); an ide linkage ( CH2CH(S)HNHCH 2- or -CH2NHCH(S) amino-, isopropyl-, or O-allyl-containing phenylalanine ana CH2-). For example, Ledu et al. (Proc Natl Acad. Sci. logue; a dopa, O-methyl-L-tyrosine; a glycosylated amino 100: 11263-78, 2003) describe methods for preparing lactam acid; a p-(propargyloxy)phenylalanine; dimethyl-Lysine; and amide cross-links. Exemplary GCRA peptides which hydroxy-proline; mercaptopropionic acid; methyl-lysine; include a lactam bridge include for example SP-370. US 2016/0235807 A1 Aug. 18, 2016

0091. The GCRA peptides can have one or more conven 0095 Preferred variants are those that have conservative tional polypeptide bonds replaced by an alternative bond. amino acid Substitutions made at one or more predicted non Such replacements can increase the stability of the polypep essential amino acid residues. A "conservative amino acid tide. For example, replacement of the polypeptide bond substitution' is one in which the amino acid residue is between a residue amino terminal to an aromatic residue (e.g. replaced with an amino acid residue having a similar side Tyr, Phe, Trp) with an alternative bond can reduce cleavage by chain. Families of amino acid residues having similar side carboxypeptidases and may increase half-life in the digestive chains have been defined in the art. These families include tract. Bonds that can replace polypeptide bonds include: a amino acids with basic side chains (e.g., lysine, arginine, retro-inverso bond (C(O)—NH instead of NH C(O); a histidine), acidic side chains (e.g., aspartic acid, glutamic reduced amide bond (NH-CH2); a thiomethylene bond acid), uncharged polar side chains (e.g., glycine, asparagine, (S CH2 or CH2-S); an oxomethylene bond (0-CH 2 or glutamine, serine, threonine, tyrosine, cysteine), nonpolar CH2-O); an ethylene bond (CH2-CH2); a thioamide bond side chains (e.g., alanine, Valine, leucine, isoleucine, proline, (C(S) NH); a trans-olefine bond (CH=CH); a fiuoro sub phenylalanine, methionine, tryptophan), beta-branched side stituted trans-olefine bond (CF=CH); a ketomethylene bond chains (e.g., threonine, Valine, isoleucine) and aromatic side (C(O) CHR or CHR C(O) wherein R is H or CH3; and a chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). fluoro-ketomethylene bond (C(O) CFR or CFR C(O) Thus, a predicted nonessential amino acid residue in a GCRA wherein R is H or F or CH3. polypeptide is replaced with anotheramino acid residue from 0092. The GCRA peptides can be modified using standard the same side chain family. Alternatively, in another embodi modifications. Modifications may occur at the amino (N-), ment, mutations can be introduced randomly along all or part carboxy (C-) terminus, internally or a combination of any of of a GCRA coding sequence, such as by Saturation mutagen the preceding. In one aspect described herein, there may be esis, and the resultant mutants can be screened to identify more than one type of modification on the polypeptide. Modi mutants that retain activity. fications include but are not limited to: acetylation, amida (0096. The GCRA peptides of the invention also include tion, biotinylation, cinnamoylation, farnesylation, formyla analogs that contain an O-aminoadipic acid (Aad), preferably tion, myristoylation, palmitoylation, phosphorylation (Ser, at the 3rd position from the N-terminus of each peptide or at Tyr or Thr), Stearoylation, Succinylation, Sulfurylation and the position to the N-terminal side next to the first cysteine cyclisation (via disulfide bridges or amide cyclisation), and (“Cys”) residue. See, for example, US 2014/0256762 filed modification by Cys3 or Cys5. The GCRA peptides described Mar. 13, 2014 which is hereby incorporated by reference in its herein may also be modified by 2, 4-dinitrophenyl (DNP), entirety for all purposes. DNP-lysine, modification by 7-Amino-4-methyl-coumarin (0097. The GCRA peptides of the invention also include (AMC), flourescein, NBD (7-Nitrobenz-2-Oxa-1,3-Diazole), anoalogs, where 5-aminosalicylic acid (“5-ASA'; also called p-nitro-anilide, rhodamine B, EDANS (5-((2-aminoethyl) mesalamine or mesalazine) or its derivative or pharmaceuti amino)naphthalene-1-sulfonic acid), dabcyl, dabsyl, dansyl, cally acceptable salt thereof is covalently linked to the N texas red, FMOC, and Tamra (Tetramethylrhodamine). The terminus and/or the C terminus of a GCRA peptide (referred GCRA peptides described herein may also be conjugated to, herein “5-ASA GCRA analog peptide') (see, US for example, polyethylene glycol (PEG); alkyl groups (e.g., 20140256762, filed Mar. 13, 2014 which is hereby incorpo C1-C20 straight or branched alkyl groups); fatty acid radi rated in its entirety for all purposes). These peptides are cals; combinations of PEG, alkyl groups and fatty acid radi biologically inactive or biologically less active than a GCRA cals (See, U.S. Pat. No. 6,309,633; Soltero et al., 2001 Inno peptide alone. However, upon cleavage of the glycosidic bond vations in Pharmaceutical Technology 106-110); BSA and between peptide and sugar residues of the 5-ASA molecule or KLH (Keyhole Limpet Hemocyanin). The addition of PEG the PEG molecule by sugar hydrolases produced by colon and other polymers which can be used to modify polypeptides bacteria, released GCRA peptide and 5-ASA molecule then of the invention is described in US2006.019347 section IX. produce a colon-specific synergistic effect to stimulate coMP 0093. Also included in the invention are peptides that bio production, to induce apoptosis, and/or to enhance anti-in logically or functional equivalent to the peptides described flammation. Such 5-ASA GCRA analog peptides also pre herein. The term “biologically equivalent” or functional vent or reduce the potential side effect of a GCRA peptide equivalent is intended to mean that the compositions of the before reaching to colon. In some embodiments, 5-ASA or its present invention are capable of demonstrating some or all of derivative or pharmaceutically acceptable salt thereof is the coMP production modulatory effects. covalently linked to the N terminus and/or the C terminus of 0094 GCRA peptides can also include derivatives of a GCRA peptide (referred herein “5-ASA GCRA analog GCRA peptides which are intended to include hybrid and peptide'). Preferably, the derivative is sulfasalazine. modified forms of GCRA peptides in which certain amino 0098. In some embodiments, the 5-ASA GCRA analog acids have been deleted or replaced and modifications such as peptide includes: where one or more amino acids have been changed to a modified amino acid or unusual amino acid and modifications (0099 5-ASA-GCRA (formula A), Such as glycosylation so long the modified form retains the 0100 GCRA-5-ASA (formula B) or biological activity of GCRA peptides. By retaining the bio logical activity, it is meant that cOMP and or apoptosis is 0101 5-ASA-GCRA-5-ASA (formula C). induced by the GCRA peptide, although not necessarily at the 0102) A skilled artisan would readily recognize that the same level of potency as that of a naturally-occurring GCRA N-terminus of the peptide is on the left side and the C-termi peptide identified. nus of the peptide is on the right side in these formulas. US 2016/0235807 A1 Aug. 18, 2016 11

0103) In a merely illustrative embodiment, a 5-ASA GCRA analog peptide of the invention has the following formula:

(Formula i) (Formula ii) HOOC COOH

HO N=N-GCRA peptide, GCRA peptide-Y-X-N=N OH or

(Formula iii) HOOC COOH

HO N=N-GCRA peptide-Y-X-N=N OH,

0104 wherein X is absent, aryl or alkyl and Y is absent or ileum, terminal ileum, or ascending colon). The formulation any function group that reacts with the carboxyl group of the may contain an inert carrier coated with 5-ASA GCRA ana GCRA peptide. A skilled artisan could readily determine the log peptides and an enteric coating which releases the pep function groups that can react with the carboxyl group of the tides at a specific pH (such as pH5 or pH7). Preferred pH for GCRA peptide. In certain embodiments, when the last amino duodenum or jejunum release is pH 4.5-5.5 or pH 5.5-6.5. acid (i.e., the amino acid at the most c-terminus end) in the Preferred pH for ileum, terminal ileum, or ascending colon GCRA peptide contains a free NH group in its side chain (for release is pH 5.5-6.5 or pH 6.5-7.5. Preferably, the inert example, lysine), X and Y can be absent. 5-ASA GCRA carrier is a selected from mannitol, lactose, a microcrystalline analog peptides described herein are biologically inactive or cellulose, or starch. biologically less active thana GCRA peptide alone. However, 0106 The term “consisting essentially of includes pep upon cleavage of the glycosidic bond between peptide and tides that are identical to a recited sequence (any one from sugar residues of the 5-ASA molecule or the PEG molecule Tables 1-7) and other sequences that do not differ substan by Sugar hydrolases produced by colon bacteria, released tially in terms of either structure or function. For the purpose GCRA peptide and 5-ASA molecule then produce a colon of the present application, a peptide differs substantially if its specific synergistic effect to stimulate coMP production, to structure varies by more than three amino acids from a pep induce apoptosis, and/or to enhance anti-inflammation. Such tide of any one from Tables 1-7 or if its activation of cellular 5-ASA GCRA analog peptides also prevent or reduce the cGMP production is reduced or enhanced by more than 50%. potential side effect of a GCRA peptide before reaching to Preferably, substantially similar peptides should differ by no colon. more than two amino acids and not differ by more than about 0105. In some embodiments, the 5-ASA GCRA analog 25% with respect to activating c(3MP production. peptides described herein are formulated in a pH dependent 0107 Also included within the meaning of substantially release form. Alternatively, such analog peptides are formu homologous is any GCRA peptide which may be isolated by lated in a form that releases the peptides at a specific region of virtue of cross-reactivity with antibodies to the GCRA pep the gastrointestinal (GI) tract (e.g., duodenum, jejunum, tide. TABL E 1 GCRA Peptides (SP-304 and Derivatives) Position of SEQ Name Disulfide bonds Structure NO ID SP-3O4 C4: C12, C7: C15 Asn'-Asp-Glu-Cys'-Glu-Leu-Cys-Val-Asn-Val'-Ala'-Cys'- 1. Thr-Gly-Cys-Leu SP-326 C3: C11, C6: C14 Asp-Glu-Cys-Glu-Leu-Cys-Val-Asn-Val-Ala'-Cys-Thr- 2 Gly-Cys-Leu" SP-327 C3: C11, C6: C14 Asp-Glu-Cys-Glu-Leu-Cys-Val-Asn-Val-Ala'-Cys-Thr- 3 Gly-Cys4 SP-328 C2 : C1 O C5: C13 Glu'-Cys’-Glu-Leu-Cys-Val-Asn-Val-Ala-Cys'-Thr-Gly- 4. Cys-Leu' SP-329 C2 : C1 O C5: C13 Glu'-Cys’-Glu-Leu-Cys-Val-Asn-Val-Ala-Cys'-Thr-Gly- 5 Cys SP-33 O C1: C9, C4 : C12 Cys-Glu-Leu-Cyst-Val-Asn-Val-Ala- Cys-Thr-Gly-Cys- 6 Lieu"

US 2016/0235807 A1 Aug. 18, 2016

TABLE 7-continued ST Peptide and Analogues

Position of SEQ Name Disulfide bonds Structure ID NO N163 C3: C8, C4 : C12, Asn-Phe-Cys-Cys-Glu-Thr-Cys"- Cys-Asn-Pro-Alla'-Cys'?-Thr- 245 C7 : C1s Gly-Cys-Tyr-PEG3 N164 C3: C8, C4 : C12, Asn-Phe-Cys- Cys'-Glu-Tyr- Cys 7-Cys-Asn-Pro-Ala-Cys-Thr- 246 C7 : C1s Gly'-Cys-Tyr16 N165 C3: C8, C4 : C12, dAsn-Phe-Cys-Cys'-Glu-Tyr-Cys 7-Cys-Asn-Pro-Ala-Cys-Thr- 247 C7 : C1s Gly14- Cys-dTyr16 N166 C3: C8, C4 : C12, Asn-Phe-Cys-Cys-Glu-Tyr-Cys"- Cys-Asn-Pro-Alla'-Cys'?-Thr- 248 C7 : C1s Gly14- Cys-dTyr16 N167 C3: C8, C4 : C12, dAsn-Phe-Cys-Cys'-Glu-Tyr-Cys"- Cys-Asn-Pro'-Ala-Cys-Thr- 249 C7 : C1s Gly'-Cys-Tyr16

0108 Preparation of GCRA Peptides 0113 Acetylation of the N-terminal can be accomplished 0109 GCRA peptides are easily prepared using modern by reacting the final peptide with acetic anhydride before cloning techniques, or may be synthesized by Solid state cleavage from the resin. C-amidation is accomplished using methods or by site-directed mutagenesis. A GCRA peptide an appropriate resin Such as methylbenzhydrylamine resin may include dominant negative forms of a polypeptide. using the Boc technology. 0110 Chemical synthesis may generally be performed 0114. Alternatively the GCRA peptides are produced by using standard solution phase or Solid phase peptide synthesis modern cloning techniques. For example, the GCRA peptides techniques, in which a peptide linkage occurs through the are produced either in bacteria including, without limitation, direct condensation of the amino group of one amino acid E. coli, or in other existing systems for polypeptide or protein with the carboxy group of the other amino acid with the production (e.g., Bacillus subtilis, baculovirus expression elimination of a water molecule. Peptide bond synthesis by systems using Drosophila Sf9 cells, yeast or filamentous direct condensation, as formulated above, requires Suppres fungal expression systems, mammalian cell expression sys sion of the reactive character of the amino group of the first tems), or they can be chemically synthesized. If the GCRA and of the carboxyl group of the second amino acid. The peptide or variant peptide is to be produced in bacteria, e.g., E. masking Substituents must permit their ready removal, with coli, the nucleic acid molecule encoding the polypeptide may out inducing breakdown of the labile peptide molecule. also encode a leader sequence that permits the secretion of the 0111. In solution phase synthesis, a wide variety of cou mature polypeptide from the cell. Thus, the sequence encod pling methods and protecting groups may be used (See, Gross ing the polypeptide can include the pre sequence and the pro and Meienhofer, eds., “The Peptides: Analysis, Synthesis, sequence of for example, a naturally-occurring bacterial ST Biology. Vol. 1-4 (Academic Press, 1979); Bodansky and polypeptide. The secreted, mature polypeptide can be puri Bodansky, “The Practice of Peptide Synthesis. 2d ed. fied from the culture medium. (Springer Verlag, 1994)). In addition, intermediate purifica 0115 The sequence encoding a GCRA peptide described tion and linear scale up are possible. Those of ordinary skill in herein can be inserted into a vector capable of delivering and the art will appreciate that Solution synthesis requires consid maintaining the nucleic acid molecule in a bacterial cell. The eration of main chain and side chain protecting groups and DNA molecule may be inserted into an autonomously repli activation method. In addition, careful segment selection is cating vector (Suitable vectors include, for example, necessary to minimize racemization during segment conden pGEM37 and pcDNA3, and derivatives thereof). The vector sation. Solubility considerations are also a factor. Solid phase nucleic acid may be a bacterial or bacteriophage DNA such as peptide synthesis uses an insoluble polymer for Support dur bacteriophage lambda or M13 and derivatives thereof. Con ing organic synthesis. The polymer-Supported peptide chain struction of a vector containing a nucleic acid described permits the use of simple washing and filtration steps instead hereincan be followed by transformation of a host cell such as of laborious purifications at intermediate steps. Solid-phase a bacterium. Suitable bacterial hosts include but are not lim peptide synthesis may generally be performed according to ited to, E. coli, B subtilis, Pseudomonas, Salmonella. The the method of Merrifield et al., J. Am. Chem. Soc., 1963, genetic construct also includes, in addition to the encoding 85:2149, which involves assembling a linear peptide chain on nucleic acid molecule, elements that allow expression, Such a resin Support using protected amino acids. Solid phase as a promoter and regulatory sequences. The expression vec peptide synthesis typically utilizes either the Boc or Fmoc tors may contain transcriptional control sequences that con strategy, which is well known in the art. trol transcriptional initiation, Such as promoter, enhancer, 0112 Those of ordinary skill in the art will recognize that, operator, and repressor sequences. in Solid phase synthesis, deprotection and coupling reactions 0116 A variety of transcriptional control sequences are must go to completion and the side-chain blocking groups well known to those in the art. The expression vector can also must be stable throughout the synthesis. In addition, solid include a translation regulatory sequence (e.g., an untrans phase synthesis is generally most Suitable when peptides are lated 5' sequence, an untranslated 3' sequence, or an internal to be made on a small scale. ribosome entry site). The vector can be capable of autono US 2016/0235807 A1 Aug. 18, 2016 24 mous replication or it can integrate into host DNA to ensure I0123 Exemplary src inhibitors include, without limita stability during polypeptide production. tion, Small molecules, chemical compounds and nucleic acid 0117 The protein coding sequence that includes a GCRA molecules which function to down regulate expression of peptide described herein can also be fused to a nucleic acid target genes and inhibit the function of direct and indirect encoding a polypeptide affinity tag, e.g., glutathione S-trans c-Src Substrates, such as the focal adhesion kinase, signal ferase (GST), maltose E binding protein, protein A, FLAG transducer and activator of transcription 3 (STAT3), vascular tag, hexa-histidine, myc tag or the influenza HA tag, in order endothelial growth factor (VEGF), paxillin, Cas, to facilitate purification. The affinity tag or reporter fusion p190RhoGAP, RRas, E-cadherin, c-Jun amino-terminal joins the reading frame of the polypeptide of interest to the kinase, NEDD9, and others. Exemplary agents include dasa reading frame of the gene encoding the affinity tag such that tinib, SU6656, and AZD05530. Src inhibitors are also avail a translational fusion is generated. Expression of the fusion able from Wyeth and include for example, 4-(2,4-Dichloro gene results in translation of a single polypeptide that 5-methoxyphenyl)amino-7-3-(4-ethyl-1-piperazinyl) includes both the polypeptide of interest and the affinity tag. propo-Xyl-6-methoxy-3-quinolinecarbonitrile; 4-(2,4- In some instances where affinity tags are utilized, DNA Dichloro-5-methoxyphenyl)amino-6-methoxy-7-2-(4- sequence encoding a protease recognition site will be fused methyl-1-pipera-Zinyl)ethoxy-3-quinolinecarbonitrile; between the reading frames for the affinity tag and the 4-(2,4-Dichloro-5-methoxyphenyl)amino-7-2-(4-ethyl-1- polypeptide of interest. piperazinyl)ethox-y-6-methoxy-3-quinolinecarbonitrile; 0118 Genetic constructs and methods suitable for produc 4-(2,4-Dichloro-5-methoxyphenyl)amino-6-methoxy-7- tion of immature and mature forms of the GCRA peptides and (1-methylpiperidin-4-yl)methoxy-3-quinolinecarbonitrile; variants described herein in protein expression systems other 4-(2,4-Dichloro-5-methoxyphenyl)amino-6-methoxy-7- than bacteria, and well known to those skilled in the art, can 2-(1-methylpiperidin-4-yl)ethoxy-3-quinolinecarbonitrile; also be used to produce polypeptides in a biological system. 4-(2,4-Dichloro-5-methoxyphenyl)amino-6-methoxy-7- 0119 The peptides disclosed herein may be modified by 3-(1-methylpiperidin-4-yl)propoxyquinoline-3-carboni attachment of a second molecule that confers a desired prop trile: 4-(2,4-Dichloro-5-methoxyphenyl)amino-7-(1-eth erty upon the peptide. Such as increased half-life in the body, ylpiperidin-4-yl)methoxy--6-methoxyquinoline-3- for example, pegylation. Such modifications also fall within carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino)-6- the scope of the term “variant” as used herein. ethoxy-7-3-(4-methylpiperazin-1-yl)propoxyquinoline-3- 0120 Compositions carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino-6- 0121 The present invention provides a composition ethoxy-7-(1-methylpiperidin-4-yl)methoxyquinoline-3- including at least one GC-C peptide (i.e., GCRA peptide). carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino)-6- The composition may further include other therapeutic ethoxy-7-3-(4-ethylpiperazin-1-yl)propoxyquinoline-3- agents, including, but not limited to, a NF-kB inhibitor, a carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino)-6- c-Src inhibitor, c-Myc inhibitors, Ikk inhibitors, an anti-in ethoxy-7-3-(1-methylpiperidin-4-yl)propoxyquinoline-3- flammatory agent, an analgesic, a chemotherapeutic, or a carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino)-6- combination thereof. ethoxy-7-2-(4-methyl-1-piperazinyl)ethoxyquinoline-3- carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino)-6- 0122 Exemplary NF-kB inhibitors include, but are not ethoxy-7-2-(1-methylpiperidin-4-yl)ethoxyquinoline-3- limited to, Small molecules, chemical compounds and nucleic carbonitrile; or 4-(2,4-Dichloro-5-methoxyphenyl)amino acid molecules which function to downregulate expression of 6-methoxy-7-3-(4-propyl-1-pipera-Zinyl)propoxy-3- target genes and inhibit the function of direct and indirect NF-KB signaling pathway, proteasome inhibitors, inhibitors quinolinecarbonitrile; and pharmaceutically acceptable salts of ubiquitin conjugation, inhibitors of proteasome peptidases, thereof. and protease inhibitors. Additionally, the use of antisense 0.124 Suitable compounds possessing inhibitory activity oligonucleotides to control the expression of cellular compo against the Src family of non-receptor tyrosine kinases nents is known in the art, and may be utilized in the present include the quinazoline derivatives disclosed in International invention to reduce the expression of NFkB or its subunits. Patent Applications WO 01/94341, WO 02/16352, WO (Antisense oligonucleotides that hybridize to NF-kB mRNA, 02/30924, WO 02/30926, WO 02/34744, WO 02/085895, and their therapeutic use to Suppress processes that depend on WO 02/092577 (arising from PCT/GB 02/02117), WO activation of NF-kB, are described in WO95/35032). Exem 02/092578 (arising from PCT/GB 02/02124) and WO plary NF-kB inhibitors include, but are not limited to, inhibi 02/092579 (arising from PCT/GB 02/02128), the quinoline tors of chymotrypsin-like and trypsin-like proteases, and derivatives described in WO 03/008409 (arising from PCT/ inhibitors of thiol (or cysteine) and serine proteases; natural GB 02/03177), WO 03/047584 and WO 03/048159 and the and chemical protease inhibitors (such as peptides containing quinazoline derivatives described in European Patent Appli an C-diketone or an O-keto ester, peptide chloromethyl cations 02292736.2 (filed 4 Nov. 2002) and 03290900.4 (filed ketones, isocoumarins, peptide Sulfonyl fluorides, peptidyl 10 Apr. 2003). boronates, peptide epoxides, and peptidyl diazomethanes); pyrrolidine dithiocarbamate (PTDC); glucocorticoids, pre 0.125. It is disclosed in Journal Medicinal Chemistry, donsone, prednisolone, methyl prednisolone, dexametha 2001, 44, 822-833 and 3965-3977 that certain 4-anilino-3- Sone, prednisone, deoxycorticosterone, cortisone, hydrocor cyanoquinoline derivatives are useful for the inhibition of tisone, nonglucocorticoid lazaroids, novel amides that are Src-dependent cell proliferation. The 4-anilino-3-cyano inhibitors of NF-kB DNA binding (WO97/23457), antisense quinoline Src inhibitor known as SKI 606 is described in oligonucleotides that hybridize to NF-kB mRNA (WO95/ Cancer Research, 2003, 63,375. 35032). In a preferred embodiment, a NF-kB inhibitor is 0.126 Other compounds which possess Src kinase inhibi PTDC. tory properties are described in, for example, International US 2016/0235807 A1 Aug. 18, 2016

Patent Applications WO 96/10028, WO 97/07131, WO chostatin-A), protein synthesis inhibitors (e.g. anisomycin, 97/08193, WO 97/16452, WO 97/28161, WO 97/32879 and cycloheximide), kinase inhibitors (e.g. staaurosporine), 20 WO 97/497O6. S-proteasome chymotrypsin inhibitors (e.g. Gliotoxin), 0127. Other compounds which possess Src kinase inhibi topoisomerase I inhibitors (e.g. Camptothecin, 10-Hydroxy tory properties are described in, for example, J Bone Mineral camptothecin), Topoisomerase II inhibitors (e.g. Etoposide), Research, 1999, 14 (Suppl. 1), S487, Molecular Cell, 1999,3, tubulin inhibitors (e.g. podophyllotoxin, Vinblastine), RNA 639-647, Journal Medicinal Chemistry, 1997, 40,2296-2303, synthesis inhibitors (e.g. Actinomycin D), HSP-90 inhibitors Journal Medicinal Chemistry, 1998, 41, 3276-3292 and (e.g. 17-allylamino-geldanamycin), and DNA polymerase Bioorganic & Medicinal Chemistry Letters, 2002, 12, 1361 inhibitors (e.g. Aphicicolin). and 3153. 0140 Exemplary Ikk inhibitors include, but are not lim 0128 Particular Src kinase inhibitors include the follow ited to, NEMO Binding Domain Peptide, anti-inflammatory ing: agents (e.g. aspirin, sodium salicylate, Sulindac), thalido 0129 (i) 4-amino-5-(3-methoxyphenyl)-7-(4-2-(2- mide, cyclopentenone prostaglandins, Arsenic trioxide, methoxyethylamino)ethox-yl)phenyl)-3-pyrrolo[2,3-dipyri quinazoline analogues (e.g. SPC 839), ATP analogs, B-car midine and 4-amino-5-(3-methoxyphenyl)-7-(4-(2-di-(2- bolines (e.g. PS-1145, ML120B), aminothiophenecarboxim methoxyethyl)aminolethoxyphe-nyl)pyrrolo[2,3-d ide (e.g. SC514, BMS-34541), ureidocarboximide pyrimidine which are obtainable by methods described in thiophenes, pyridooxazin derivatives, and Small molecule International Patent Application WO96/10028; inhibitors. 0130 (ii) 4-amino-7-tert-butyl-5-(4-tolyl)pyrazolo 3,4-d 0.141. In some embodiments, the compositions described pyrimidine which is also known as PP1 and is described in herein are formulated in a pH dependent release form. Alter Molecular Cell, 1999, 3, 639-648: natively, Such compositions are formulated in a form that 0131 (iii) 2-(2,6-dichloroanilino)-6,7-dimethyl-1,8-dihy releases the peptides at a specific region of the gastrointestinal droimidazo 4.5-hisoquinolin-9-one and 2-(2,6-dichloroa (GI) tract (e.g., duodenum, jejunum, ileum, terminal ileum, or nilino)-7-(E)-3-diethylaminoprop-1-enyl-6-met-hyl-1.8- ascending colon). The formulation may contain an inert car dihydroimidazo[4,5-hisoquinolin-9-one which a rier coated with a composition and an enteric coating which obtainable by methods described in Journal Medicinal Chem releases the peptides at a specific pH (such as pH5 or pH7). istry, 2002, 45,3394; Preferred pH for duodenum or jejunum release is pH 4.5-5.5 (0132 (iv) 1-6-(2,6-dichlorophenyl)-2-(4-diethylami or pH 5.5-6.5. Preferred pH for ileum, terminal ileum, or nobutyl)pyrido 2,3-dipyrimidin-7-yl)-3-ethylurea which is ascending colon release is pH 5.5-6.5 or pH 6.5-7.5. Prefer obtainable by methods described in Journal Medicinal Chem ably, the inert carrier is a selected from mannitol, lactose, a istry, 1997, 40,2296-2303 and Journal Medicinal Chemistry, microcrystalline cellulose, or starch. 2001, 44, 1915; 0.142 Methods 0.133 (v) 6-(2,6-dichlorophenyl)-2-[4-(2-diethylaminoet 0143. The invention relates in part to the use of GC-C hoxy)anilino-8-me-thyl-8H-pyrido2,3-dipyrimidin-7-one agonists to inhibit activation of NF-kB, to reduce production which is also known as PD166285 and is described in J. of pro-inflammatory cytokines/chemokines, and/or to Pharmacol. Exp. Ther., 1997, 283, 1433-1444; increase secretion of anti-inflammatory cytokines. 0134 (vi) the compound known as PD 162531 which is 0144. Accordingly, the present invention provides meth described in Mol. Biol. Cell, 2000, 11, 51-64: ods of treating a disorder that is mediated by guanylate 0135 (vii) the compound known as PD166326 which is cyclase receptor agonists by administering to a subject in described in Biochem Pharmacol., 2000, 60, 885-898; and need thereofan effective amount of a GCRA peptide or phar 0.136 (viii) the compound known as PD173955 which is maceutical composition thereof or a composition described described in Cancer Research, 1999, 59, 61.45-6152. herein, where the effective amount is sufficient to inhibit 0.137 Other compounds which may possess Src kinase NF-kB activation. Disorders mediated by the guanylate inhibitory properties are described in, for example, Interna cyclase receptoragonists include lipid metabolism disorders, tional Patent Applications WO 02/0791.92, WO 03/000188, biliary disorders, gastrointestinal disorders, inflammatory WO 03/000266, WO 03/000705, WO 02/083668, WO disorders, lung disorders, cancer, cardiac disorders including 02/092573, WO 03/004492, WO 00/49018, WO 03/013541, cardiovascular disorders, eye disorders, oral disorders, blood WO 01/00207, WO 01/00213 and WO 01/00214. Particular disorders, liver disorders, skin disorders, prostate disorders, Src inhibitors include those provided in International Patent endocrine disorders, increasing gastrointestinal motility and Application WO 01/94341. Further particular Src inhibitors obesity. Lipid metabolism disorders include, but not limited include the following compounds from International Patent to, dyslipidemia, hyperlipidemia, hypercholesterolemia, Application WO 02/16352, WO 02/30924, WO 02/30926 and hypertriglyceridemia, sitosterolemia, familial hypercholes WO O2/34744. terolemia, Xanthoma, combined hyperlipidemia, lecithin cho 0.138. In a preferred embodiment, a c-Src tyrosine kinase lesterol acyltransferase deficiency, tangier disease, abetalipo inhibitor is: N-benzyl-2-(5-(4-(2-morpholinoethoxy)phenyl) proteinemia, erectile dysfunction, fatty liver disease, and pyridin-2-yl)acetamide (also called KX2-391) or PP2 (pro hepatitis. Billary disorders include gallbladder disorders such tein phosphatase 2). as for example, gallstones, gallbladder cancer cholangitis, or 0139 Exemplary c-Myc inhibitors include, but are not primary Sclerosing cholangitis; or bile duct disorders such as limited to Myc inhibitors, include, but are not limited to, for example, cholecystitis, bile duct cancer or fascioliasis. Omomyc transgene, Small-molecule disruptors of MYC: Gastointestinal disorders include for example, irritable bowel MAX heterodimerization, small molecules (e.g. 10058-F4 syndrome (IBS), non-ulcer dyspepsia, chronic intestinal (Huang et al. 2006)), BET bromodomain inhibitors (e.g. pseudo-obstruction, functional dyspepsia, colonic pseudo JQ1), alkylating agents (e.g. Mitomycin C), DHFR inhibitors obstruction, duodenogastric reflux, gastroesophageal reflux (e.g. Methotrexate), histone deacetylase inhibitors (e.g. Tri disease (GERD), ileus inflammation (e.g., post-operative US 2016/0235807 A1 Aug. 18, 2016 26 illeus), gastroparesis, heartburn (high acidity in the GI tract), 0149 Pro-inflammatory cytokines include, but are not constipation (e.g., IBS-associated constipation, constipation limited to, IL-1, IL-2, TNF, IL-12p40, IL-17, and IL-23. associated with use of medications such as opioids, osteoar Chemokines include, but are not limited to, IL-8, RANTES thritis drugs, osteoporosis drugs; post Surgical constipation, and MIP-1C. Anti-inflammatory cytokines include, but are constipation associated with neuropathic disorders). Inflam not limited to, IL-10. matory disorders include tissue and organ inflammation Such 0150. In some embodiments, GCRA peptides inhibit the as kidney inflammation (e.g., nephritis), gastrointestinal sys nuclear localization of NF-kB. tem inflammation (e.g., chronic inflammatory bowel disease, 0151. In some embodiments, GCRA peptides mediate Crohn's disease, colitis, and ulcerative colitis); necrotizing inhibition of NF-kB activating factors. NF-kB activating fac enterocolitis (NEC); pancreatic inflammation (e.g., pancre tors are, without being limited to the examples herein, are atis), lung inflammation (e.g., bronchitis or asthma) or skin cytokines such as tumor necrosis factor (TNF) and interleukin inflammation (e.g., psoriasis, eczema). Lung disorders (IL)-1, lipopolysaccharides, bacterial and viral infections, include for example chronic obstructive pulmonary disease activators of protein kinase C, and oxidants. The inhibition of (COPD), and fibrosis. Cancer includes tissue and organ car NF-kB may result in reduced production of cytokines (such cinogenesis including metatases Such as for example gas as, without being limited to the examples herein, TNF, IL-1, trointestinal cancer (e.g., gastric cancer, esophageal cancer, IL-2, IL-6, IL-8, IL-12p40, IL-17, and IL-23), adhesion mol pancreatic cancer, colorectal cancer including colorectal ecules (such as ICAM-1, VCAM-1, E-selectin, and MAd metastasis, intestinal cancer, anal cancer, liver cancer, gall CAM-1), and enzymes that are involved in inflammation, bladder cancer, or colon cancer); lung cancer, thyroid cancer, Such as inducible nitric oxide synthase and cyclooxygenase skin cancer (e.g., melanoma); oral cancer, urinary tract cancer 2. In some embodiments, the invention may provide inhibi (e.g. bladder cancer or kidney cancer); blood cancer (e.g. tion of proteins whose genes are switched on by NF-KB, such myeloma or leukemia) or prostate cancer. Cardiac disorders as, without being limited to the examples herein, TNF and include for example, congestive heart failure, trachea cardia IL-1. hypertension, high cholesterol, or high tryglycerides. Cardio 0152. In some embodiments, the inhibition of NF-KB acti vascular disorders include for example aneurysm, angina, Vation with GC-C agonists may prevent IkB degradation and atherosclerosis, cerebrovascular accident (stroke), cere may attenuate chronic inflammation associated with Crohn's brovasculardisease, congestive heart failure, coronary artery disease. In another embodiment, inhibition of p65 subunit of disease, myocardial infarction (heart attack), or peripheral NF-kB may effectively abrogate colonic inflammation asso vascular disease. Liver disorders include for example cirrho ciated with colitis. sis and fibrosis. In addition, GC-C agonist may also be useful 0153. In another embodiment of the current invention, to facilitate liver regeneration in liver transplant patients. Eye inhibition of NF-kB with GC-C agonists may prevent disorders include for example increased intra-ocular pres mucosal NF-kB activation in ulcerative colitis patients. In Sure, glaucoma, dry eyes retinal degeneration, disorders of particular the GC-C agonists of the current invention may tear glands or eye inflammation. Skin disorders include for inhibit NF-kB activation in macrophages. example xerosis. Oral disorders include for example dry 0154) In some embodiments, GC-C agonists mediated mouth (xerostomia), Sjögren's syndrome, gum diseases (e.g., enhancement or stimulation of c(GMP signaling pathway periodontal disease), or salivary gland duct blockage or mal results in the inhibition of NF-kB. According to some function. Prostate disorders include for example benign pro embodiments, the GC-C agonist mediated enhancement or static hyperplasia (BPH). Endocrine disorders include for stimulation of c(GMP may result in the activation of the cyclic example diabetes mellitus, hyperthyroidism, hypothyroid dependent protein kinase (PKG). The cyclic GMP-dependent ism, and cystic fibrosis. kinase (PKG) is an important mediator of signal transduction 0145. In a preferred embodiment, the disorder mediated that may induce gene expression through cAMP response by the guanylate cyclase receptor agonists is a GI inflamma element binding protein (CREB). tory disorder, such as chronic inflammatory bowel disease, (O155 By “inhibiting or “inhibition” or “reduce”, it Crohn's disease, colitis, ulcerative colitis, DSS- and TNBS means the GCRA peptide decreases the activity and/or pro induced colitis, inflammation-induced colonic cancer, or col duction of a protein by at least about 5%, at least about 10%, orectal metastasis. at least about 15%, at least about 20%, at least about 25%, at 0146 The present invention also provides a method of least about 30%, at least about 35%, at least about 40%, at modulating NF-kB induction in a cell by contacting the cell least about 45%, at least about 50%, at least about 55%, at with an effective amount of a GCRA peptide or pharmaceu least about 60%, at least about 65%, at least about 70%, at tical composition thereof, or a composition described herein. least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, or 0147 The present invention also provides a method of more relative to the activity and/or production of the protein modulating NF-KB-dependent target gene expression in a cell without the GCRA peptide. by contacting the cell with an effective amount of a GCRA 0156 By “induce”, it means the GCRA peptide increases peptide or pharmaceutical composition thereof or a compo the activity and/or production of a protein by at least about sition described herein, where the GCRA peptide or the com 5%, at least about 10%, at least about 15%, at least about 20%, position inhibits NF-kB activation. NF-kB-dependent target at least about 25%, at least about 30%, at least about 35%, at gene includes, for example, TNF. least about 40%, at least about 45%, at least about 50%, at 0148 NF-KB may include one or more transcription factor least about 55%, at least about 60%, at least about 65%, at of the NF-KB family, for example without being limited to the least about 70%, at least about 75%, at least about 80%, at list herein, NF-kB1 (p50), NF-kB2 (p52), p65 (RelA), c-Rel, least about 85%, at least about 90%, at least about 95%, or at and RelB, or any protein that share a commonstructural motif least about 100%, or more relative to the activity and/or called the Rel homology domain. production of the protein without the GCRA peptide. US 2016/0235807 A1 Aug. 18, 2016 27

(O157. The term “treatment” or “treating refers to reduc corticoids, predonsone, prednisolone, methyl prednisolone, ing or alleviating symptoms in a Subject, preventing Symp dexamethasone, prednisone, deoxycorticosterone, cortisone, toms from worsening or progressing, and/or preventing dis hydrocortisone, nonglucocorticoid lazaroids, novel amides ease in a Subject who is free therefrom. For a given subject, that are inhibitors of NF-kB DNA binding (WO 97/23457), improvement, worsening, regression, or progression of a antisense oligonucleotides that hybridize to NF-kB mRNA symptom may be determined by any objective or Subjective (WO95/35032). In a preferred embodiment, a NF-kB inhibi measure. Efficacy of the treatment may be measured as an tor is PTDC. improvement in morbidity or mortality (e.g., lengthening of 0163 Exemplary src inhibitors include, without limita survival curve for a selected population). Thus, effective tion, Small molecules, chemical compounds and nucleic acid treatment would include therapy of existing disease, control molecules which function to down regulate expression of of disease by slowing or stopping its progression, prevention target genes and inhibit the function of direct and indirect of disease occurrence, reduction in the number or severity of c-Src Substrates, such as the focal adhesion kinase, signal symptoms, or a combination thereof. The effect may be transducer and activator of transcription 3 (STAT3), vascular shown in a controlled study using one or more statistically endothelial growth factor (VEGF), paxillin, Cas, significant criteria. p190RhoGAP, RRas, E-cadherin, c-Jun amino-terminal 0158. The term “prevention” in relation to a given disease kinase, NEDD9, and others. Exemplary agents include dasa or disorder means: preventing the onset of disease develop tinib, SU6656, and AZD05530. Src inhibitors are also avail ment if none had occurred, preventing the disease or disorder able from Wyeth and include for example, 4-(2,4-Dichloro from occurring in a subject that may be predisposed to the 5-methoxyphenyl)amino-7-3-(4-ethyl-1-piperazinyl) disorder or disease but has not yet been diagnosed as having propo-Xyl-6-methoxy-3-quinolinecarbonitrile; 4-(2,4- the disorder or disease, and/or preventing further diseasef Dichloro-5-methoxyphenyl)amino-6-methoxy-7-2-(4- disorder development if already present. methyl-1-pipera-Zinyl)ethoxy-3-quinolinecarbonitrile; 0159 Intracellular cGMP produced by exposing, e.g., 4-(2,4-Dichloro-5-methoxyphenyl)amino-7-2-(4-ethyl-1- contacting a tissue (e.g., gastrointestinals tissue) or cell with piperazinyl)ethox-y-6-methoxy-3-quinolinecarbonitrile; GCRA agonists. By inducing is meant an increase in coiMP 4-(2,4-Dichloro-5-methoxyphenyl)amino-6-methoxy-7- production compared to a tissue or cell that has not been in (1-methylpiperidin-4-yl)methoxy-3-quinolinecarbonitrile; contact with GCRA peptide or variant. Tissues or cells are 4-(2,4-Dichloro-5-methoxyphenyl)amino-6-methoxy-7- directly contacted with a GCRA peptide or variant. Alterna 2-(1-methylpiperidin-4-yl)ethoxy-3-quinolinecarbonitrile; tively, the GCRA peptide or variant is administered systemi 4-(2,4-Dichloro-5-methoxyphenyl)amino-6-methoxy-7- cally. GCRA peptide or variant are administered in an amount 3-(1-methylpiperidin-4-yl)propoxyquinoline-3-carboni sufficient to increase intracellular cGMP concentration. trile: 4-(2,4-Dichloro-5-methoxyphenyl)amino-7-(1-eth cGMP production is measured by a cell-based assay known in ylpiperidin-4-yl)methoxy--6-methoxyquinoline-3- the art (25). carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino)-6- 0160 Disorders are treated, prevented or alleviated by ethoxy-7-3-(4-methylpiperazin-1-yl)propoxyquinoline-3- administering to a Subject, e.g., a mammal Such as a human in carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino)-6- need thereof, a therapeutically effective dose of a GCRA ethoxy-7-(1-methylpiperidin-4-yl)methoxyquinoline-3- peptide. The GCRA peptides may be in a pharmaceutical carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino)-6- composition in unit dose form, together with one or more ethoxy-7-3-(4-ethylpiperazin-1-yl)propoxyquinoline-3- pharmaceutically acceptable excipients. The term “unit dose carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino)-6- form” refers to a single drug delivery entity, e.g., a tablet, ethoxy-7-3-(1-methylpiperidin-4-yl)propoxyquinoline-3- capsule, solution or inhalation formulation. The amount of carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino)-6- peptide present should be sufficient to have a positive thera ethoxy-7-2-(4-methyl-1-piperaz-inyl)ethoxyquinoline-3- peutic effect when administered to a patient (typically, carbonitrile, 4-(2,4-Dichloro-5-methoxyphenyl)amino)-6- between 10 ug and 3 g). What constitutes a “positive thera ethoxy-7-2-(1-methylpiperidin-4-yl)ethoxyquinoline-3- peutic effect” will depend upon the particular condition being carbonitrile; or 4-(2,4-Dichloro-5-methoxyphenyl)amino treated and will include any significant improvement in a 6-methoxy-7-3-(4-propyl-1-pipera-Zinyl)propoxy-3- condition readily recognized by one of skill in the art. quinolinecarbonitrile; and pharmaceutically acceptable salts 0161 The GCRA peptides can be administered alone or in thereof. combination with other agents. For example the GCRA pep 0164 Suitable compounds possessing inhibitory activity tides can be administered in combination with a NF-KB against the Src family of non-receptor tyrosine kinases inhibitor, a Src inhibitor, 5-ASA, or inhibitors of c(GMP include the quinazoline derivatives disclosed in International dependent phosphodiesterase, such as, for example, Suldinac Patent Applications WO 01/94341, WO 02/16352, WO Sulfone, Zaprinast, motapizone, Vardenafilor sildenifil; one or 02/30924, WO 02/30926, WO 02/34744, WO 02/085895, more other chemotherapeutic agents; or anti-inflammatory WO 02/092577 (arising from PCT/GB 02/02117), WO drugs such as, for example, steroids or non-steroidal anti 02/092578 (arising from PCT/GB 02/02124) and WO inflammatory drugs (NSAIDS), such as aspirin. 02/092579 (arising from PCT/GB 02/02128), the quinoline 0162 Exemplary NF-kB inhibitors include, but are not derivatives described in WO 03/008409 (arising from PCT/ limited to, inhibitors of chymotrypsin-like and trypsin-like GB 02/03177), WO 03/047584 and WO 03/048159 and the proteases, and inhibitors of thiol (or cysteine) and serine quinazoline derivatives described in European Patent Appli proteases; natural and chemical protease inhibitors (such as cations 02292736.2 (filed 4 Nov. 2002) and 03290900.4 (filed peptides containing an O-diketone oran C-keto ester, peptide 10 Apr. 2003). chloromethyl ketones, isocoumarins, peptide Sulfonyl fluo 0.165. It is disclosed in Journal Medicinal Chemistry, rides, peptidylboronates, peptide epoxides, and peptidyl dia 2001, 44, 822-833 and 3965-3977 that certain 4-anilino-3- Zomethanes); pyrrolidine dithiocarbamate (PTDC); gluco cyanoquinoline derivatives are useful for the inhibition of US 2016/0235807 A1 Aug. 18, 2016 28

Src-dependent cell proliferation. The 4-anilino-3-cyano Concurrent administration may be achieved with a formula quinoline Src inhibitor known as SKI 606 is described in tion in which two or more active agents are mixed, or with Cancer Research, 2003, 63,375. simultaneous administration of two or more active agents 0166 Other compounds which possess Src kinase inhibi formulated independently. Sequential administration of two tory properties are described in, for example, International or more active agents may be achieved with two or more Patent Applications WO 96/10028, WO 97/07131, WO active agents, formulated independently, administered in 97/08193, WO 97/16452, WO 97/28161, WO 97/32879 and sequence with one agent administered first followed by the WO 97/497O6. second agent administered seconds, minutes, hours, or days 0167. Other compounds which possess Src kinase inhibi after the first agent. tory properties are described in, for example, J Bone Mineral 0180 Combination therapy can be achieved by adminis Research, 1999, 14 (Suppl. 1), S487, Molecular Cell, 1999,3, tering two or more agents, e.g., a GCRA peptide described 639-647, Journal Medicinal Chemistry, 1997, 40,2296-2303, herein or a composition described herein and another com Journal Medicinal Chemistry, 1998, 41, 3276-3292 and pound, each of which is formulated and administered sepa Bioorganic & Medicinal Chemistry Letters, 2002, 12, 1361 rately, or by administering two or more agents in a single and 3153. formulation. Other combinations are also encompassed by 0168 Particular Src kinase inhibitors include the follow combination therapy. For example, two agents can be formu ing: lated together and administered in conjunction with a sepa (0169 (i) 4-amino-5-(3-methoxyphenyl)-7-(4-2-(2- rate formulation containing a third agent. While the two or methoxyethylamino)ethox-yphenyl)-3-pyrrolo[2,3-dipyri more agents in the combination therapy can be administered midine and 4-amino-5-(3-methoxyphenyl)-7-(4-(2-di-(2- simultaneously, they need not be. For example, administra methoxyethyl)aminolethoxyphe-nyl)pyrrolo[2,3-d tion of a first agent (or combination of agents) can precede pyrimidine which are obtainable by methods described in administration of a second agent (or combination of agents) International Patent Application WO96/10028; by minutes, hours, days, or weeks. Thus, the two or more 0170 (ii) 4-amino-7-tert-butyl-5-(4-tolyl)pyrazolo 3,4-d agents can be administered within minutes of each other or pyrimidine which is also known as PP1 and is described in within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other or Molecular Cell, 1999, 3, 639-648: within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days of each other 0171 (iii) 2-(2,6-dichloroanilino)-6,7-dimethyl-1,8-dihy or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of each other. In droimidazo 4.5-hisoquinolin-9-one and 2-(2,6-dichloroa Some cases even longer intervals are possible. While in many nilino)-7-(E)-3-diethylaminoprop-1-enyl-6-met-hyl-1.8- cases it is desirable that the two or more agents used in a dihydroimidazo[4,5-hisoquinolin-9-one which a combination therapy be present in within the patient’s body at obtainable by methods described in Journal Medicinal Chem the same time, this need not be so. istry, 2002, 45,3394; 0172 (iv) 1-6-(2,6-dichlorophenyl)-2-(4-diethylami 0181. The combination therapies featured in the present nobutyl)pyrido 2,3-dipyrimidin-7-yl)-3-ethylurea which is invention can result in a synergistic effect in the treatment of obtainable by methods described in Journal Medicinal Chem a disease or cancer. A “synergistic effect” is defined as where istry, 1997, 40,2296-2303 and Journal Medicinal Chemistry, the efficacy of a combination of therapeutic agents is greater 2001, 44, 1915; than the Sum of the effects of any of the agents given alone. A 0173 (v) 6-(2,6-dichlorophenyl)-2-[4-(2-diethylaminoet synergistic effect may also be an effect that cannot be hoxy)anilino-8-me-thyl-8H-pyrido2,3-dipyrimidin-7-one achieved by administration of any of the compounds or other which is also known as PD166285 and is described in J. therapeutic agents as single agents. Pharmacol. Exp. Ther., 1997, 283, 1433-1444; 0182. In some embodiments, the term “synergistic effect” (0174 (vi) the compound known as PD 162531 which is means the combination of a GCRA peptide and a selected described in Mol. Biol. Cell, 2000, 11, 51-64: compound described herein reduces about 5%, about 10%, (0175 (vii) the compound known as PD166326 which is about 20%, about 30%, about 40%, about 50%, about 75%, described in Biochem Pharmacol., 2000, 60, 885-898; and about 90% or more inflammation compared to a GCRA pep (0176 (viii) the compound known as PD173955 which is tide alone or a selected compound alone. described in Cancer Research, 1999, 59, 61.45-6152. 0183 In some embodiments, the term “synergistic effect” 0177. Other compounds which may possess Src kinase means the combination of a GCRA peptide and a selected inhibitory properties are described in, for example, Interna compound described herein down regulates about 5%, about tional Patent Applications WO 02/0791.92, WO 03/000188, 10%, about 20%, about 30%, about 40%, about 50%, about WO 03/000266, WO 03/000705, WO 02/083668, WO 75%, about 90% or more NF-KB signaling compared to a 02/092573, WO 03/004492, WO 00/49018, WO 03/013541, GCRA peptide alone or a selected compound alone. WO 01/00207, WO 01/00213 and WO 01/00214. Particular Src inhibitors include those provided in International Patent 0184. In some embodiments, the term “synergistic effect” Application WO 01/94341. Further particular Src inhibitors means the combination of a GCRA peptide and a selected include the following compounds from International Patent compound described herein induces about 5%, about 10%, Application WO 02/16352, WO 02/30924, WO 02/30926 and about 20%, about 30%, about 40%, about 50%, about 75%, WO O2/34744. about 90% or more secretion of anti-inflammatory cytokines 0178. In a preferred embodiment, a c-Src tyrosine kinase compared to a GCRA peptide alone or a selected compound inhibitor is: N-benzyl-2-(5-(4-(2-morpholinoethoxy)phenyl) alone. pyridin-2-yl)acetamide (also called KX2-391) or PP2 (pro 0185. In some embodiments, the term “synergistic effect” tein phosphatase 2). means the combination of a GCRA peptide and a selected 0179 The term “combination therapy” means administer compound described herein reduces about 5%, about 10%, ing two or more active agents concurrently or sequentially. about 20%, about 30%, about 40%, about 50%, about 75%, US 2016/0235807 A1 Aug. 18, 2016 29 about 90% or more production of pro-inflammatory ctyok (e.g., inhalation), transdermal (topical), transmucosal, and ines compared to a GCRA peptide alone or a selected com rectal administration. Solutions or Suspensions used for pound alone. parenteral, intradermal, or Subcutaneous application can 0186. In some embodiments, the term “synergistic effect” include the following components: a sterile diluent Such as means the combination of a GCRA peptide and a selected water for injection, Saline Solution, fixed oils, polyethylene compound described herein induces about 5%, about 10%, glycols, glycerine, propylene glycol or other synthetic Sol about 20%, about 30%, about 40%, about 50%, about 75%, vents; antibacterial agents such as benzyl alcohol or methyl about 90% or more apoptosis compared to a GCRA peptide parabens; antioxidants such as ascorbic acid or sodium alone or a selected compound alone. bisulfite; chelating agents such as ethylenediaminetetraacetic 0187 Thus, GC-C agonists may be used either alone or in acid; buffers such as acetates, citrates or phosphates, and combination with other anti-inflammatory drugs, for agents for the adjustment of tonicity Such as Sodium chloride example, Sulfasalazine (AZulfidine) Mesalamine (Asacol, or dextrose. The pH can be adjusted with acids or bases, such Lialda), balsalazide (Colazal), olsalazine (Dipentum), Corti as hydrochloric acid or sodium hydroxide. The parenteral costeroids, immune system Suppressors, for example, AZa preparation can be enclosed in ampoules, disposable syringes thioprine (AZasan, Imuran) and mercaptopurine (Purinethol), or multiple dose vials made of glass or plastic. Cyclosporine (Gengraf, Neoral, Sandimmune), Infliximab 0194 Pharmaceutical compositions suitable for injectable (Remicade), and/or immunomodulatory agents (such as use include: Sterile aqueous solutions (where water Soluble) 6-mercaptopurine and methotrexate). or dispersions and sterile powders for the extemporaneous 0188 The GCRA peptides described herein may be com preparation of sterile injectable solutions or dispersion. For bined with phosphodiesterase inhibitors, e.g., Sulindae Sul intravenous administration, Suitable carriers include physi fone, Zaprinast, sildenafil. Vardenafil or tadalafil to further ological saline, bacteriostatic water, Cremophor EL (BASF, enhance levels of c(3MP in the target tissues or organs. Parsippany, N.J.) or phosphate buffered saline (PBS). In all 0189 Combination therapy can also include two or more cases, the composition must be sterile and should be fluid to administrations of one or more of the agents used in the the extent that easy syringeability exists. It must be stable combination. For example, if agent X and agent Y are used in under the conditions of manufacture and storage and must be a combination, one could administer them sequentially in any preserved against the contaminating action of microorgan combination one or more times, e.g., in the order X-Y-X. isms such as bacteria and fungi. The carrier can be a solvent X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc. or dispersion medium containing, for example, water, etha 0.190 Combination therapy can also include the adminis nol, polyol (for example, glycerol, propylene glycol, and tration of one of the GC-C agonist with azothioprine and/or liquid polyethylene glycol, and the like), and Suitable mix other immunomodulating agents. The immunomodulating tures thereof. The proper fluidity can be maintained, for agents may include Small molecule drugs and biologics Such example, by the use of a coating Such as lecithin, by the as Remicade, Humaira, Cimzia etc. maintenance of the required particle size in the case of dis 0191 Combination therapy can also include the adminis persion and by the use of surfactants. Prevention of the action tration of two or more agents via different routes or locations. of microorganisms can be achieved by various antibacterial For example, (a) one agent is administered orally and another and antifungal agents, for example, parabens, chlorobutanol, agents is administered intravenously or (b) one agent is phenol, ascorbic acid, thimerosal, and the like. In many cases, administered orally and another is administered locally. In it will be preferable to include isotonic agents, for example, each case, the agents can either simultaneously or sequen Sugars, polyalcohols such as manitol, Sorbitol, Sodium chlo tially. Approximated dosages for Some of the combination ride in the composition. Prolonged absorption of the inject therapy agents described herein are found in the “BNF Rec able compositions can be brought about by including in the ommended Dose” column of tables on pages 11-17 of WO01/ composition an agent which delays absorption, for example, 76632 (the data in the tables being attributed to the March aluminum monostearate and gelatin. 2000 British National Formulary) and can also be found in 0.195 Sterile injectable solutions can be prepared by other standard formularies and other drug prescribing direc incorporating the active compound (e.g., a GCRA agonist) in tories. For some drugs, the customary prescribed dose for an the required amount in an appropriate solvent with one or a indication will vary somewhat from country to country. combination of ingredients enumerated above, as required, 0.192 The GCRA peptides, alone or in combination, can followed by filtered sterilization. Generally, dispersions are be combined with any pharmaceutically acceptable carrier or prepared by incorporating the active compound into a sterile medium. Thus, they can be combined with materials that do vehicle that contains a basic dispersion medium and the not produce an adverse, allergic or otherwise unwanted reac required other ingredients from those enumerated above. In tion when administered to a patient. The carriers or mediums the case of sterile powders for the preparation of sterile inject used can include solvents, dispersants, coatings, absorption able solutions, methods of preparation are vacuum drying and promoting agents, controlled release agents, and one or more freeze-drying that yields a powder of the active ingredient inert excipients (which include starches, polyols, granulating plus any additional desired ingredient from a previously ster agents, microcrystalline cellulose (e.g. celphere, Celphere ile-filtered solution thereof. Beads(R), diluents, lubricants, binders, disintegrating agents, 0.196 Oral compositions generally include an inert diluent and the like), etc. If desired, tablet dosages of the disclosed or an edible carrier. Such as mannitol, fructooligosaccha compositions may be coated by Standard aqueous or non rides, polyethylene glycol and other excepients. They can be aqueous techniques. enclosed in gelatin capsules or compressed into tablets. For 0193 A pharmaceutical composition of the invention is the purpose of oral therapeutic administration, the active formulated to be compatible with its intended route of admin compound can be incorporated with excipients and used in istration. Examples of routes of administration include the form of tablets, troches, or capsules. Oral compositions parenteral, e.g., intravenous, intradermal, Subcutaneous, oral can also be prepared using a fluid carrier for use as a mouth US 2016/0235807 A1 Aug. 18, 2016 30 wash, wherein the compound in the fluid carrier is applied 0203 Compositions of the present invention may also orally and Swished and expectorated or swallowed. Pharma optionally include other therapeutic ingredients, anti-caking ceutically compatible binding agents, and/or adjuvant mate agents, preservatives, Sweetening agents, colorants, flavors, rials can be included as part of the composition. The tablets, desiccants, plasticizers, dyes, glidants, anti-adherents, anti pills, capsules, troches and the like can contain any of the static agents, surfactants (wetting agents), anti-oxidants, following ingredients, or compounds of a similar nature: a film-coating agents, and the like. Any Such optional ingredi binder Such as microcrystalline cellulose, gum tragacanth or ent must be compatible with the compound described herein gelatin; an excipient Such as starch or lactose, a disintegrating to insure the stability of the formulation. agent such as alginic acid, Primogel, or corn starch; a lubri 0204 The composition may contain other additives as cant such as magnesium Stearate or Sterotes; a glidant Such as needed, including for example lactose, glucose, fructose, colloidal silicon dioxide; a Sweetening agent Such as Sucrose galactose, trehalose, Sucrose, maltose, raffnose, maltitol, or saccharin; or a flavoring agent Such as peppermint, methyl melezitose, stachyose, lactitol, palatinite, starch, Xylitol, salicylate, or orange flavoring. mannitol, myoinositol, and the like, and hydrates thereof, and 0.197 For administration by inhalation, the compounds amino acids, for example alanine, glycine and betaine, and are delivered in the form of an aerosol spray from pressured polypeptides and proteins, for example albumen. container or dispenser which contains a Suitable propellant, 0205 Examples of excipients for use as the pharmaceuti e.g., a gas such as carbon dioxide, or a nebulizer. cally acceptable carriers and the pharmaceutically acceptable 0198 Systemic administration can also be by transmu inert carriers and the aforementioned additional ingredients cosal or transdermal means. For transmucosal or transdermal include, but are not limited to binders, fillers, disintegrants, administration, penetrants appropriate to the barrier to be lubricants, anti-microbial agents, and coating agents such as: permeated are used in the formulation. Such penetrants are BINDERS: corn starch, potato starch, other starches, gelatin, generally known in the art, and include, for example, for natural and synthetic gums such as acacia, Xanthan, Sodium transmucosal administration, detergents, bile salts, and alginate, alginic acid, other alginates, powdered tragacanth, fusidic acid derivatives. Transmucosal administration can be guar gum, cellulose and its derivatives (e.g., ethyl cellulose, accomplished through the use of nasal sprays or Supposito cellulose acetate, carboxymethyl cellulose calcium, Sodium ries. For transdermal administration, the active compounds carboxymethyl cellulose), polyvinyl pyrrolidone (e.g., povi are formulated into ointments, salves, gels, or creams as gen done, crospovidone, copovidone, etc), methyl cellulose, erally known in the art. Methocel, pre-gelatinized starch (e.g., STARCH 1500R and 0199 The compounds can also be prepared in the form of STARCH 1500 LM(R), sold by Colorcon, Ltd.), hydroxypro Suppositories (e.g., with conventional Suppository bases Such pyl methyl cellulose, microcrystalline cellulose (FMC Cor as cocoa butter and other glycerides) or retention enemas for poration, Marcus Hook, Pa., USA), or mixtures thereof, rectal delivery. FILLERS: talc, calcium carbonate (e.g., granules or powder), 0200. In one embodiment, the active compounds are pre dibasic calcium phosphate, tribasic calcium phosphate, cal pared with carriers that will protect the compound against cium sulfate (e.g., granules or powder), microcrystalline cel rapid elimination from the body, such as a controlled release lulose, powdered cellulose, dextrates, kaolin, mannitol, formulation, including implants and microencapsulated silicic acid, Sorbitol, starch, pre-gelatinized starch, dextrose, delivery systems. Biodegradable, biocompatible polymers fructose, honey, lactose anhydrate, lactose monohydrate, lac can be used, such as ethylene vinyl acetate, polyanhydrides, tose and aspartame, lactose and cellulose, lactose and micro polyglycolic acid, collagen, polyorthoesters, and polylactic crystalline cellulose, maltodextrin, maltose, mannitol, micro acid. Methods for preparation of such formulations will be crystalline cellulose & guar gum, molasses, sucrose, or apparent to those skilled in the art. The materials can also be mixtures thereof, DISINTEGRANTS: agar-agar, alginic obtained commercially from Alza Corporation and Nova acid, calcium carbonate, microcrystalline cellulose, croscar Pharmaceuticals, Inc. Liposomal Suspensions (including mellose Sodium, crospovidone, polacrilin potassium, sodium liposomes targeted to infected cells with monoclonal antibod starch glycolate, potato or tapioca Starch, other starches, pre ies to viral antigens) can also be used as pharmaceutically gelatinized starch, clays, otheralgins, other celluloses, gums acceptable carriers. These can be prepared according to meth (like gellan), low-substituted hydroxypropyl cellulose, or mixtures thereof, LUBRICANTS: calcium stearate, magne ods known to those skilled in the art, for example, as sium Stearate, mineral oil, light mineral oil, glycerin, Sorbitol, described in U.S. Pat. No. 4,522,811, incorporated fully mannitol, polyethylene glycol, other glycols, Stearic acid, herein by reference. Sodium lauryl Sulfate, Sodium Stearyl fumarate, vegetable 0201 It is especially advantageous to formulate oral or based fatty acids lubricant, talc, hydrogenated vegetable oil parenteral compositions in dosage unit form for ease of (e.g., peanut oil, cottonseed oil, Sunflower oil, sesame oil, administration and uniformity of dosage. Dosage unit form as olive oil, corn oil and Soybean oil), Zinc Stearate, ethyl oleate, used herein refers to physically discrete units Suited as unitary ethyl laurate, agar, syloid silica gel (AEROSIL 200, W.R. dosages for the Subject to be treated; each unit containing a Grace Co., Baltimore, Md. USA), a coagulated aerosol of predetermined quantity of active compound calculated to pro synthetic silica (Deaussa Co., Piano, Tex. USA), a pyrogenic duce the desired therapeutic effect in association with the silicon dioxide (CAB-O-SIL, Cabot Co., Boston, Mass. required pharmaceutical carrier. The specification for the dos USA), or mixtures thereof, ANTI-CAKING AGENTS: cal age unit forms of the invention are dictated by and directly cium silicate, magnesium silicate, silicon dioxide, colloidal dependent on the unique characteristics of the active com silicon dioxide, talc, or mixtures thereof, ANTIMICROBIAL pound and the particular therapeutic effect to be achieved. AGENTS: benzalkonium chloride, benzethonium chloride, 0202 The pharmaceutical compositions can be included benzoic acid, benzyl alcohol, butyl paraben, cetylpyridinium in a container, pack, or dispenser together with instructions chloride, cresol, chlorobutanol, dehydroacetic acid, ethylpa for administration. raben, methylparaben, phenol, phenylethyl alcohol, phe US 2016/0235807 A1 Aug. 18, 2016 noxyethanol, phenylmercuric acetate, phenylmercuric for example Opadry(R) blue (OY-LS-20921), Opadry(R) white nitrate, potassium Sorbate, propylparaben, Sodium benzoate, (YS-2-7063), Opadry(R) white (YS-1-7040), and black ink Sodium dehydroacetate, Sodium propionate, Sorbic acid, (S-1-8 106). thimersol, thymo, or mixtures thereof, and COATING 0208. The agents either in their free form or as a salt can be AGENTS: sodium carboxymethylcellulose, cellulose acetate combined with a polymer Such as polylactic-glycoloic acid phthalate, ethylcellulose, gelatin, pharmaceutical glaze, (PLGA), poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) hydroxypropyl cellulose, hydroxypropyl methylcellulose (WO 01/12233), polyglycolic acid (U.S. Pat. No. 3,773.919), (hypromellose), hydroxypropyl methyl cellulose phthalate, polylactic acid (U.S. Pat. No. 4,767,628), poly(e-caprolac methylcellulose, polyethylene glycol, polyvinyl acetate tone) and poly(alkylene oxide) (U.S. 2003.0068384) to create phthalate, shellac, Sucrose, titanium dioxide, carnauba wax, a Sustained release formulation. Such formulations can be microcrystalline wax, gellan gum, maltodextrin, methacry used to implants that release a polypeptide or another agent lates, microcrystalline cellulose and carrageenan or mixtures over a period of a few days, a few weeks or several months thereof. depending on the polymer, the particle size of the polymer, and the size of the implant (See, e.g., U.S. Pat. No. 6,620, 0206. The formulation can also include other excipients 422). Other sustained release formulations and polymers for and categories thereof including but not limited to L-histi use in are described in EP 0467389 A2, WO 93/24150, U.S. dine, Pluronic R, Poloxamers (such as Lutrol(R) and Polox Pat. No. 5,612,052, WO 97/40085, WO 03/075887, WO amer 188), ascorbic acid, glutathione, permeability enhanc 01/01964A2, U.S. Pat. No. 5,922,356, WO 94/155587, WO ers (e.g. lipids, sodium cholate, acylcarnitine, Salicylates, 02/074247A2, WO 98/25642, U.S. Pat. No. 5,968,895, U.S. mixed bile salts, fatty acid micelles, chelators, fatty acid, Pat. No. 6,180,608, U.S. 2003.0171296, U.S. 20020176841, Surfactants, medium chain glycerides), protease inhibitors U.S. Pat. No. 5,672,659, U.S. Pat. No. 5,893,985, U.S. Pat. (e.g. soybean trypsin inhibitor, organic acids), pH lowering No. 5,134,122, U.S. Pat. No. 5,192,741, U.S. Pat. No. 5,192, agents and absorption enhancers effective to promote bio 741, U.S. Pat. No. 4,668,506, U.S. Pat. No. 4,713,244, U.S. availability (including but not limited to those described in Pat. No. 5,445,832 U.S. Pat. No. 4,931,279, U.S. Pat. No. 5,980,945, WO 02/058672, WO 97/26015, WO 97/04744, U.S. Pat. No. 6,086,918 and U.S. Pat. No. 5,912,014), creams and US20020019446. In such sustained release formulations and lotions (like maltodextrin and carrageenans); materials microparticles (Delie and Blanco-Prieto 2005 Molecule for chewable tablets (like dextrose, fructose, lactose mono 10:65-80) of polypeptide are combined with microparticles hydrate, lactose and aspartame, lactose and cellulose, malto of polymer. One or more sustained release implants can be dextrin, maltose, mannitol, microcrystalline cellulose and placed in the large intestine, the small intestine or both. U.S. guar gum, Sorbitol crystalline); parenterals (like mannitol and Pat. No. 6,011,011 and WO 94/06452 describe a sustained povidone); plasticizers (like dibutyl sebacate, plasticizers for release formulation providing either polyethylene glycols coatings, polyvinylacetate phthalate); powder lubricants (like (i.e. PEG 300 and PEG 400) or triacetin. WO 03/053401 glyceryl behenate); soft gelatin capsules (like Sorbitol special describes a formulation which may both enhance bioavail Solution); spheres for coating (like Sugar spheres); spheroni ability and provide controlled release of the agent within the Zation agents (like glyceryl behenate and microcrystalline GI tract. Additional controlled release formulations are cellulose); Suspending/gelling agents (like carrageenan, gel described in WO 02/38129, EP326151, U.S. Pat. No. 5,236, lan gum, mannitol, microcrystalline cellulose, povidone, 704, WO 02/30398, WO 98/13029; U.S. 20030064105, U.S. Sodium starch glycolate, Xanthan gum); Sweeteners (like 200301.38488A1, U.S. 20030216307A1, U.S. Pat. No. 6,667, aspartame, aspartame and lactose, dextrose, fructose, honey, 060, WO 01/49249, WO 01/49311, WO 01/49249, WO maltodextrin, maltose, mannitol, molasses, Sorbitol crystal 01/49311, and U.S. Pat. No. 5,877,224 materials which may line, Sorbitol special Solution, Sucrose); wet granulation include those described in WOO4041195 (including the seal agents (like calcium carbonate, lactose anhydrous, lactose and enteric coating described therein) and pH-sensitive coat monohydrate, maltodextrin, mannitol, microcrystalline cel ings that achieve delivery in the colon including those lulose, povidone, starch), caramel, carboxymethylcellulose described in U.S. Pat. No. 4,910,021 and WO9001329. U.S. Sodium, cherry cream flavor and cherry flavor, citric acid Pat. No. 4,910,021 describes using a pH-sensitive material to anhydrous, citric acid, confectioner's sugar, D&C Red No. coat a capsule. WO9001329 describes using pH-sensitive 33, D&C Yellow #10 Aluminum Lake, disodium edetate, coatings on beads containing acid, where the acid in the bead ethyl alcohol 15%, FD&C Yellow No. 6 aluminum lake, core prolongs dissolution of the pH-sensitive coating. U.S. FD&C Blue #1 Aluminum Lake, FD&C Blue No. 1, FD&C Pat. No. 5,175,003 discloses a dual mechanism polymer mix blue no. 2 aluminum lake, FD&C Green No. 3, FD&C Red ture composed of pH-sensitive enteric materials and film No. 40, FD&C Yellow No. 6 Aluminum Lake, FD&C Yellow forming plasticizers capable of conferring permeability to the No. 6, FD&C Yellow No. 10, glycerol palmitostearate, glyc enteric material, for use in drug-delivery systems; a matrix eryl monostearate, indigo carmine, lecithin, manitol, methyl pellet composed of a dual mechanism polymer mixture per and propyl parabens, mono ammonium glycyrrhizinate, natu meated with a drug and sometimes covering a pharmaceuti ral and artificial orange flavor, pharmaceutical glaze, poloX cally neutral nucleus; a membrane-coated pellet comprising a amer 188, Polydextrose, polysorbate 20, polysorbate 80, matrix pellet coated with a dual mechanism polymer mixture poly Vidone, pregelatinized corn starch, pregelatinized starch, envelope of the same or different composition; and a phar red iron oxide, Saccharin sodium, Sodium carboxymethyl maceutical dosage form containing matrix pellets. The matrix ether, Sodium chloride, sodium citrate, sodium phosphate, pellet releases acid-soluble drugs by diffusion in acid pH and strawberry flavor, synthetic black iron oxide, synthetic red by disintegration at pH levels of nominally about 5.0 or iron oxide, titanium dioxide, and white wax. higher. 0207 Solid oral dosage forms may optionally be treated (0209. The GCRA peptides described herein may be for with coating systems (e.g. Opadry(R) fix film coating system, mulated in the pH triggered targeted control release systems US 2016/0235807 A1 Aug. 18, 2016 32 described in WOO4052339. The agents described herein may the eventual, but sudden, delivery of the beneficial agent. beformulated according to the methodology described in any These devices enable the pH-triggered release of the benefi of WO03105812 (extruded hydratable polymers): cial agent core as a bolus by osmotic bursting. WO0243767 (enzyme cleavable membrane translocators); WO03007913 and WO03086297 (mucoadhesive systems); Exemplary Additional Agents for Combination WO02072075 (bilayer laminated formulation comprising pH Therapy lowering agent and absorption enhancer); WOO4064769 0212 Analgesic Agents (amidated polypeptides); WO05063156 (solid lipid suspen 0213. The GCRA peptides described herein can be used in sion with pseudotropic and/or thixotropic properties upon combination therapy with an analgesic agent, e.g., an analge melting); WO03035029 and WO03035041 (erodible, gastric sic compound or an analgesic polypeptide. These polypep retentive dosage forms); U.S. Pat. No. 5,007,790 and U.S. tides and compounds can be administered with the GCRA Pat. No. 5,972,389 (sustained release dosage forms); WOO41 peptides described herein (simultaneously or sequentially). 12711 (oral extended release compositions); WO05027878, They can also be optionally covalently linked or attached to WO02072033, and WO02072034 (delayed release composi an agent described herein to create therapeutic conjugates. tions with natural or synthetic gum); WO05030182 (con Among the useful analgesic agents are: Calcium channel trolled release formulations with an ascending rate of blockers, 5HT receptor antagonists (for example 5HT3, release); WO05048998 (microencapsulation system); U.S. 5HT4 and 5HT1 receptor antagonists), opioid receptor ago Pat. No. 5,952,314 (biopolymer); U.S. Pat. No. 5,108,758 nists (loperamide, fedotozine, and fentanyl), NK1 receptor (glassy amylose matrix delivery); U.S. Pat. No. 5,840.860 antagonists, CCK receptor agonists (e.g., loxiglumide), NK1 (modified starch based delivery). JP10324642 (delivery sys receptor antagonists, NK3 receptor antagonists, norepineph tem comprising chitosanand gastric resistant material Such as wheat gliadin or zein): U.S. Pat. No. 5,866.619 and U.S. Pat. rine-serotonin reuptake inhibitors (NSRI), Vanilloid and can No. 6,368,629 (saccharide containing polymer); U.S. Pat. No. nabanoid receptoragonists, and sialorphin. Analgesics agents 6.531,152 (describes a drug delivery system containing a in the various classes are described in the literature. water soluble core (Ca pectinate or other water-insoluble 0214. Among the useful analgesic polypeptides are sialor polymers) and outer coat which bursts (e.g. hydrophobic phin-related polypeptides, including those comprising the polymer-Eudragrit)); U.S. Pat. No. 6,234,464; U.S. Pat. No. amino acid sequence QHNPR (SEQID NO: 250), including: 6,403,130 (coating with polymer containing casein and high VQHNPR (SEQ ID NO: 251); VRQHNPR (SEQ ID NO: methoxy pectin; WOO174 175 (Maillard reaction product): 252): VRGQHNPR (SEQIDNO: 253); VRGPQHNPR (SEQ WO05063206 (solubility increasing formulation); WO040 ID NO: 254); VRGPRQHNPR (SEQ ID NO: 255): VRG PRRQHNPR (SEQ ID NO: 256); and RQHNPR (SEQ ID 19872 (transferring fusion proteins). NO: 257). Sialorphin-related polypeptides bind to neprilysin 0210. The GCRA peptides described herein may be for and inhibit neprilysin-mediated breakdown of substance P mulated using gastrointestinal retention system technology and Met-enkephalin. Thus, compounds or polypeptides that (GIRES: Merrion Pharmaceuticals). GIRES comprises a con are inhibitors of neprilysin are useful analgesic agents which trolled-release dosage form inside an inflatable pouch, which can be administered with the polypeptides described herein in is placed in a drug capsule for oral administration. Upon a co-therapy or linked to the polypeptides described herein, dissolution of the capsule, a gas-generating system inflates e.g., by a covalent bond. Sialophin and related polypeptides the pouch in the stomach where it is retained for 16-24 hours, are described in U.S. Pat. No. 6,589,750; U.S. 20030078200 all the time releasing agents described herein. A1; and WO 02/051435 A2. 0211. The GCRA peptides described herein can beformu 0215 Opioid receptor antagonists and agonists can be lated in an osmotic device including the ones disclosed in administered with the GCRA peptides described herein in U.S. Pat. No. 4,503,030, U.S. Pat. No. 5,609,590 and U.S. co-therapy or linked to the agent described herein, e.g., by a Pat. No. 5,358,502. U.S. Pat. No. 4,503,030 discloses an covalent bond. For example, opioid receptor antagonists Such osmotic device for dispensing a drug to certain pH regions of as naloxone, naltrexone, methyl naloZone, nalmefene, cypri the gastrointestinal tract. More particularly, the invention dime, beta funaltrexamine, naloxonazine, naltrindole, and relates to an osmotic device comprising a wall formed of a nor-binaltorphimine are thought to be useful in the treatment semi-permeable pH sensitive composition that Surrounds a of IBS. It can be useful to formulate opioidantagonists of this compartment containing a drug, with a passageway through type is a delayed and Sustained release formulation Such that the wall connecting the exterior of the device with the com initial release of the antagonist is in the mid to distal Small partment. The device delivers the drug at a controlled rate in intestine and/or ascending colon. Such antagonists are the region of the gastrointestinal tract having a pH of less than described in WO 01/32180 A2. Enkephalin pentapeptide 3.5, and the device self-destructs and releases all its drug in (HOE825: Tyr-D-Lys-Gly-Phe-L-homoserine) is an agonist the region of the gastrointestinal tract having a pH greater of the mu and delta opioid receptors and is thought to be than 3.5, thereby providing total availability for drug absorp useful for increasing intestinal motility Eur. J. Pharm. 219. tion. U.S. Pat. Nos. 5,609,590 and 5,358,502 disclose an 445, 1992), and this polypeptide can be used in conjunction osmotic bursting device for dispensing a beneficial agent to with the polypeptides described herein. Also useful is trime an aqueous environment. The device comprises a beneficial butine which is thought to bind to mu/delta/kappa opioid agent and osmagent Surrounded at least in part by a semi receptors and activate release of motilin and modulate the permeable membrane. The beneficial agent may also function release of gastrin, vasoactive intestinal polypeptide, gastrin as the osmagent. The semi-permeable membrane is perme and glucagons. Kappa opioid receptoragonists such as fedot able to water and substantially impermeable to the beneficial ozine, asimadoline, and ketocyclazocine, and compounds agent and osmagent. A trigger means is attached to the semi described in WOO3/097051 and WOO5/007626 can be used permeable membrane (e.g., joins two capsule halves). The with or linked to the polypeptides described herein. In addi trigger means is activated by a pH of from 3 to 9 and triggers tion, mu opioid receptoragonists Such as morphine, dipheny US 2016/0235807 A1 Aug. 18, 2016 loxylate, frakefamide (H-Tyr-D-Ala-Phe(F)-Phe-NH2: WO 0227 NK3 receptor antagonists such as osanetant (SR 01/019849 A1) and loperamide can be used. 142801; Sanofi-Synthelabo), SSR-241586, talnetant and 0216 Tyr-Arg (kyotorphin) is a dipeptide that acts by related compounds described in, for example, WO 02/094187 stimulating the release of met-enkephalins to elicit an anal A2, EP 876347 A1, WO 97/21680 A1, U.S. Pat. No. 6,277, gesic effect (J. Biol. Chem 262:8165, 1987). Kyotorphin can 862, WO 98/1 1090, WO 95/28418, WO 97/19927, and be used with or linked to the GCRA peptides described Boden et al. (J Med Chem. 39:1664-75, 1996) can be used herein. with or linked to the polypeptides described herein. 0217 Chromogranin-derived polypeptide (CgA 47-66: 0228 Norepinephrine-serotonin reuptake inhibitors See, e.g., Ghia et al. 2004 Regulatory polypeptides 119:199) (NSRI) such as milnacipran and related compounds can be used with or linked to the GCRA peptides described described in WO 03/077897 A1 can be used with or linked to herein. the polypeptides described herein. 0218. CCK receptor agonists such as caerulein from 0229 Vanilloid receptor antagonists such as arvanil and amphibians and other species are useful analgesic agents that related compounds described in WO 01/64212 A1 can be can be used with or linked to the GCRA peptides described used with or linked to the polypeptides described herein. herein. 0230. The analgesic polypeptides and compounds can be administered with the polypeptides and agonists described 0219 Conotoxin polypeptides represent a large class of herein (simultaneously or sequentially). The analgesic agents analgesic polypeptides that act at Voltage gated calcium chan can also be covalently linked to the polypeptides and agonists nels, NMDA receptors or nicotinic receptors. These polypep described herein to create therapeutic conjugates. Where the tides can be used with or linked to the polypeptides described analgesic is a polypeptide and is covalently linked to an agent herein. described herein the resulting polypeptide may also include at 0220 Peptide analogs of thymulin (FR Application least one trypsin cleavage site. When present within the 2830451) can have analgesic activity and can be used with or polypeptide, the analgesic polypeptide may be preceded by linked to the polypeptides described herein. (if it is at the carboxy terminus) or followed by (if it is at the 0221 CCK (CCKa or CCKb) receptor antagonists, amino terminus) a trypsin cleavage site that allows release of including loxiglumide and dexloxiglumide (the R-isomer of the analgesic polypeptide. loxiglumide) (WO 88/05774) can have analgesic activity and 0231. In addition to sialorphin-related polypeptides, anal can be used with or linked to the polypeptides described gesic polypeptides include: AspPhe, endomorphin-1, endo herein. morphin-2, nocistatin, dalargin, lupron, Ziconotide, and Sub 0222. Other useful analgesic agents include 5-HT4 ago stance P. nists such as tegaserod (Zelnorm(R), mosapride, metoclopra mide, Zacopride, cisapride, renZapride, benzimidazolone Agents to Treat Gastrointestinal Disorders derivatives such as BIMU 1 and BIMU 8, and lirexapride. 0232 Examples of additional therapeutic agents to treat Such agonists are described in: EP1321 142 A1, WO gastrointestinal and other disorders include agents to treat 03/053432A1, EP505322 A1, EP 505322 B1, U.S. Pat. No. constipation (e.g., a chloride channel activator Such as the 5,510,353, EP 507672 A1, EP 507672 B1, and U.S. Pat. No. bicylic fatty acid, Lubiprostone (formerly known as SPI 5,273,983. 0211; Sucampo Pharmaceuticals, Inc.; Bethesda, Md.), a 0223 Calcium channel blockers such as Ziconotide and laxative (e.g. a bulk-forming laxative (e.g. nonstarch polysac related compounds described in, for example, EP625162B1, charides, Colonel Tablet (polycarbophil calcium), Plantago U.S. Pat. No. 5,364,842, U.S. Pat. No. 5,587,454, U.S. Pat. OvataR), EqualactinR (Calcium Polycarbophil)), fiber (e.g. No. 5,824,645, U.S. Pat. No. 5,859,186, U.S. Pat. No. 5,994, FIBERCONR) (Calcium Polycarbophil), an osmotic laxative, 305, U.S. Pat. No. 6,087,091, U.S. Pat. No. 6,136,786, WO a stimulant laxative (such as diphenylmethanes (e.g. bisa 93/13128A1 EP 1336409 A1 EP 835126A1, EP 835126B1, codyl), anthraquinones (e.g. cascara, Senna), and Surfactant U.S. Pat. No. 5,795,864, U.S. Pat. No. 5,891,849, U.S. Pat. laxatives (e.g. castor oil, docusates), an emollient/lubricating No. 6,054,429, WO 97/01351 A1, can be used with or linked agent (such as mineral oil, glycerine, and docusates), to the polypeptides described herein. Mirallax (Braintree Laboratories, Braintree Mass.), dexloxi 0224 Various antagonists of the NK-I, NK-2, and NK-3 glumide (Forest Laboratories, also known as CR 2017 Rot receptors (for a review see Giardina et al. 2003. Drugs 6:758) tapharm (Rotta Research Laboratorium SpA)), Saline laxa can be can be used with or linked to the polypeptides tives, enemas, suppositories, and CR 3700 (Rottapharm described herein. (Rotta Research Laboratorium SpA); acid reducing agents 0225 NK1 receptor antagonists such as: aprepitant Such as proton pump inhibitors (e.g., omeprazole (Merck & Co Inc), Vofopiitant, ezlopitant (Pfizer, Inc.), R-673 (PrilosecR), esomeprazole (Nexium(R), lansoprazole (Pre (Hoffmann-La Roche Ltd), SR-48968 (Sanofi Synthelabo), vacidR), pantoprazole (Protonix(R) and rabeprazole (Aci CP-122,721 (Pfizer, Inc.), GW679769 (GlaxoSmith Kline), pheX(R)) and Histamine H2-receptor antagonist (also known TAK-637 (Takeda/Abbot), SR-14033, and related com as H2 receptor blockers including cimetidine, ranitidine, pounds described in, for example, EP 873753 A1, US famotidine and nizatidine); prokinetic agents including ito 20010006972 A1, US 2003.0109417 A1, WO 01/52844 A1, pride, octreotide, bethanechol, metoclopramide (ReglanR), can be used with or linked to the polypeptides described domperidone (Motilium(R), erythromycin (and derivatives herein. thereof) or cisapride (Propulsider); Prokineticin polypeptides 0226 NK-2 receptor antagonists such as nepadutant (Me homologs, variants and chimeras thereof including those narini Ricerche SpA), saredutant (Sanofi-Synthelabo), described in U.S. Pat. No. 7,052,674 which can be used with GW5975.99 (Glaxo Smith Kline), SR-144190 (Sanofi-Syn or linked to the polypeptides described herein; pro-motility thelabo) and UK-290795 (Pfizer Inc) can be used with or agents such as the vasostatin-derived polypeptide, chromog linked to the polypeptides described herein. ranin A (4-16) (See, e.g., Ghia et al. 2004 Regulatory US 2016/0235807 A1 Aug. 18, 2016 34 polypeptides 121:31) or motilin agonists (e.g., GM-611 or polypeptide RDP58 (Genzyme, Inc.; Cambridge, Mass.), and mitemcinal fumarate) or nociceptin/Orphanin FQ receptor TRAFICET-ENTM (ChemoCentryx, Inc.; San Carlos, Calif); modulators (US20050169917); other peptides which can agents that treat gastrointestinal or visceral pain, agents that bind to and/or activate GC-C including those described in increase coMP levels (as described in US20040121994) like US20050287067; completeorpartial 5HT (e.g. 5HT1,5HT2, adrenergic receptor antagonists, dopamine receptor agonists 5HT3, 5HT4) receptor agonists or antagonists (including and PDE (phosphodiesterase) inhibitors including but not 5HT1A antagonists (e.g. AGI-OOI (AGI therapeutics), limited to those disclosed herein; purgatives that draw fluids 5HT2B antagonists (e.g. PGN 1091 and PGN1 164 (Pharma to the intestine (e.g., VISICOLR, a combination of sodium gene Laboratories Limited), and 5HT4 receptor agonists phosphate monobasic monohydrate and Sodium phosphate (such as tegaserod (ZELNORMR), prucalopride, mosapride, dibasic anhydrate); Corticotropin Releasing Factor (CRF) metoclopramide, Zacopride, cisapride, renZapride, benzimi receptor antagonists (including NBI-34041 (Neurocrine Bio dazolone derivatives such as BIMU 1 and BIMU 8, and lir sciences, San Diego, Calif.), CRH9-41, astressin, R121919 exapride). Such agonists/modulators are described in: (Janssen Pharmaceutica), CP154,526, NBI-27914, Anta EP1321142 A1, WO 03/053432A1 EP 505322 A1, EP larmin, DMP696 (Bristol-Myers Squibb) CP-316,311 505322 B1, U.S. Pat. No. 5,510,353, EP 507672 A1, EP (Pfizer, Inc.), SB723620 (GSK), GW876008 (Neurocrine/ 507672B1, U.S. Pat. No. 5,273,983, and U.S. Pat. No. 6,951, Glaxo Smith Kline), ONO-2333Ms (Ono Pharmaceuticals), 867): 5HT3 receptor agonists such as MKC-733; and 5HT3 TS-041 (Janssen), AAG561 (Novartis) and those disclosed in receptor antagonists such as DDP-225 (MCI-225; Dynogen U.S. Pat. No. 5,063,245, U.S. Pat. No. 5,861,398, Pharmaceuticals, Inc.), cilansetron (CalmactinR), alosetron US20040224964, US20040198726, US20040176400, (Lotronex(R), Ondansetron HCl (ZofranR), Dolasetron (AN US2004.0171607, US20040110815, US20040006066, and ZEMETR), palonosetron (Aloxi(R), Granisetron (KytrilR), US20050209253); glucagon-like polypeptides (glp-1) and YMO60 (ramosetron; Astellas Pharma Inc.; ramosetron may be given as a daily dose of 0.002 to 0.02 mg as described in analogues thereof (including exendin-4 and GTP-010 (Gas EP01588707) and ATI-7000 (Aryx Therapeutics, Santa Clara trotech Pharma A)) and inhibitors of DPP-IV (DPP-IV medi Calif.); muscarinic receptor agonists; anti-inflammatory ates the inactivation of glp-1); tofisopam, enantiomerically agents; antispasmodics including but not limited to anticho pure R-tofisopam, and pharmaceutically-acceptable salts linergic drugs (like dicyclomine (e.g. ColimeX(R), FormuleX(R), thereof (US 20040229867); tricyclic anti-depressants of the Lomine(R), Protylol R, Visceral(R), Spasmoban R., Bentyl(R), dibenzothiazepine type including but not limited to BentylolR), hyoscyamine (e.g. IB-StatR, NulevR, Levsin(R), Dextofisopam R. (Vela Pharmaceuticals), tianeptine LevbidR), Levsinex Timecaps.(R), Levsin/SL(R), Anaspaz(R), (Stablon R) and other agents described in U.S. Pat. No. 6,683, A-Spas S/L(R), CystospaZR), Cystospaz-M(R), Donnamar R, 072; (E)-4 (1.3bis(cyclohexylmethyl)-1,2,34-tetrahydro-2, Colidrops Liquid Pediatric R, Gastrosed R, Hyco Elixir R, 6-diono-9H-purin-8-yl)cinnamic acid nonaethylene glycol Hyosol R, Hyospaz(R), Hyosyne(R), Losamine(R), MedispaZR, methyl ether ester and related compounds described in WO Neosol(R), Spacol R, SpasdelR), Symax(R), Symax SL(R), Don 02/067942; the probiotic PROBACTRIXR (The BioBalance natal (e.g. Donnatal Extentabs(R), clidinium (e.g. Quarzan, in Corporation; New York, N.Y.) which contains microorgan combination with Librium=Librax), methantheline (e.g. Ban isms useful in the treatment of gastrointestinal disorders; thine), MepenZolate (e.g. Cantil), homatropine (e.g. hycodan, antidiarrheal drugs including but not limited to loperamide Homapin), Propantheline bromide (e.g. Pro-Banthine), Gly (Imodium, Pepto Diarrhea), diphenoxylate with atropine (Lo copyrrolate (e.g. Robinul R. Robinul ForteR), scopolamine motil, Lomocot), cholestyramine (Questran, Cholybar), atro (e.g. Transderm-ScopR, Transderm-V(R), hyosine-N-butyl pine (Co-Phenotrope, Diarsed, Diphenoxylate, Lofene, bromide (e.g. Buscopan R), Pirenzepine (e.g. Gastrozepin(R) Propantheline Bromide (e.g. Propanthel(R), dicycloverine Logen, Lonox, Vi-Atro, atropine Sulfate injection) and (e.g. Merbenty1R), glycopyrronium bromide (e.g. Glycopy Xifaxan R (rifaximin; Salix Pharmaceuticals Ltd), TZP-201 rrolate(R), hyoscine hydrobromide, hyoscine methobromide, (Tranzyme Pharma Inc.), the neuronal acetylcholine receptor methanthelinium, and octatropine); peppermint oil; and (nAChR) blocker AGI-004 (AGI therapeutics), and bismuth direct Smooth muscle relaxants like cimetropium bromide, Subsalicylate (Pepto-bismol); anxiolytic drugs including but mebeverine (DUSPATAL(R), DUSPATALINR, COLOFAC not limited to Ativan (lorazepam), alprazolam (Xanax(R), MRR), COLOTAL(R), otilonium bromide (octilonium), chlordiazepoxide/clidinium (Librium(R), Librax(R), clon pinaverium (e.g. Dicetel(R) (pinaverium bromide: Solvay S. azepam (Klonopin(R), cloraZepate (Tranxene.R.), diazepam A.)), SpasfonR (hydrated phloroglucinol and trimethylphlo (Valium(R), estazolam (ProSomr), flurazepam (Dalmane(R). roglucinol) and trimebutine (including trimebutine maleate oxazepam (SeraX(R), prazepam (CentraX(R), temazepam (Re (Modulon(R); antidepressants, including but not limited to storil(R), triazolam (Halcion(R); Bedelix(R) (Montmorillonite those listed herein, as well as tricyclic antidepressants like beidellitic; Ipsen Ltd), Solvay SLV332 (ArCule Inc), YKP amitriptyline (Elavil(R), desipramine (Norpramin R), imi (SKPharma), Asimadoline (Tioga Pharmaceuticals/Merck), pramine (TofranilR), amoxapine (AsendinR), nortriptyline; AGI-003 (AGI Therapeutics); neurokinin antagonists includ the selective serotonin reuptake inhibitors (SSRTs) like par ing those described in US20060040950; potassium channel oxetine (Paxil(R), fluoxetine (ProzacR), sertraline (Zoloft(R), modulators including those described in U.S. Pat. No. 7.002, and citralopram (CelexaR); and others like doxepin (Sine 015; the serotonin modulator AZD7371 (AstraZeneca PIc); quan R) and traZodone (DesyrelR); centrally-acting analgesic M3 muscarinic receptor antagonists such as darifenacin (En agents such as opioid receptor agonists, opioid receptor ablex; Novartis AG and Zamifenacin (Pfizer); herbaland natu antagonists (e.g., naltrexone); agents for the treatment of ral therapies including but not limited to acidophilus, chamo Inflammatory bowel disease; agents for the treatment of mile tea, evening primrose oil, fennel seeds, wormwood, Crohn's disease and/or ulcerative colitis (e.g., alequel (Enzo comfrey, and compounds of Bao-Ji-Wan (magnolol. Biochem, Inc., Farmingsale, N.Y.), the anti-inflammatory honokiol, imperatorin, and isoimperatorin) as in U.S. Pat. No. US 2016/0235807 A1 Aug. 18, 2016

6,923,992; and compositions comprising lysine and an anti tyrphostin AG 34, tyrphostin AG 879, urinary trypsin inhibi stress agent for the treatment of irritable bowel syndrome as tor fragment, valproic acid (2-propylpentanoic acid), and described in EPO 1550443. XK469. 0240 Representative gene regulators include 5-aza-2'- Agents to Treat Gastrointestinal Cancers deoxycytidine, 5-azacytidine, cholecalciferol (Vitamin D3), 0233. The GCRA peptides described herein can be used in ciglitiZone, cyproterone acetate, 15-deoxy-D. Sup.12, 14 combination with one or more antitumor agents including but prostaglandin J.Sub.2, epitestosterone, flutamide, glycyr not limited to alkylating agents, epipodophyllotoxins, rhizic acid ammonium salt (glycyrrhizin), 4-hydroxytamox nitrosoureas, anti-metabolites, Vinca alkaloids, anthracycline ifen, mifepristone, procainamide hydrochloride, raloxifene antibiotics, nitrogen mustard agents, and the like. Particular hydrochloride, all trans-retinal (vitamin Aaldehyde), retinoic antitumor agents include tamoxifen, taxol, etoposide, and acid (vitamin A acid), 9-cis-retinoic acid, 13-cis-retinoic 5-fluorouracil. In one embodiment, the GCRA peptides are acid, retinoic acid p-hydroxyanilide, retinol (Vitamin A), used in combination with an antiviral agent or a monoclonal tamoxifen, tamoxifen citrate salt, tetradecylthioacetic acid, antibody. and troglitaZone. 0234 Non-limiting examples of antitumor agents that can 0241 Representative HSP-90 inhibitors include 17-(ally be used in combination with the GCRA peptides of the inven lamino)-17-demethoxygeldanamycin and geldanamycin. tion for the treatment of colon cancer include anti-prolifera 0242 Representative microtubule inhibitors include tive agents, agents for DNA modification or repair, DNA colchicines, dolastatin 15, nocodazole, taxanes and in par synthesis inhibitors, DNA/RNA transcription regulators, ticular paclitaxel, podophyllotoxin, rhizoxin, vinblastine Sul RNA processing inhibitors, agents that affect protein expres fate salt, Vincristine Sulfate salt, and vindesine Sulfate salt and Sion, synthesis and Stability, agents that affect protein local vinorelbine (Navelbine) ditartrate salt. ization or their ability to exert their physiological action, 0243 Representative agents for performing phototherapy agents that interfere with protein-protein or protein-nucleic include photoactive porphyrin rings, hypericin, 5-methoxyp acid interactions, agents that act by RNA interference, recep Soralen, 8-methoxypsoralen, psoralen and urSodeoxycholic tor binding molecules of any chemical nature (including acid. Small molecules and antibodies), targeted toxins, enzyme 0244 Representative agents used as therapy adjuncts activators, enzyme inhibitors, gene regulators, HSP-90 include amifostine, 4-amino-1.8-naphthalimide, brefeldin A, inhibitors, molecules interfering with microtubules or other cimetidine, phosphomycin disodium salt, leuprolide (leupro cytoskeletal components or cell adhesion and motility, agents relin) acetate salt, luteinizing hormone-releasing hormone for phototherapy, and therapy adjuncts. (LH-RH) acetate salt, lectin, papaverine hydrochloride, 0235 Representative anti-proliferative agents include pifithrin-a, (-)-scopolamine hydrobromide, and thapsigargin. N-acetyl-D-sphingosine (C. Sub.2 ceramide), apigenin, ber 0245. The agents can also be anti-VEGF (vascular endot berine chloride, dichloromethylenediphosphonic acid diso helial growth factor) agents, as such are known in the art. dium salt, loe-emodine, emodin, HA 14-1, N-hexanoyl-D- Several antibodies and small molecules are currently in clini sphingosine (C. sub.6 ceramide), 7b-hydroxycholesterol, cal trials or have been approved that function by inhibiting 25-hydroxycholesterol, hyperforin, parthenolide, and rapa VEGF, such as Avastin (Bevacizumab), SU5416, SU11248 mycin. and BAY 43-9006. The agents can also be directed against 0236 Representative agents for DNA modification and growth factor receptors such as those of the EGF/Erb-B fam repair include aphidicolin, bleomycin Sulfate, carboplatin, ily such as EGF Receptor (Iressa or Gefitinib, and Tarceva or carmustine, chlorambucil, cyclophosphamide monohydrate, Erlotinib), Erb-B2, receptor (Herceptin or Trastuzumab), cyclophosphamide monohydrate ISOPAC(R), cis-diammine other receptors (such as Rituximab or Rituxan/MabThera), platinum(II) dichloride (Cisplatin), esculetin, melphalan, tyrosine kinases, non-receptor tyrosine kinases, cellular methoxyamine hydrochloride, mitomycin C, mitoxantrone serine/threonine kinases (including MAP kinases), and vari dihydrochloride, Oxaliplatin, and streptozocin. ous other proteins whose deregulation contribute to oncogen 0237 Representative DNA synthesis inhibitors include esis (such as Small/Ras family and large/heterotrimeric G (..+-.)amethopterin (methotrexate), 3-amino-1,2,4-benzotri proteins). Several antibodies and Small molecules targeting azine 1,4-dioxide, aminopterin, cytosine b-D-arabinofurdno those molecules are currently at various stages of develop side (Ara-C), cytosine b-D-arabinofuranoside (Ara-C) ment (including approved for treatment or in clinical trials). hydrochloride, 2-fluoroadenine-9-b-D-arabinofuranoside 0246. In a preferred embodiment, the invention provides a (Fludarabine des-phosphate; F-ara-A), 5-fluoro-5'-deox method for treating colon cancer in a Subject in need thereof yuridinc, 5-fluorouracil, ganciclovir, hydroxyurea, 6-mer by administering to the Subject a GCRA peptide or a compo captopurine, and 6-thioguanine. sition described herein in combination with one or more 0238 Representative DNA/RNA transcription regulators antitumor agent selected from the group consisting of pacli include actinomycin D, daunorubicin hydrochloride, 5,6- taxel, docetaxel, tamoxifen, Vinorelbine, gemcitabine, cispl dichlorobenzimidazole 1-b-D-ribofuranoside, doxorubicin atin, etoposide, topotecan, irinotecan, anastrozole, rituximab, hydrochloride, homoharringtonine, and idarubicin hydro trastuzumab, fludarabine, cyclophosphamide, gentuZumab, chloride. carboplatin, interferons, and doxorubicin. In a particular 0239 Representative enzyme activators and inhibitors embodiment the antitumor agent is paclitaxel. In a further include forskolin, DL-aminoglutethimide, apicidin, Bow embodiment, the method further comprises an antitumor man-Birk Inhibitor, butein, (S)-(+)-camptothecin, curcumin, agent selected from the group consisting of 5-FU, doxorubi (-)-deguelin, (-)-depudecin, doxycycline hyclate, etoposide, cin, Vinorelbine, Cytoxan, and cisplatin. formestane, fostriecin Sodium salt, hispidin, 2-limino-1-imi Agents that Treat Crohn's Disease dazolidineacetic acid (Cyclocreatine), oxamflatin, 4-phenyl 0247. In one embodiment, a GCRA peptide of the inven butyric acid, roscovitine, sodium valproate, trichostatin A, tion is administered as part of a combination therapy with one US 2016/0235807 A1 Aug. 18, 2016 36 or more additional therapeutic agents for the treatment of Agents for the Treatment of Postoperative Ileus Crohn's disease. Non-limiting examples of the one or more additional therapeutic agents include SulfaSalazine and other 0252. The GCRA peptides described herein can also be mesalamine-containing drugs, generally known as 5-ASA used in combination therapy with agents (e.g., Entereg M agents, such as Asacol, Dipentum, or Pentasa, Salofalk.R., (alvimopan; formerly called adolor/ADL 8-2698), conivap sulfasalazine, Salazopyrin R, Salazopyrin En-tabs(R), or tan and related agents describe in U.S. Pat. No. 6,645,959) generics thereof or infliximab (REMICADE). In certain used for the treatment of postoperative ileus and other disor embodiments, the one or more additional agents is a corticos ders. teroid oran immunosuppressive agent such as 6-mercaptopu rine or azathioprine. In another embodiment, the one or more Anti-Hypertensive Agents additional agents are antidiarrheal agents such as diphenoxy late, loperamide, or codeine. 0253) The GCRA peptides described herein can be used in Agents that Treat Ulcerative Colitis combination therapy with an anti-hypertensive agent includ 0248. In one embodiment, a GCRA peptide of the inven ing but not limited to: (1) diuretics, such as thiazides, includ tion is administered as part of a combination therapy with one ing chlorthalidone, chlorthiazide, dichlorophenamide, or more additional therapeutic agents for the treatment of hydroflumethiazide, indapamide, polythiazide, and hydro ulcerative colitis. The agents that are used to treat ulcerative chlorothiazide; loop diuretics, such as bumetanide, colitis overlap with those used to treat Crohn's Disease. Non ethacrynic acid, furosemide, and torsemide; potassium spar limiting examples of the one or more additional therapeutic ing agents, such as amiloride, and triamterene; carbonic agents that can be used in combination with a GCRA peptide anhydrase inhibitors, osmotics (such as glycerin) and aldos of the invention include aminosalicylates (drugs that contain terone antagonists, such as spironolactone, epirenone, and the 5-aminosalicyclic acid (5-ASA)) such as Sulfasalazine, like; (2) beta-adrenergic blockers such as acebutolol. olsalazine, mesalamine, and balsalazide. Other therapeutic atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, car agents that can be used include corticosteroids, Such as pred teolol, carvedilol, celiprolol, esmolol, indenolol, metaprolol. nisone and hydrocortisone, immunomodulators, such as aza nadolol, nebivolol, penbutolol, pindolol, propanolol, Sotalol, thioprine, 6-mercapto-purine (6-MP), cytokines, interleu tertatolol, tilisolol, and timolol, and the like; (3) calcium channel blockers such as amlodipine, aranidipine, azelnid kins, and lymphokines, and anti-TNF-alpha agents, including ipine, barnidipine, benidipine, bepridil, cinaldipine, clevid the thiazolidinediones or glitaZones Such as rosiglitaZone and ipine, diltiazem, efonidipine, felodipine, gallopamil, israd pioglitaZone. In one embodiment, the one or more additional ipine, lacidipine, lemildipine, lercanidipine, nicardipine, therapeutic agents include both cyclosporine A and 6-MP or nifedipine, nilvadipine, nimodepine, nisoldipine, nitren azathioprine for the treatment of active, severe ulcerative dipine, manidipine, pranidipine, and Verapamil, and the like; colitis. (4) angiotensin converting enzyme (ACE) inhibitors such as Agents that Treat Constipation/Irritable Bowel Syndrome benazepril; captopril; ceranapril; cilaZapril; delapril; enala 0249. In one embodiment, a GCRA peptide of the inven pril; enalopril; fosinopril; imidapril; lisinopril; losinopril; tion is administered as part of a combination therapy with one moexipril; quinapril; quinaprilat, ramipril; perindopril; per or more additional therapeutic agents for the treatment of indropril; quanipril; spirapril; tenocapril; trandolapril, and constipation, such as that associated with irritable bowel syn Zofenopril, and the like; (5) neutral endopeptidase inhibitors drome. Non-limiting examples of the one or more additional Such as omapatrilat, cadoxatril and ecadotril, fosidotril, Sam therapeutic agents include laxatives such as SENNA, patrilat, AVE7688, ER4030, and the like: (6) endothelin MIRALAX, LACTULOSE, PEG, or calcium polycarbophil), antagonists such as tezosentan, A3081.65, andYM62899, and stool softeners (such as mineral oil or COLACE), bulking the like; (7) vasodilators such as hydralazine, clonidine, agents (such as METAMUCIL or bran), agents such as ZEL minoxidil, and nicotinyl alcohol, and the like; (8) angiotensin NORM (also called tegaserod), and anticholinergic medica II receptor antagonists such as aprosartan, candesartan, epro tions such as BENTYL and LEVSIN. Sartan, irbesartan, losartan, olmesartan, pratosartan, tasosar Insulin and Insulin Modulating Agents tan, telmisartan, Valsartan, and EXP-3137, FI6828K, and RNH6270, and the like; (9) C/B adrenergic blockers such as 0250. The GCRA peptides described herein can be used in nipradillol, arotinolol and amosulalol, and the like: (10) alpha combination therapy with insulin and related compounds 1 blockers, such as teraZosin, urapidil, prazosin, tamsulosin, including primate, rodent, or rabbit insulin including biologi bunaZosin, trimaZosin, doxazosin, naftopidil, indoramin, cally active variants thereof including allelic variants, more WHP 164, and XENO1O, and the like: (11) alpha 2 agonists preferably human insulin available in recombinant form. Such as lofexidine, tiamenidine, moxonidine, rillmenidine and Sources of human insulin include pharmaceutically accept guanobenz, and the like; (12) aldosterone inhibitors, and the able and sterile formulations such as those available from Eli like; and (13) angiopoietin-2-binding agents such as those Lilly (Indianapolis, Ind. 46285) as Humulin TM (human insu disclosed in WO03/030833. Specific anti-hypertensive lin rDNA origin). See, the THE PHYSICIAN'S DESK REF agents that can be used in combination with polypeptides and ERENCE, 55.sup.th Ed. (2001) Medical Economics, Thom agonists described herein include, but are not limited to: son Healthcare (disclosing other Suitable human insulins). diuretics, such as thiazides (e.g., chlorthalidone, cyclothiaz 0251. The GCRA peptides described herein can also be ide (CAS RN 2259-96-3), chlorothiazide (CAS RN 72956 used in combination therapy with agents that can boost insu 09-3, which may be prepared as disclosed in U.S. Pat. No. lin effects or levels of a Subject upon administration, e.g. 2.809,194), dichlorophenamide, hydroflumethiazide, inda glipizide and/or rosiglitaZone. The polypeptides and agonists pamide, polythiazide, bendroflumethazide, methyclothazide, described herein can be used in combitherapy with SYM polythiazide, trichlormethazide, chlorthalidone, indapamide, LINR (pramlintide acetate) and Exenatide(R) (synthetic exen metolazone, quinethazone, althiazide (CAS RN 5588-16-9, din-4; a 39 aa polypeptide). which may be prepared as disclosed in British Patent No. US 2016/0235807 A1 Aug. 18, 2016 37

902,658), benzthiazide (CAS RN 91-33-8, which may be tis), pamatolol Sulfate (Carbamic acid, 2-4-2-hydroxy-3- prepared as disclosed in U.S. Pat. No. 3,108,097), buthiazide (1-methylethyl)aminopropoxylphenyl-ethyl-, methyl (which may be prepared as disclosed in British Patent Nos. ester, (+) sulfate (salt) (2:1), CAS RN 59954-01-7), penb 861.367), and hydrochlorothiazide), loop diuretics (e.g. utolol sulfate (2-Propanol, 1-(2-cyclopentylphenoxy)-3-1,1- bumetanide, ethacrynic acid, furosemide, and torasemide), dimethyle-thyl)amino1, (S)-, sulfate (2:1) (salt), CAS RN potassium sparing agents (e.g. amiloride, and triamterene 38363-32-5), practolol (Acetamide, N-4-2-hydroxy-3-(1- (CAS Number 396-01-O)), and aldosterone antagonists (e.g. methylethyl)amino-propoxyphenyl-, CAS RN 6673-35 spironolactone (CAS Number 52-01-7), epirenone, and the 4:) tiprenolol hydrochloride (Propanol, 1-(1-methylethyl) like); B-adrenergic blockers such as Amiodarone (Cordarone, amino-3-2-(methylthio)-phenoxy-, hydrochloride, (t), Pacerone), bunolol hydrochloride (CAS RN 31969-05-8, CAS RN39832-43-4), tolamolol (Benzamide, 4-2-2-hy Parke-Davis), acebutolol (N-3-Acetyl-4-2-hydroxy-3-(1 droxy-3-(2-methylphenoxy)-propylaminoethoxyl-, CAS methylethyl)aminopropoxyphenyl-butanamide, or (t)-3'- RN 38.103-61-6), bopindolol, indenolol, pindolol, pro Acetyl-4-2-hydroxy-3-(isopropylamino) propoxybutyra panolol, tertatolol, and tilisolol, and the like; calcium channel nilide), acebutolol hydrochloride (e.g. Sectral R, Wyeth-Ay blockers such as besylate salt of amlodipine (such as 3-ethyl erst), alprenolol hydrochloride (CAS RN 13707-88-5 see 5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1, Netherlands Patent Application No. 6,605,692), atenolol (e.g. 4-dihydro-6-methyl-3,5-pyridinedicarboxylate benzene Tenormin(R), AstraZeneca), carteolol hydrochloride (e.g. Car Sulphonate, e.g., Norvasc(R), Pfizer), clentiazem maleate (1.5- trolR Filmtab(R), Abbott), Celiprolol hydrochloride (CASRN Benzothiazepin-4(5H)-one, 3-(acetyloxy)-8-chloro-5-2- 57470-78-7, also see in U.S. Pat. No. 4,034,009), cetamolol (dimethylamino)ethyl-2,3-dihydro-2-(4-methoxyphenyl)- hydrochloride (CAS RN 77590-95-5, see also U.S. Pat. No. (2S-cis)-, (Z)-2-butenedioate (1:1), see also U.S. Pat. No. 4,059,622), labetalol hydrochloride (e.g. Normodyne.R., 4,567,195), isradipine (3.5-Pyridinedicarboxylic acid, 4-(4- Schering), esmolol hydrochloride (e.g. BreviblocR, Baxter), benzofurazanyl)-1,4-dihydro-2,6-dimethyl-, methyl 1-meth levobetaxolol hydrochloride (e.g. BetaxonTM Ophthalmic ylethyl ester, (+)-4-(4-benzofurazanyl)-1,4-dihydro-2,6-dim Suspension, Alcon), levobunolol hydrochloride (e.g. Beta ethyl-3,5-pyridinedicarboxylate, see also U.S. Pat. No. 4.466, gan R. LiquifilmR) with CCAP(R) Compliance Cap, Allergan), 972); nimodipine (such as is isopropyl (2-methoxyethyl) 1, nadolol (e.g. Nadolol, Mylan), practolol (CAS RN 6673-35 4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-di 4, see also U.S. Pat. No. 3,408,387), propranolol hydrochlo carboxylate, e.g. NimotopR, Bayer), felodipine (such as ethyl ride (CAS RN 318-98-9), sotalol hydrochloride (e.g. Beta methyl 4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3, pace AFTM, Berlex), timolol (2-Propanol, 1-(1,1- 5-pyridinedicarboxylate-, e.g. Plendil R. Extended-Release, dimethylethyl)amino-3-4-4-(4-morpholinyl)-1,2,5- AstraZeneca LP), nilvadipine (3.5-Pyridinedicarboxylic thiadiazol-3-yl)oxy-, hemihydrate, (S)- CAS RN91524-16 acid, 2-cyano-1,4-dihydro-6-methyl-4-(3-nitrophenyl)-3- 2), timolol maleate (S)-I-(1,1-dimethylethyl)amino-3-4- methyl 5-(1-methylethyl) ester, also see U.S. Pat. No. 3,799, (4-morpholinyl)-1,2,5-thiadiazol-3-yloxy-2-propanol (Z)- 934), nifedipine (such as 3, 5-pyridinedicarboxylic acid, 1,4- 2-butenedioate (1:1) salt, CAS RN 26921-17-5), bisoprolol dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester, (2-Propanol. 1-4-2-(1-methylethoxy)ethoxy-methylphe e.g., Procardia XLR) Extended Release Tablets, Pfizer), dilt noxyl)-3-(1-meth-ylethyl)amino-, (+), CAS RN 66722-44 iazem hydrochloride (such as 1.5-Benzothiazepin-4 (5H)- 9), bisoprolol fumarate (such as (+)-1-4-2-(1-Methyl one.3-(acetyloxy)-52-(dimethylamino)ethyl-2-3-dihydro ethoxy) ethoxymethylphenoxy-3-(1-methylethyl) 2(4-methoxyphenyl)-, monohydrochloride, (+)-cis, e.g., amino-2-propanol (E)-2-butenedioate (2:1) (salt), e.g., TiazacR, Forest), Verapamil hydrochloride (such as benzene ZebetaTM, Lederle Consumer), nebivalol (2H-1-Benzopyran acetronitrile, (alpha)-3-2-(3,4-dimethoxyphenyl) ethyl 2-methanol, O.C.'-iminobis(methylene)bis 6-fluoro-3,4-di methylaminopropyl-3,4-dimethoxy-(alpha)-(1-methyl hydro-, CAS RN 992.00-09-6 see also U.S. Pat. No. 4,654, ethyl) hydrochloride, e.g., Isoptin R. SR, Knoll Labs), 362), cicloprolol hydrochloride, such 2-Propanol. 1-4-2- teludipine hydrochloride (3.5-Pyridinedicarboxylic acid, (cyclopropylmethoxy)ethoxyphenoxy-3-1-methylethyl) 2-(dimethylamino)methyl4-2-(1E)-3-(1,1-dimethyl amino-, hydrochloride, A.A.S. RN 63686-79-3), ethoxy)-3-oxo-1-propenylphenyl-1,4-dihydro-6-methyl-, dexpropranolol hydrochloride (2-Propanol. 1-1-methyl diethyl ester, monohydrochloride) CAS RN 108700-03-4), ethyl)-amino-3-(1-naphthalenyloxy)-hydrochloride (CAS belfosdil (Phosphonic acid, 2-(2-phenoxy ethyl)-1,3-pro RN 13071-11-9), diacetolol hydrochloride (Acetamide, pane-diyl)bis-, tetrabutyl ester CAS RN 103486-79–9), foste N-3-acetyl-4-2-hydroxy-3-(1-methyl-ethyl)aminopro dil (Phosphonic acid, 4-(2-benzothiazolyl)phenylme poxyphenyl-, monohydrochloride CAS RN 69796-04-9), thyl-, diethyl ester CAS RN 75889-62-2), aranidipine, dilevalol hydrochloride (Benzamide, 2-hydroxy-5-1-hy aZelnidipine, barnidipine, benidipine, bepridil, cinaldipine, droxy-2-1-methyl-3-phenylpropyl)aminoethyl-, monohy clevidipine, efonidipine, gallopamil, lacidipine, lemildipine, drochloride, CAS RN 75659-08-4), exaprolol hydrochloride lercanidipine, monatepil maleate (1-Piperazinebutanamide, (2-Propanol, 1-(2-cyclohexylphenoxy)-3-(1-methylethyl) N-(6, 11-dihydrodibenzo(b.e)thiepin-11-yl)-(4-fluorophe amino-, hydrochloride CAS RN 59333-90-3), flestolol sul nyl)-, (+)-, (Z)-2-butenedioate (1:1) (+)-N-(6,11-Dihydrod fate (Benzoic acid, 2-fluro-3-2-aminocarbonyl)amino ibenzo(b.e)thiep-in-11-yl)-4-(p-fluorophenyl)-1-pipera dimethylethylamino-2-hydroxypropyl ester, (+)-sulfate Zinebutyramide maleate (1:1) CAS RN 132046-06-1), (1:1) (salt), CAS RN 88844-73-9; metalol hydrochloride nicardipine, nisoldipine, nitrendipine, manidipine, pranid (Methanesulfonamide, N-(4-1-hydroxy-2-(methylamino) ipine, and the like; T-channel calcium antagonists such as propylphenyl-, monohydrochloride CAS RN 7701-65-7), mibefradil; angiotensin converting enzyme (ACE) inhibitors metoprolol 2-Propanol. 1-4-(2-methoxyethyl)phenoxy-3- such as benazepril, benazepril hydrochloride (such as 3-1- 1-methylethyl)amino-: CAS RN 37350-58-6), metoprolol (ethoxycarbonyl)-3-phenyl-(1S)-propylamino-2,3,4,5-tet tartrate (such as 2-Propanol. 1-4-(2-methoxyethyl)phe rahydro-2-oxo-1H-1-(3S)-benzazepine-1-acetic acid mono noxy-3-(1-methylethyl)amino-, e.g., Lopressor R, Novar hydrochloride, e.g., Lotrel (R), Novartis), captopril (such as US 2016/0235807 A1 Aug. 18, 2016

1-(2S)-3-mercapto-2-methylpropionyl-L-proline, C.9. RN 4205-91-8), catapres, minoxidil (loniten), nicotinyl alco Captopril, Mylan, CAS RN 62571-86-2 and others disclosed hol (roniacol), diltiazem hydrochloride (such as 1.5-Ben in U.S. Pat. No. 4,046,889), ceranapril (and others disclosed Zothiazepin-4 (5H)-one.3-(acetyloxy)-52-(dimethylamino) in U.S. Pat. No. 4,452.790), cetapril (alacepril, Dainippon ethyl-2,-3-dihydro-2(4-methoxyphenyl)- disclosed in Eur. Therap. Res. 39:671 (1986): 40:543 (1986)), monohydrochloride, (+)-cis, e.g., TiaZac R. Forest), isosor cilazapril (Hoffman-LaRoche) disclosed in J. Cardiovasc. bide dinitrate (such as 1.4:3,6-dianhydro-D-glucitol 2,5-dini Pharmacol. 9:39 (1987), indalapril (delapril hydrochloride trate e.g., Isordil(R) Titradose.R., Wyeth-Ayerst), sosorbide (2H-1,2,4-Benzothiadiazine-7-sulfonamide, 3-bicyclo[2.2. mononitrate (such as 1.4:3,6-dianhydro-D-glucito-1,5-ni 1 hept-5-en-2-yl-6-chloro-3,4-dihydro-, 1,1-dioxide CAS trate, an organic nitrate, e.g., Ismo R, Wyeth-Ayerst), nitro RN2259-96-3); disclosed in U.S. Pat. No. 4,385,051), enala glycerin (such as 2.3 propanetriol trinitrate, e.g., Nitrostat(R) pril (and others disclosed in U.S. Pat. No. 4.374,829), enalo Parke-Davis), Verapamil hydrochloride (such as benzeneac pril, enaloprilat, fosinopril, ((such as L-proline, 4-cyclo etonitrile, (+)-(alpha)3-2-(3.4 dimethoxypheny 1)ethyl hexyl-1-(2-methyl-1-(1-oxopropoxy) propoxy (4- methylaminopropyl-3,4-dimethoxy-(alpha)-(1-methyl phenylbutyl) phosphinyl)acetyl-, Sodium salt, e.g., ethyl) hydrochloride, e.g., Covera HSR) Extended-Release, Monopril, Bristol-Myers Squibb and others disclosed in U.S. Searle), chromonar (which may be prepared as disclosed in Pat. No. 4,168.267), fosinopril sodium (L-Proline, 4-cyclo U.S. Pat. No. 3,282,938), clonitate (Annalen 1870 155), dro hexyl-1-(R)-(1S)-2-methyl-1-(1-ox-opropoxy)propox), premilamine (which may be prepared as disclosed in imidapril, indolapril (Schering, disclosed in J. Cardiovasc. DE252 1113), lidoflazine (which may be prepared as dis Pharmacol. 5:643,655 (1983)), lisinopril (Merck), losinopril, closed in U.S. Pat. No. 3.267,104); prenylamine (which may moexipril, moexipril hydrochloride (3-Isoquinolinecarboxy be prepared as disclosed in U.S. Pat. No. 3,152.173), propatyl lic acid, 2-(2S)-2-(1S)-1-(ethoxycarbonyl)-3-phenylpro nitrate (which may be prepared as disclosed in French Patent pylamino-1-oxopropyl-1-2,3,4-tetrahydro-6,7- No. 1,103,113), mioflazine hydrochloride (1-Piperazineac dimethoxy-, monohydrochloride, (3S)-CAS RN 82586-52 etamide, 3-(aminocarbonyl)-4.4-bis(4-fluorophenyl)bu 5), quinapril, quinaprilat, ramipril (Hoechsst) disclosed in EP tyl-N-(2,6-dichlorophenyl)-, dihydrochloride CAS RN 79022 and Curr. Ther. Res. 40:74 (1986), perindopril erbu 83898-67-3), mixidine (Benzeneethanamine, 3,4- mine (such as 2S,3aS,7aS-1-(S)-N-(S)-1-Carboxybutyl dimethoxy-N-(1-methyl-2-pyrrolidinylidene)-Pyrrolidine, jalanylhexahydro-indolinecarboxylic acid, 1-ethyl ester, 2-(3,4-dimethoxyphenethyl)imino-1-methyl-1-Methyl-2- compound with tert-butylamine (1:1), e.g., Aceon(R), Solvay), (3, 4-dimethoxyphenethyl)iminolpyrrolidine CAS RN perindopril (Servier, disclosed in Eur. J. din. Pharmacol. 27737-38-8), molsidomine (1,2,3-Oxadiazolium, 31:519 (1987)), quanipril (disclosed in U.S. Pat. No. 4.344, 5-(ethoxycarbonyl)amino-3-(4-morpholinyl)-, inner salt 949), spirapril (Schering, disclosed in Acta. Pharmacol. Toxi CAS RN 25717-80-0), isosorbide mononitrate (D-Glucitol, col. 59 (Supp. 5): 173 (1986)), tenocapril, trandolapril, 14:3,6-dianhydro-, 5-nitrate CAS RN 16051-77-7), erythri Zofenopril (and others disclosed in U.S. Pat. No. 4.316,906), tyl tetranitrate (1,2,3,4-Butanetetrol, tetranitrate, (2R,3S)- rentiapril (fentiapril, disclosed in Clin. Exp. Pharmacol. rel-CAS RN 7297-25-8), clonitrate(1,2-Propanediol, Physiol. 10:131 (1983)), pivopril, YS980, teprotide (Brady 3-chloro-, dinitrate (7CI, 8CI, 9CI) CAS RN 2612-33-1), kinin potentiator BPP9a CAS RN35115-60-7), BRL 36,378 dipyridamole Ethanol, 2.2.2".2"-(4,8-di-1-piperidinylpy (SmithKline Beecham, see EP80822 and EP60668), MC-838 rimido5,4-dipyrimidine-2,6-diyl)dinitrilotetrakis-CAS RN (Chugai, see CA. 102:72588v and Jap. J. Pharmacol. 40:373 58-32-2), nicorandil (CAS RN 65141-46-03-), pyridinecar (1986), CGS 14824 (Ciba-Geigy, 3-(1-ethoxycarbonyl-3- boxamide (N-2-(nitrooxy)ethyl-Nisoldipine3.5-Py phenyl-(1S)-propylamino)-2,3,4,5-tetrahydro-2-ox-o-1- ridinedicarboxylic acid, 1,4-dihydro-2,6-dimethyl-4-(2-ni (3S)-benzazepine-1 acetic acid HCl, see U.K. Patent No. trophenyl)-, methyl 2-methylpropyl ester CAS RN63675-72 2103614), CGS 16,617 (Ciba-Geigy, 3(S)-(1S)-5-amino-1- 9), nifedipine3.5-Pyridinedicarboxylic acid, 1,4-dihydro-2, carboxypentyl)amino-2,3,4,-5-tetrahydro-2-oxo-1H-1-ben 6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester CAS RN zazepine-1-ethanoic acid, see U.S. Pat. No. 4.473,575), Ru 21829-25-4), perhexiline maleate (Piperidine, 2-(2,2-dicy 44570 (Hoechst, see Arzneimittelforschung 34:1254 (1985)), clohexylethyl)-, (2Z)-2-butenedioate (1:1) CAS RN 6724 R 31-2201 (Hoffman-LaRoche see FEBS Left. 165:201 53-4), oxprenolol hydrochloride (2-Propanol, 1-(1-methyl (1984)), CI925 (Pharmacologist 26:243, 266 (1984)), ethyl)amino-3-2-(2-propenyloxy)phenoxy-, WY-44221 (Wyeth, see J. Med. Chem. 26:394 (1983)), and hydrochloride CAS RN 6452-73-9), pentrinitrol (1,3-Pro those disclosed in US2003006922 (paragraph 28), U.S. Pat. panediol. 2.2-bis(nitrooxy)methyl-, mononitrate (ester) No. 4,337,201, U.S. Pat. No. 4,432,971 (phosphonamidates): CAS RN 1607-17-6), Verapamil (Benzeneacetonitrile, C-3- neutral endopeptidase inhibitors such as omapatrilat (Van 2-(3,4-dimethoxyphenyl)ethyl-methylaminopropyl-3,4- lev(R), CGS 30440, cadoxatril and ecadotril, fasidotril (also dimethoxy-O-(1-methylethyl)-CAS RN 52-53-9) and the known as aladotril oralatriopril), Sampatrilat, mixanpril, and like; angiotensin II receptor antagonists such as, aprosartan, gemopatrilat, AVE7688, ER4030, and those disclosed in U.S. Zolasartan, olmesartan, pratosartan, FI6828K, RNH6270, Pat. No. 5,362,727, U.S. Pat. No. 5,366,973, U.S. Pat. No. candesartan (1H-Benzimidazole-7-carboxylic acid, 5,225,401, U.S. Pat. No. 4,722,810, U.S. Pat. No. 5,223,516, 2-ethoxy-1-2'-(1H-tetrazol-5-yl) 1,1'-biphenyl 4-yl)me U.S. Pat. No. 4,749,688, U.S. Pat. No. 5,552,397, U.S. Pat. thyl-CAS RN 139481-59-7), candesartan cilexetil ((+/-)-1- No. 5,504,080, U.S. Pat. No. 5,612,359, U.S. Pat. No. 5,525, (cyclohexylcarbonyloxy)ethyl-2-ethoxy-1-2'-(1H-tetrazol 723, EP0599444, EP0481522, EP0599444, EP0595610, 5-yl)biphenyl-4-yl)-1H-benzimidazole carboxylate, CAS EP0534363, EP534396, EP534492, EP0629627; endothelin RN 145040-37-5, U.S. Pat. No. 5,703,110 and U.S. Pat. No. antagonists such as tezosentan, A3081.65, andYM62899, and 5,196,444), eprosartan (3-1-4-carboxyphenylmethyl)-2-n- the like; vasodilators such as hydralazine (apresoline), cloni butyl-imidazol-5-yl)-(2-thienylmethyl) propenoic acid, U.S. dine (clonidine hydrochloride (1H-Imidazol-2-amine, N-(2, Pat. No. 5,185.351 and U.S. Pat. No. 5,650,650), irbesartan 6-dichlorophenyl)4,5-dihydro-, monohydrochloride CAS (2-n-butyl-3-2'-(lh-tetrazol-5-yl)biphenyl-4-yl)methyl 1,

US 2016/0235807 A1 Aug. 18, 2016 40

No. 5,187,159, U.S. Pat. No. 5,198,438, U.S. Pat. No. 5,182, 32295-184); adrenergic stimulants such as guanfacine hydro 288, U.S. Pat. No. 5,036,048, U.S. Pat. No. 5,140,036, U.S. chloride (such as N-amidino-2-(2,6-dichlorophenyl)aceta Pat. No. 5,087,634, U.S. Pat. No. 5,196,537, U.S. Pat. No. mide hydrochloride, e.g., Tenex R. Tablets available from 5,153,347, U.S. Pat. No. 5,191,086, U.S. Pat. No. 5,190,942, Robins); methyldopa-hydrochlorothiazide (such as levo-3- U.S. Pat. No. 5,177,097, U.S. Pat. No. 5,212,177, U.S. Pat. (3,4-dihydroxyphenyl)-2-methylalanine) combined with No. 5,208,234, U.S. Pat. No. 5,208,235, U.S. Pat. No. 5,212, Hydrochlorothiazide (such as 6-chloro-3,4-dihydro-2H-1.2. 195, U.S. Pat. No. 5,130,439, U.S. Pat. No. 5,045,540, U.S. 4-benzothiadiazine-7-sulfonamide 1,1-dioxide, e.g., the Pat. No. 5,041,152, and U.S. Pat. No. 5,210,204, and phar combination as, e.g., Aldoril R. Tablets available from Merck), maceutically acceptable salts and esters thereof; C/B adren methyldopa-chlorothiazide (such as 6-chloro-2H-1,2,4-ben Zothiadiazine-7-sulfonamide 1,1-dioxide and methyldopa as ergic blockers such as nipradillol, arotinolol, amoSulalol, described above, e.g., Aldoclor(R), Merck), clonidine hydro bretylium tosylate (CAS RN: 61-75-6), dihydroergtamine chloride (such as 2-(2,6-dichlorophenylamino)-2-imidazo mesylate (such as ergotaman-3',6', 18-trione.9,-10-dihydro line hydrochloride and chlorthalidone (such as 2-chloro-5-(1- 12'-hydroxy-2-methyl-5'-(phenylmethyl)-(5'(O.))-. hydroxy-3-oxo-1-isoindolinyl) benzenesulfonamide), e.g., monomethanesulfonate, e.g., DHE 45(R) Injection, Novartis), Combipres(R), Boehringer Ingelheim), clonidine hydrochlo carvedilol (such as (+)-1-(Carbazol-4-yloxy)-3-2-(o-meth ride (Such as 2-(2,6-dichlorophenylamino)-2-imidazoline oxyphenoxy)ethylamino-2-propanol, e.g., Coreg R, Smith hydrochloride, e.g., Catapres(R), Boehringer Ingelheim), Kline Beecham), labetalol (such as 5-1-hydroxy-2-(1-me clonidine (1H-Imidazol-2-amine, N-(2,6-dichlorophenyl)4, thyl-3-phenylpropyl) aminoethylsalicylamide 5-dihydro-CAS RN 4205-90-7), Hyzaar (Merck; a combina monohydrochloride, e.g., Normodyne R, Schering), brety tion of losartan and hydrochlorothiazide), Co-Diovan (No lium tosylate (Benzenemethanaminium, 2-bromo-N-ethyl vartis; a combination of Valsartan and hydrochlorothiazide, N,N-dimethyl-, salt with 4-methylbenzenesulfonic acid (1:1) Lotrel (Novartis; a combination of benazepril and amlo CAS RN 61-75-6), phentolamine mesylate (Phenol, 3-(4.5- dipine) and Caduet (Pfizer; a combination of amlodipine and dihydro-1H-imidazol-2-yl)methyl(4-methylphenyl) atorvastatin), and those agents disclosed in US20030069221. amino-, monomethanesulfonate (salt) CAS RN 65-28-1), solypertine tartrate (5H-1,3-Dioxolo4.5-findole, 7-2-4- (2-methoxyphenyl)-1-piperazinylethyl-, (2R,3R)-2,3-di Agents for the Treatment of Respiratory Disorders hydroxybutanedioate (1:1) CAS RN 5591-43-5), Zolertine 0254 The GCRA peptides described herein can be used in hydrochloride (Piperazine, 1-phenyl4-2-(1H-tetrazol-5-yl) combination therapy with one or more of the following agents ethyl-, monohydrochloride (8Cl, 9CI) CAS RN 7241-94-3) useful in the treatment of respiratory and other disorders and the like; O. adrenergic receptor blockers, such as alfuZosin including but not limited to: (1) B-agonists including but not (CAS RN: 81403-68-1), terazosin, urapidil, prazosin (Mini limited to: albuterol (PROVENTIL(R), S ALBUT AMOIR, press.(R), tamsulosin, bunaZosin, trimaZosin, doxazosin, naf VENTOLINR), bambuterol, bitoterol, clenbuterol, fenoterol, topidil, indoramin, WHP 164, XENO1O, fenspiride hydro formoterol, isoetharine (BRONKOSOL(R), BRONKOME chloride (which may be prepared as disclosed in U.S. Pat. No. TER(R), metaproterenol (ALUPENTR), METAPREL(R), pir 3.399,192), proroxan (CAS RN33743-96-3), and labetalol buterol (MAXAIRR), reproterol, rimiterol, salmeterol, terb hydrochloride and combinations thereof. C.2 agonists such as utaline (BRETHAIRE(R), BRETHINE(R), BRICANYL(R), methyldopa, methyldopa HCL, lofexidine, tiamenidine, adrenalin, isoproterenol (ISUPRELR), epinephrine bitartrate moXonidine, rillmenidine, guanobenz, and the like; aldoster (PRIMATENER), ephedrine, orciprenline, fenoterol and iso one inhibitors, and the like; renin inhibitors including etharine; (2) steroids, including but not limited to beclom Aliskiren (SPP100; Novartis/Speedel); angiopoietin-2-bind ethasone, beclomethasone dipropionate, betamethasone, ing agents such as those disclosed in WO03/030833; anti budesonide, bunedoside, butiXocort, dexamethasone, angina agents such as ranolazine (hydrochloride 1-Pipera flunisolide, fluocortin, fluticaSone, hydrocortisone, methyl zineacetamide, N-(2,6-dimethylphenyl)-4-2-hydroxy-3-(2- prednisone, mometaSone, predonisolone, predonisone, tipre methoxyphenoxy)propyl-, dihydrochloride CAS RN95635 dane, tiXocortal, triamcinolone, and triamcinolone acetonide; 56-6), betaxolol hydrochloride (2-Propanol. 1-4-2 (3) B2-agonist-corticosteroid combinations e.g., Salmeterol (cyclopropylmethoxy)ethylphenoxy-3-(1-methylethyl) fluticasone (AD V AIRR), formoterol-budesonid (SYM amino-, hydrochloride CAS RN 63659-19-8), butoprozine BICORTR); (4) leukotriene D4 receptor antagonists/leukot hydrochloride (Methanone, 4-3(dibutylamino)propoxy riene antagonists/LTD4 antagonists (i.e., any compound that phenyl (2-ethyl-3-indolizinyl)-, monohydrochloride CAS is capable of blocking, inhibiting, reducing or otherwise RN 62134-34-3), cinepazet maleatel-Piperazineacetic acid, interrupting the interaction between leukotrienes and the Cys 4-1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl-, ethyl LTI receptor) including but not limited to: Zafhiukast, mon ester, (2Z)-2-butenedioate (1:1) CAS RN 50679-07-7), telukast, montelukast sodium (SINGULAIRR), pranlukast, tosifen (Benzenesulfonamide, 4-methyl-N-(1S)-1-methyl iralukast, pobilukast, SKB-106,203 and compounds 2-phenylethylaminocarbonyl-CAS RN32295-184), Vera described as having LTD4 antagonizing activity described in pamilhydrochloride (Benzeneacetonitrile, C-3-2-(3,4- U.S. Pat. No. 5,565,473; (5) 5-lipoxygenase inhibitors and/or dimethoxyphenyl)ethylmethylaminopropyl-3,4- leukotriene biosynthesis inhibitors e.g., Zileuton and dimethoxy-O-(1-methylethyl)-, monohydrochloride CAS BAY1005 (CA registry 128253-31-6); (6) histamine H1 RN 152-114), molsidomine (1,2,3-Oxadiazolium, receptor antagonists/antihistamines (i.e., any compound that 5-(ethoxycarbonyl)amino-3-(4-morpholinyl)-, inner salt is capable of blocking, inhibiting, reducing or otherwise CAS RN25717-80-0), and ranolazine hydrochloride (1-Pip interrupting the interaction between histamine and its recep erazineacetamide, N-(2,6-dimethylphenyl)-2-hydroxy-3- tor) including but not limited to: astemizole, acrivastine, anta (2-meth-oxyphenoxy)propyl-, dihydrochloride CAS RN Zoline, azatadine, azelastine, astamizole, bromopheniramine, 95635-56-6); tosifen (Benzenesulfonamide, 4-methyl-N- bromopheniramine maleate, carbinoxamine, carebastine, (1S)-1-methyl-2-phenylethylaminocarbonyl-CAS RN cetirizine, chlorpheniramine, chloropheniramine maleate, US 2016/0235807 A1 Aug. 18, 2016 cimetidine clemastine, cyclizine, cyproheptadine, descar and Zomepirac); and (21) aerosolized antioxidant therapeu boethoxyloratadine, dexchlorpheniramine, dimethindene, tics such as S-Nitrosoglutathione. diphenhydramine, diphenylpyraline, doxylamine Succinate, doxylamine, ebastine, efletirizine, epinastine, famotidine, Anti-Obesity Agents fexofenadine, hydroxy Zine, hydroxy Zine, ketotifen, levo (0255. The GCRA peptides described herein can be used in cabastine, levocetirizine, levocetirizine, loratadine, mecliz combination therapy with an anti-obesity agent. Suitable ine, mepyramine, meduitazine, methdilazine, mianserin, such agents include, but are not limited to: 113 HSD-I (11 mizolastine, noberastine, norasternizole, noraZtemizole, beta hydroxy steroid dehydrogenase type 1) inhibitors, such phenindamine, pheniramine, picumast, promethazine, pyn as BVT 3498, BVT 2733, 3-(1-adamantyl)-4-ethyl-5-(eth lamine, pyrilamine, ranitidine, temelastine, terfenadine, tri ylthio)-4H-1,2,4-triazole, 3-(1-adamantyl)-5-(3,4,5-tri meprazine, tripelenamine, and triprolidine; (7) an anticholin methoxyphenyl)-4-methyl-4H-1,2,4-triazole, 3-adamanta ergic including but not limited to: atropine, benztropine, nyl-4,5,6,7,8,9, 10,11,12.3a-decahydro-1,2,4-triazolo 4.3-a biperiden, flutropium, hyoscyamine (e.g. Levsin(R); LevbidR; 11 annulene, and those compounds disclosed in WO01/ Levsin/SL(R), AnaspazR), Levsinex Timecaps.(R), NuLev(R), 90091, WO01/90090, WO01/90092 and WO02/072084; ilutropium, ipratropium, ipratropium bromide, methScopola 5HT antagonists such as those in WO03/037871, WO03/ mine, oxybutinin, rispenzepine, Scopolamine, and tiotro 037887, and the like; 5HTIa modulators such as carbidopa, pium; (8) an anti-tussive including but not limited to: dex benserazide and those disclosed in U.S. Pat. No. 6,207,699, tromethorphan, codeine, and hydromorphone; (9) a WO03/031439, and the like; 5HT2c (serotonin receptor 2c) decongestant including but not limited to: pseudoephedrine agonists, such as BVT933, DPCA37215, IK264, PNU 22394, and phenylpropanolamine; (10) an expectorant including but WAY 161503, R-1065, SB 243213 (GlaxoSmith Kline) and not limited to: guafenesin, guaicolsulfate, terpin, ammonium YM 348 and those disclosed in U.S. Pat. No. 3,914,250, chloride, glycerol guaicolate, and iodinated glycerol; (11) a WO00/77010, WO02/36596, WO02/48124, WO02/10169, WO01/66548, WO02/44152, WO02/51844, WO02/40456, bronchodilator including but not limited to: theophylline and and WO02/40457; 5HT6 receptor modulators, such as those aminophylline; (12) an anti-inflammatory including but not in WO03/030901, WO03/035061, WO03/039547, and the limited to: fluribiprofen, diclophenac, indomethacin, keto like; acyl-estrogens, such as oleoyl-estrone, disclosed in del profen, S-ketroprophen, tenoxicam; (13) a PDE (phosphodi Mar-Grasa, M. et al., Obesity Research, 9:202-9 (2001) and esterase) inhibitor including but not limited to those disclosed Japanese Patent Application No. JP 2000256190; anorectic herein; (14) a recombinant humanized monoclonal antibody bicyclic compounds such as 1426 (Aventis) and 1954 (Aven e.g. Xolair (also called omalizumab), rhuMab, and tali tis), and the compounds disclosed in WO00/18749, WO01/ Zumab); (15) a humanized lung Surfactant including recom 32638, WO01/62746, WO01/62747, and WO03/015769; CB binant forms of surfactant proteins SP-B, SP-C or SP-D e.g. 1 (cannabinoid-1 receptor) antagonist/inverse agonists such SURFAXINR), formerly known as disc-104 (Discovery Labo as rimonabant (Acomplia; Sanofi), SR-147778 (Sanofi), ratories), (16) agents that inhibit epithelial Sodium channels SR-141716 (Sanofi), BAY 65-2520 (Bayer), and SLV 319 (ENaC) such as amiloride and related compounds: (17) anti (Solvay), and those disclosed in patent publications U.S. Pat. microbial agents used to treat pulmonary infections such as No. 4,973,587, U.S. Pat. No. 5,013,837, U.S. Pat. No. 5,081, acyclovir, amikacin, amoxicillin, doxycycline, trimethoprin 122, U.S. Pat. No. 5,112,820, U.S. Pat. No. 5,292,736, U.S. Sulfamethoxazole, amphotericin B, azithromycin, clarithro Pat. No. 5,532,237, U.S. Pat. No. 5,624,941, U.S. Pat. No. mycin, roXithromycin, clarithromycin, cephalosporins (cef 6,028,084, U.S. Pat. No. 6,509,367, U.S. Pat. No. 6,509,367, foXitin, cefimetazole etc), ciprofloxacin, ethambutol, genti WO96/33159, WO97/29079, WO98/31227, WO98/33765, mycin, ganciclovir, imipenem, isoniazid, itraconazole, WO98/37061, WO98/41519, WO98/43635, WO98/43636, penicillin, ribavirin, rifampin, rifabutin, amantadine, riman WO99/02499, WO00/10967, WO00/10968, WO01/09120, tidine, Streptomycin, tobramycin, and Vancomycin; (18) WO01/58869, WO01/64.632, WO01/64633, WO01/64634, agents that activate chloride secretion through Ca++ depen WO01/70700, WO01/96330, WO02/076949, WO03/ dent chloride channels (such as purinergic receptor (P2Y(2) 006007, WO03/007887, WO03/020217, WO03/026647, agonists); (19) agents that decrease sputum viscosity, such as WO03/026648, WO03/027069, WO03/027076, WO03/ human recombinant DNase 1. (Pulmozyme(R); (20) nonste 027114, WO03/037332, WO03/04.01.07, WO03/086940, roidal anti-inflammatory agents (acemetacin, acetami WO03/084943 and EP658546; CCK-A (cholecystokinin-A) nophen, acetyl salicylic acid, alclofenac, alminoprofen, apa agonists, such as AR-R 15849, GI 181771 (GSK), JMV-180, Zone, aspirin, benoxaprofen, bezpiperylon, bucloxic acid, A-71378, A-71623 and SR146131 (Sanofi), and those carprofen, clidanac, diclofenac, diclofenac, diflunisal, diflu described in U.S. Pat. No. 5,739,106; CNTF (Ciliary neu sinal, etodolac, fenbufen, fenbufen, fenclofenac, fenclozic rotrophic factors), such as GI-181771 (Glaxo-SmithKline), acid, fenoprofen, fentiazac, feprazone, flufenamic acid, SR146131 (Sanofi Synthelabo), butabindide, PD 170,292, flufenisal, flufenisal, fluprofen, flurbiprofen, flurbiprofen, and PD 149164 (Pfizer); CNTF derivatives, such as AXok furofenac, ibufenac, ibuprofen, indomethacin, indomethacin, ineR (Regeneron), and those disclosed in WO94/09134, indoprofen, isoxepac, isoxicam, ketoprofen, ketoprofen, WO98/22128, and WO99/43813; dipeptidyl peptidase IV ketorolac, meclofenamic acid, meclofenamic acid, mefe (DP-IV) inhibitors, such as isoleucine thiazolidide, valine namic acid, mefenamic acid, miroprofen, mofebutaZone, pyrrolidide, NVP-DPP728, LAF237, P93/01, P3298, TSL nabumetone oxaprozin, naproxen, naproxen, niflumic acid, 225 (tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic oxaprozin, oXpinac, oxyphenbutaZone, phenacetin, phenylb acid; disclosed byYamada et al. Bioorg. & Med. Chem. Lett. utaZone, phenylbutaZone, piroxicam, piroxicam, pirprofen, 8 (1998) 1537-1540), TMC-2A/2B/2C, CD26 inhibitors, FE pranoprofen, Sudoxicam, tenoxican, SulfaSalazine, Sulindac, 999011, P9310/K364, VIP 0177, SDZ 274-444, 2-cyanopy Sulindac. Suprofen, tiaprofenic acid, tiopinac, tioxaprofen, rrolidides and 4-cyanopyrrolidides as disclosed by Ashworth tolfenamic acid, tolmetin, tolmetin, Zidometacin, Zomepirac, et al. Bioorg. & Med. Chem. Lett. Vol. 6, No. 22, pp 1163 US 2016/0235807 A1 Aug. 18, 2016 42

1166 and 2745-2748 (1996) and the compounds disclosed WO02/051809, WO02/06245, WO02/076929, WO02/ patent publications. WO99/38501, WO99/46272, WO99/ 076947, WO02/04433, WO02/.51809, WO02/083134, 67279 (Probiodrug), WO99/67278 (Probiodrug), WO99/ WO02/094799, WO03/004027, WO03/13574, WO03/ 61431 (Probiodrug), WO02/083128, WO02/062764, WO03/ 15769, WO03/028641, WO03/035624, WO03/033476, 000180, WO03/000181, WO03/000250, WO03/002530, WO03/033480, JP13226269, and JP1437059: mGluR5 WO03/002531, WO03/002553, WO03/002593, WO03/ modulators such as those disclosed in WO03/029210, WO03/ 004498, WO03/004496, WO03/017936, WO03/024942, 047581, WO03/048137, WO03/051315, WO03/051833, WO03/024965, WO03/033524, WO03/037327 and WO03/053922, WO03/059904, and the like: serotoninergic EP1258476; growth hormone secretagogue receptor ago agents, such as fenfluramine (such as Pondimin R (BenZe nists/antagonists, such as NN703, hexarelin, MK-0677 neethanamine, N-ethyl-alpha-methyl-3-(trifluoromethyl)- (Merck), SM-130686, CP-424391 (Pfizer), LY 444,711 (Eli hydrochloride), Robbins), dexfenfluramine (such as Redux(R) Lilly), L-692,429 and L-163.255, and such as those disclosed (Benzeneethanamine, N-ethyl-alpha-methyl-3-(trifluorom in U.S. Ser. No. 09/662,448, U.S. provisional application ethyl)-, hydrochloride), Interneuron) and sibutramine ((Me 60/203,335, U.S. Pat. No. 6,358,951, US20020491.96, ridia(R, Knoll/ReductiltM) including racemic mixtures, as US2002/022637, WO01/56592 and WO02/32888; H3 (his optically pure isomers (+) and (-), and pharmaceutically tamine H3) antagonist/inverse agonists, such as thioperam acceptable salts, solvents, hydrates, clathrates and prodrugs ide, 3-(1H-imidazol-4-yl)propyl N-(4-pentenyl)carbamate), thereof including sibutramine hydrochloride monohydrate clobenpropit, iodophenpropit, imoproxifan, GT2394 (Gliat salts thereof, and those compounds disclosed in U.S. Pat. No. ech), and A331440, O-3-(1H-imidazol-4-yl)propanol car 4,746,680, U.S. Pat. No. 4,806,570, and U.S. Pat. No. 5,436, bamates (Kiec-Kononowicz, K. et al., Pharmazie, 55:349-55 272, US20020006964, WO01/27068, and WO01/62341; NE (2000)), piperidine-containing histamine H3-receptor (norepinephrine) transport inhibitors, such as GW 320659, antagonists (Lazewska, D. et al., Pharmazie, 56:927-32 despiramine, talsupram, and nomifensine; NPY 1 antago (2001), benzophenone derivatives and related compounds nists, such as BIBP3226, J-115814, BIBO 3304, LY-357897, (Sasse, A. et al., Arch. Pharm. (Weinheim) 334:45-52 CP-671906, GI-264879A, and those disclosed in U.S. Pat. (2001)), substituted N-phenylcarbamates (Reidemeister, S. et No. 6,001836, WO96/14307, WO01/23387, WO99/51600, al., Pharmazie, 55:83-6 (2000)), and proxifan derivatives WO01/85690, WO01/85098, WO01/85173, and WO01/ (Sasse, A. et al., J. Med. Chem. 43:3335-43 (2000)) and 89528: NPY5 (neuropeptide YY5) antagonists, such as 152, histamine H3 receptor modulators such as those disclosed in 804, GW-569180A, GW-594884A, GW-587081X, WO02/15905, WO03/024928 and WO03/024929; leptin GW-548118X, FR235208, FR226928, FR240662, derivatives, such as those disclosed in U.S. Pat. No. 5,552, FR252384, 1229U91, GI-264879A, CGP71683A, 524, U.S. Pat. No. 5,552,523, U.S. Pat. No. 5,552,522, U.S. LY-377897, LY-366377, PD-160170, SR-120562A, Pat. No. 5,521,283, WO96/23513, WO96/23514, WO96/ SR-120819A, JCF-104, and H409/22 and those compounds 23515, WO96/23516, WO96/23517, WO96/23518, WO96/ disclosed in patent publications U.S. Pat. No. 6,140,354, U.S. 23519, and WO96/23520; leptin, including recombinant Pat. No. 6, 191,160, U.S. Pat. No. 6,218,408, U.S. Pat. No. human leptin (PEG-OE, Hoffman La Roche) and recombi 6,258,837, U.S. Pat. No. 6,313,298, U.S. Pat. No. 6,326,375, nant methionyl human leptin (Amgen); lipase inhibitors, such U.S. Pat. No. 6,329,395, U.S. Pat. No. 6,335,345, U.S. Pat. as tetrahydrolipstatin (orlistat/Xenical(R), Triton WR1 339, No. 6,337,332, U.S. Pat. No. 6,329,395, U.S. Pat. No. 6,340, RHC80267, lipstatin, teasaponin, diethylumbelliferyl phos 683, EP01010691, EP-01044970, WO97/19682, WO97/ phate, FL-386, WAY-121898, Bay-N-3176, valilactone, 20820, WO97/20821, WO97/20822, WO97/20823, WO98/ esteracin, ebelactone A, ebelactone B, and RHC 80267, and 27063, WO00/107409, WO00/185714, WO00/185730, those disclosed in patent publications WO01/77094, U.S. Pat. WO00/64880, WO00/68197, WO00/69849, WO/0113917, No. 4,598,089, U.S. Pat. No. 4,452,813, U.S. Pat. No. 5,512, WO01/09120, WO01/14376, WO01/85714, WO01/85730, 565, U.S. Pat. No. 5,391,571, U.S. Pat. No. 5,602,151, U.S. WO01/07409, WO01/02379, WO01/23388, WO01/23389, Pat. No. 4,405,644, U.S. Pat. No. 4,189,438, and U.S. Pat. No. WO01/44201, WO01/62737, WO01/62738, WO01/09120, 4,242.453; lipid metabolism modulators such as maslinic WO02/20488, WO02/22592, WO02/48152, WO02/49648, acid, erythrodiol, ursolic acid uvaol, betulinic acid, betulin, WO02/051806, WO02/094789, WO03/009845, WO03/ and the like and compounds disclosed in WO03/01 1267; O14083, WO03/022849, WO03/028726 and Norman et al., J. Mc4r (melanocortin 4 receptor) agonists, such as Med. Chem. 43:4288-4312 (2000); opioid antagonists, such CHIR86036 (Chiron), ME-10142, ME-10145, and HS-131 as nalmefene (REVEXR), 3-methoxynaltrexone, methylnal (Melacure), and those disclosed in PCT publication Nos. trexone, naloxone, and naltrexone (e.g. PT901; Pain Thera WO99/64002, WO00/74679, WO01/991752, WO01/25192, peutics, Inc.) and those disclosed in US20050004155 and WO01/52880, WO01/74844, WO01/70708, WO01/70337, WO00/21509; orexin antagonists, such as SB-334867-A and WO01/91752, WO02/059095, WO02/059107, WO02/ those disclosed in patent publications WO01/96302, WO01/ 059108, WO02/059117, WO02/06276, WO02/12166, 68609, WO02/44172, WO02/51232, WO02/51838, WO02/ WO02/11715, WO02/12178, WO02/15909, WO02/38544, 089800, WO02/090355, WO03/023561, WO03/032991, and WO02/068387, WO02/068388, WO02/067869, WO02/ WO03/037847; PDE inhibitors (e.g. compounds which slow 081430, WO03/06604, WO03/007949, WO03/009847, the degradation of cyclic AMP (cAMP) and/or cyclic GMP WO03/009850, WO03/013509, and WO03/031410; Mc5r (cGMP) by inhibition of the phosphodiesterases, which can (melanocortin 5 receptor) modulators, such as those dis lead to a relative increase in the intracellular concentration of closed in WO97/19952, WO00/15826, WO00/15790, cAMP and coMP: possible PDE inhibitors are primarily US20030092041; melanin-concentrating hormone 1 receptor those Substances which are to be numbered among the class (MCHR) antagonists, such as T-226296 (Takeda), SB consisting of the PDE3 inhibitors, the class consisting of the 568849, SNP-7941 (Synaptic), and those disclosed in patent PDE4 inhibitors and/or the class consisting of the PDE5 publications WO01/21169, WO01/82925, WO01/87834, inhibitors, in particular those Substances which can be desig US 2016/0235807 A1 Aug. 18, 2016

nated as mixed types of PDE3/4 inhibitors or as mixed types (NPY2) agonists include but are not limited to: polypeptide of PDE3/4/5 inhibitors) such as those disclosed in patent YY and fragments and variants thereof (e.g. YY3-36 (PYY3 publications DE 1470341, DE2108438, DE2123328, 36)(N. Engl.J.Med. 349:941, 2003; IKPEAPGE DASPEEL DE2305339, DE2305575, DE2315801, DE2402908, NRY YASLRHYLNL VTRQRY (SEQ ID NO: 2 of WO DE2413935, DE2451417, DE2459090, DE2646469, 2009080608, which is herein incorporated by reference in its DE2727481, DE2825048, DE2837161, DE2845220, entirety)) and PYYagonists such as those disclosed in WO02/ DE2847621, DE2934747, DE3021792, DE3038166, 47712, WO03/026591, WO03/057235, and WO03/027637; DE3044568, EP000718, EP0008.408, EP0010759, serotonin reuptake inhibitors, such as, paroxetine, fluoxetine EP0059948, EP0075436, EP00965.17, EPO1 12987, EPO1 (ProzacTM), fluvoxamine, sertraline, citalopram, and imi 16948, EPO150937, EP0158380, EPO161632, EPO161918, pramine, and those disclosed in U.S. Pat. No. 6,162,805, U.S. EP0167121, EP019.9127, EP0220044, EP0247725, Pat. No. 6,365,633, WO03/00663, WO01/27060, and WO01/ EP0258191 EP0272910, EP0272914, EP0294647, 162341; thyroid hormone B agonists, such as KB-2611 EPO300726, EPO335386, EPO357788, EPO38.9282, (KaroBioBMS), and those disclosed in WO02/15845, WO97/ EPO406958, EPO426180, EPO428302, EPO435811, 21993, WO99/00353, GB98/284425, U.S. Provisional Appli EPO470805, EPO482208, EPO490823, EP0506194, cation No. 60/183,223, and Japanese Patent Application No. EP0511865, EP0527117, EP0626939, EP0664289, JP 2000256190; UCP-I (uncoupling protein-1), 2, or 3 acti EP0671389, EP0685474, EP0685475, EP0685479, vators, such as phytanic acid, 4-(E)-2-(5,6,7,8-tetrahydro JP92234389, JP94329652, JP950.10875, U.S. Pat. No. 4,963, 5,5,8,8-tetramethyl-2-napthalenyl)-1-propenylbenzoic acid 561, U.S. Pat. No. 5,141,931, WO9117991, WO9200968, (TTNPB), retinoic acid, and those disclosed in WO99/00123; WO9212961, WO9307146, WO9315044, WO9315045, B3 (beta adrenergic receptor 3) agonists, such as AJ9677/ WO9318024, WO9319068, WO9319720, WO931.9747, TAK677 (Dainippon/Takeda), L750355 (Merck), CP331648 WO9319749, WO9319751, WO9325517, WO9402465, (Pfizer), CL-316,243, SB 418790, BRL-37344, L-796568, WO9406423, WO9412461, WO9420455, WO9422852, BMS-196085, BRL-35135A, CGP12177A, BTA-243, GW WO9425437, WO9427947, WO9500,516, WO950.1980, 427353, Trecadrine, Zeneca D7114, N-5984 (Nisshin WO9503794, WO9504045, WO9504046, WO9505386, Kyorin), LY-377604 (Lilly), SR 59119A, and those disclosed WO9508534, WO9509623, WO9509624, WO9509627, in U.S. Pat. No. 5,541,204, U.S. Pat. No. 5,770,615, U.S. Pat. WO9509836, WO9514667, WO9514680, WO9514681, No. 5,491,134, U.S. Pat. No. 5,776,983, U.S. Pat. No. 488, WO9517392, WO9517399, WO9519362, WO9522520, O64, U.S. Pat. No. 5,705,515, U.S. Pat. No. 5,451,677, WO9524381, WO9527692, WO9528926, WO9535281, WO94/18161, WO95/2915.9, WO97/.46556, WO98/04526 WO9535282, WO9600218, WO9601825, WO9602541, and WO98/32753, WO01/74782, WO02/32897, WO03/ WO961 1917, DE3142982, DE1 116676, DE2162096, 014113, WO03/016276, WO03/016307, WO03/024948, EP0293063, EPO463756, EPO482208, EP0579496, WO03/024953 and WO03/037881; noradrenergic agents EP0667345 U.S. Pat. No. 6,331,543, US20050004222 (in including, but not limited to, diethylpropion (Such as Tenu cluding those disclosed in formulas I-XIII and paragraphs ate R (1-propanone, 2-(diethylamino)-1-phenyl-, hydrochlo 37-39, 85-0545 and 557-577), WO9307124, EP0163965, ride), Merrell), dextroamphetamine (also known as dextro EPO393500, EP0510562, EP0553174, WO9501338 and amphetamine Sulfate, dexamphetamine, dexedrine, WO9603399, as well as PDE5 inhibitors (such as RX-RA-69, Dexampex, Ferndex, Oxydess II, Robese, Spancap #1), SCH-51866, KT-734, vesnarinone, Zaprinast, SKF-96231, mazindol (or 5-(p-chlorophenyl)-2,5-dihydro-3H-imidazo ER-21355, BF/GP-385, NM-702 and sildenafil (ViagraTM)), 2,1-aisoindol-5-01) such as Sanorex R, Novartis or Maza PDE4 inhibitors (such as etazolate, IC163197, RP73401, nor R, Wyeth Ayerst), phenylpropanolamine (or Benzen imazolidinone (RO-20-1724), MEM 1414 (R1533/R1500; emethanol, alpha-(1-aminoethyl)-, hydrochloride), Pharmacia Roche), denbufylline, rolipram, oxagrelate, phentermine (or Phenol, 3-4,5-duhydro-1H-imidazol-2- nitraquaZone, Y-590, DH-6471, SKF-94120, motapizone, yl)ethyl(4-methylpheny-1)amino, monohydrochloride) lixazinone, indolidan, olprinone, atizoram, KS-506-G, such as Adipex-PR), Lemmon, FASTINR, Smith-Kline Bee dipamfylline, BMY-43351, atizoram, arofylline, filaminast, cham and Ionamin(R), Medeva), phendimetrazine ((or (2S, PDB-093, UCB-29646, CDP-840, SKF-107806, piclamilast, 3S)-3,4-Dimethyl-2phenylmorpholine L-(+)-tartrate (1:1)) RS-17597, RS-25344-000, SB-207499, TIBENELAST, such as Metra(R) (Forest), Plegine(R) (Wyeth-Ayerst), Prelu-2(R) SB-210667, SB-211572, SB-211600, SB-212066, (Boehringer Ingelheim), and StatobeXR (Lemmon), phen SB-212179, GW-3600, CDP-840, mopidamol, anagrelide, damine tartrate (such as ThephorinR (2.3.4.9-Tetrahydro-2- ibudilast, amrinone, pimobendan, cilostaZol, quaZinone and methyl-9-phenyl-1H-indenol.2.1-cpyridine L-(+)-tartrate N-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluo (1:1)), Hoffmann-LaRoche), methamphetamine (such as romethoxybenzamide, PDE3 inhibitors (such as IC 1153, DesoxynR), Abbot (S)-N, (alpha)-dimethylbenzeneetha 100, bemorandane (RWJ 22867), MCI-154, UD-CG 212, namine hydrochloride)), and phendimetrazine tartrate (such Sulmazole, ampiZone, cilostamide, carbaZeran, piroXimone, as Bontril.R Slow-Release Capsules, Amarin (-3,4-Dim imaZodan, CI-930, SiguaZodan, adibendan, Saterinone, SKF ethyl-2-phenylmorpholine Tartrate); fatty acid oxidation 95654, SDZ-MKS-492, 349-U-85, emoradan, EMD-53998, upregulator/inducers such as Famoxin R (Genset); monamine EMD-57033, NSP-306, NSP-307, revizinone, NM-702, oxidase inhibitors including but not limited to befloxatone, WIN-62582 and WIN-63291, enoximone and milrinone, moclobemide, brofaromine, phenoxathine, esuprone, befol. PDE3/4 inhibitors (such as benafentrine, trequinsin, ORG toloxatone, pirlindol, amiflamine, Sercloremine, bazinaprine, 3.0029, Zardaverine, L-686398, SDZ-ISQ-844, ORG-20241, lazabemide, millacemide, caroxazone and other certain com EMD-54622, and tolafentrine) and other PDE inhibitors pounds as disclosed by WO01/12176; and other anti-obesity (such as Vinpocetin, papaverine, enprofylline, cilomilast, agents such as 5HT-2 agonists, ACC (acetyl-CoA carboxy fenoximone, pentoxifylline, roflumilast, tadalafil (Cialis(R), lase) inhibitors such as those described in WO03/072197, theophylline, and Vardenafil (Levitra(R); Neuropeptide Y2 alpha-lipoic acid (alpha-LA), AOD9604, appetite suppres US 2016/0235807 A1 Aug. 18, 2016 44 sants such as those in WO03/40107, ATL-962 (Alizyme NF-kB activation described herein can be used in therapeutic PLC), benzocaine, benzphetamine hydrochloride (Didrex), combination with one or more anti-diabetic agents, including bladderwrack (focus vesiculosus), BRS3 (bombesin receptor but not limited to: PPARY agonists such as glitazones (e.g., Subtype 3) agonists, bupropion, caffeine, CCK agonists, chi WAY-120,744, AD 5075, balaglitazone, ciglitazone, dargli tosan, chromium, conjugated linoleic acid, corticotropin-re tazone (CP-86325, Pfizer), englitazone (CP-68722, Pfizer), leasing hormone agonists, dehydroepiandrosterone, DGAT1 isaglitazone (MIT/J&J), MCC-555 (Mitsibishi disclosed in (diacylglycerol acyltransferase 1) inhibitors, DGAT2 (dia U.S. Pat. No. 5,594,016), pioglitazone (such as such as cylglycerol acyltransferase 2) inhibitors, dicarboxylate trans ActosTM pioglitazone: Takeda), rosiglitazone (AvandiaTM: porter inhibitors, ephedra, exendin-4 (an inhibitor of glp-1) FAS (fatty acid synthase) inhibitors (such as Cerulenin and Smith Kline Beecham), rosiglitazone maleate, troglitazone C75), fat resorption inhibitors (such as those in WO03/ (Rezulin(R), disclosed in U.S. Pat. No. 4,572,912), rivoglita 053451, and the like), fatty acid transporter inhibitors, natural Zone (CS-O1 1, Sankyo), GL-262570 (Glaxo Welcome), water Soluble fibers (such as psyllium, plantago, guar, oat, BRL49653 (disclosed in WO98/05331), CLX-0921, pectin), galanin antagonists, galega (Goat's Rue, French 5-BTZD, GW-0207, LG-100641, JJT-501 (JPNT/P&U), Lilac), garcinia cambogia, germander (teucrium L-895.645 (Merck), R-1 19702 (Sankyo/Pfizer), NN-2344 chamaedrys), ghrelin antibodies and ghrelin antagonists (Dr. Reddy/NN), YM-440 (Yamanouchi), LY-300512, (such as those disclosed in WO01/87335, and WO02/08250), LY-519818, R483 (Roche), T131 (Tularik), and the like and polypeptide hormones and variants thereof which affect the compounds disclosed in U.S. Pat. No. 4,687,777, U.S. Pat. islet cell secretion, such as the hormones of the secretin/ No. 5,002,953, U.S. Pat. No. 5,741,803, U.S. Pat. No. 5,965, gastric inhibitory polypeptide (GIP)/vasoactive intestinal 584, U.S. Pat. No. 6,150,383, U.S. Pat. No. 6,150,384, U.S. polypeptide (VIP)/pituitary adenylate cyclase activating Pat. No. 6,166,042, U.S. Pat. No. 6,166,043, U.S. Pat. No. polypeptide (PACAP)/glucagon-like polypeptide II (GLP 6,172,090, U.S. Pat. No. 6,211,205, U.S. Pat. No. 6,271,243, II)/glicentin/glucagon gene family and/or those of the U.S. Pat. No. 6,288,095, U.S. Pat. No. 6,303,640, U.S. Pat. adrenomedullin/amylin/calcitonin gene related polypeptide No. 6,329.404, U.S. Pat. No. 5,994,554, WO97/10813, (CGRP) gene family including GLP-1 (glucagon-like WO97/27857, WO97/28115, WO97/28137, WO97/27847, polypeptide 1) agonists (e.g. (1) exendin-4, (2) those GLP-I WO00/76488, WO03/000685, WO03/027112, WO03/ molecules described in US20050130891 including GLP-1 (7- 035602, WO03/048130, WO03/055867, and pharmaceuti 34), GLP-1 (7-35), GLP-1 (7-36) or GLP-1 (7-37) in its C-ter cally acceptable salts thereof; biguanides such as metformin minally carboxylated or amidated form or as modified GLP-I hydrochloride (N,N-dimethylimidodicarbonimidic diamide polypeptides and modifications thereof including those hydrochloride, such as GlucophageTM, Bristol-Myers described in paragraphs 17-44 of US20050130891, and Squibb); metformin hydrochloride with glyburide, such as derivatives derived from GLP-1-(7-34)COOH and the corre GlucovanceTM, Bristol-Myers Squibb); buformin (Imidodi sponding acid amide are employed which have the following carbonimidic diamide, N-butyl-): etoformine (1-Butyl-2-eth general formula: R NH-HAEGTFTSDVSYLEGQAAKE ylbiguanide, Schering A. G.); other metformin salt forms FIAWLVK-CONH wherein R=H or an organic compound (including where the salt is chosen from the group of acetate. having from 1 to 10 carbonatoms. Preferably, R is the residue benzoate, citrate, ftimarate, embonate, chlorophenoxyac of a carboxylic acid. Particularly preferred are the following etate, glycolate, palmoate, aspartate, methanesulphonate. carboxylic acid residues: formyl, acetyl, propionyl, isopro maleate, parachlorophenoxyisobutyrate, formate, lactate, pionyl, methyl, ethyl, propyl, isopropyl. n-butyl, sec-butyl, Succinate, Sulphate, tartrate, cyclohexanecarboxylate, hex tert-butyl.) and glp-1 (glucagon-like polypeptide-1), gluco anoate, octanoate, decanoate, hexadecanoate, octodecanoate, corticoid antagonists, glucose transporter inhibitors, growth benzenesulphonate, trimethoxybenzoate, paratoluene hormone secretagogues (such as those disclosed and specifi Sulphonate, adamantanecarboxylate, glycoxylate, glutamate, cally described in U.S. Pat. No. 5,536,716), interleukin-6 pyrrolidonecarboxylate, naphthalenesulphonate, 1-glucose (IL-6) and modulators thereof (as in WO03/057237, and the phosphate, nitrate, Sulphite, dithionate and phosphate), and like), L-carnitine, Mc3r (melanocortin 3 receptor) agonists, phenformin; protein tyrosine phosphatase-IB (PTP-IB) MCH2R (melanin concentrating hormone 2R) agonist/an inhibitors, such as A-401,674, KR 61639, OC-060062, tagonists, melanin concentrating hormone antagonists, mel OC-83839, OC-297962, MC52445, MC52453, ISIS 113715, anocortinagonists (such as Melanotan II or those described in and those disclosed in WO99/585521, WO99/58518, WO99/ WO99/64002 and WO00/74679), nomame herba, phosphate 58522, WO99/61435, WO03/032916, WO03/032982, transporter inhibitors, phytopharm compound 57 (CP 644, WO03/041729, WO03/055883, WO02/26707, WO02/ 673), pyruvate, SCD-I (stearoyl-CoA desaturase-1) inhibi 26743, JP2002114768, and pharmaceutically acceptable tors, T71 (Tularik, Inc., Boulder Colo.), Topiramate (Topi salts and esters thereof, sulfonylureas such as acetohexamide max R, indicated as an anti-convulsant which has been shown (e.g. Dymelor, Eli Lilly), carbutamide, chlorpropamide (e.g. to increase weight loss), transcription factor modulators (such Diabinese(R, Pfizer), gliamilide (Pfizer), gliclazide (e.g. as those disclosed in WO03/026576), B-hydroxy steroid Diamcron, Servier Canada Inc), glimepiride (e.g. disclosed in dehydrogenase-1 inhibitors (B-HSD-I), B-hydroxy-B-meth U.S. Pat. No. 4.379,785, such as Amaryl, Aventis), glipentide, glipizide (e.g. Glucotrol or Glucotrol XL Extended Release, ylbutyrate, p57 (Pfizer), Zonisamide (ZonegranTM, indicated Pfizer), gliquidone, glisolamide, glyburide/glibenclamide as an anti-epileptic which has been shown to lead to weight (e.g. Micronase or Glynase Prestab, Pharmacia & Upjohn and loss), and the agents disclosed in US200301 19428 para Diabeta, Aventis), tolazamide (e.g. Tolinase), and tolbuta graphs 20-26. mide (e.g. Orinase), and pharmaceutically acceptable salts Anti-Diabetic Agents and esters thereof meglitinides such as repaglinide (e.g. Pranidin R, Novo Nordisk), KAD1229 (PF/Kissei), and 0256 Patients with diabetic nephropathy may have higher nateglinide (e.g. Starlix R, Novartis), and pharmaceutically NF-kB activity. Thus, to the GCRA peptides for inhibiting acceptable salts and esters thereof a glucoside hydrolase US 2016/0235807 A1 Aug. 18, 2016

inhibitors (or glucoside inhibitors) such as acarbose (e.g. esters thereof; other insulin sensitizing drugs; VPAC2 recep PrecoseTM, Bayer disclosed in U.S. Pat. No. 4,904.769), tor agonists; GLK modulators, such as those disclosed in miglitol (such as GLYSETTM, Pharmacia & Upjohn disclosed WO03/015774; retinoid modulators such as those disclosed in U.S. Pat. No. 4,639.436), camiglibose (Methyl 6-deoxy-6- in WO03/000249; GSK313/GSK3 inhibitors such as 4-2- (2R.3R.4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperi (2-bromophenyl)-4-(4-fluorophenyl-1H-imidazol-5-ylpyri dino-alpha-D-glucopyranoside, Marion Merrell Dow), dine and those compounds disclosed in WO03/024447, Voglibose (Takeda), adiposine, emiglitate, pradimicin-Q, sal WO03/037869, WO03/037877, WO03/037891, WO03/ bostatin, CKD-711, MDL-25,637, MDL-73,945, and MOR 068773, EP1295.884, EP1295.885, and the like: glycogen phosphorylase (HGLPa) inhibitors such as CP-368,296, 14, and the compounds disclosed in U.S. Pat. No. 4,062,950, CP-316,819, BAYR3401, and compounds disclosed in U.S. Pat. No. 4,174,439, U.S. Pat. No. 4,254.256, U.S. Pat. WO01/94300, WO02/20530, WO03/037864, and pharma No. 4,701,559, U.S. Pat. No. 4,639,436, U.S. Pat. No. 5,192, ceutically acceptable salts or esters thereof; ATP consump 772, U.S. Pat. No. 4,634,765, U.S. Pat. No. 5,157,116, U.S. tion promotors such as those disclosed in WO03/007990; Pat. No. 5,504,078, U.S. Pat. No. 5,091,418, U.S. Pat. No. TRB3 inhibitors; vanilloid receptor ligands such as those 5,217.877 and WO01/47528 (polyamines); C-amylase disclosed in WO03/049702; hypoglycemic agents such as inhibitors such as tendamistat, trestatin, and A1-3688, and the those disclosed in WO03/015781 and WO03/040114; glyco compounds disclosed in U.S. Pat. No. 4.451,455, U.S. Pat. gen synthase kinase 3 inhibitors such as those disclosed in No. 4,623,714, and U.S. Pat. No. 4,273,765: SGLT2 inhibi WO03/035663 agents such as those disclosed in WO99/ tors including those disclosed in U.S. Pat. No. 6,414,126 and 51225, US2003.0134890, WO01/24786, and WO03/059870; U.S. Pat. No. 6,515,117; an aP2 inhibitor such as disclosed in insulin-responsive DNA binding protein-1 (IRDBP-I) as dis U.S. Pat. No. 6,548,529; insulin secreatagogues such as closed in WO03/057827, and the like; adenosine A2 antago linogliride, A-4166, forskilin, dibutyrl cAMP isobutylmeth nists such as those disclosed in WO03/035639, WO03/ ylxanthine (IBMX), and pharmaceutically acceptable salts 035640, and the like: PPARö agonists such as GW 501516, and esters thereof, fatty acid oxidation inhibitors, such as GW 590735, and compounds disclosed in JP10237049 and clomoxir, and etomoxir, and pharmaceutically acceptable WO02/14291; dipeptidyl peptidase IV (DP-IV) inhibitors, salts and esters thereof; A2 antagonists, such as midaglizole, such as isoleucine thiazolidide, NVP-DPP728A (1-2-(5- isaglidole, deriglidole, idazoxan, earoxan, and fluparoxan, cyanopyridin-2-yl)aminoethylaminoacetyl-2-cyano-(S)- and pharmaceutically acceptable salts and esters thereof. pyrrolidine, disclosed by Hughes etal, Biochemistry, 38(36), insulin and related compounds (e.g. insulin mimetics) such as 11597-11603, 1999), P32/98, NVP-LAF-237, P3298, biota, LP-100, novarapid, insulin detemir, insulin lispro, insu TSL225 (tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-car lin glargine, insulin Zinc suspension (lente and ultralente), boxylic acid, disclosed by Yamada et al. Bioorg. & Med. Lys-Pro insulin, GLP-I (1-36) amide, GLP-I (73-7) (insulin Chem. Lett. 8 (1998) 1537-1540), valine pyrrolidide, TMC tropin, disclosed in U.S. Pat. No. 5,614,492), LY-315902 2A/2B/2C, CD-26 inhibitors, FE999011, P9310/K364, VIP (Lilly), GLP-I (7-36)-NH2), AL-401 (Autoimmune), certain 0.177, DPP4, SDZ 274-444, 2-cyanopyrrolidides and 4-cyan compositions as disclosed in U.S. Pat. No. 4.579.730, U.S. opyrrolidides as disclosed by Ashworth etal, Bioorg. & Med. Pat. No. 4,849,405, U.S. Pat. No. 4,963,526, U.S. Pat. No. Chem. Lett. Vol. 6, No. 22, pp 1163-1166 and 2745-2748 5,642,868, U.S. Pat. No. 5,763,396, U.S. Pat. No. 5,824,638, (1996), and the compounds disclosed in U.S. Pat. No. 6,395, U.S. Pat. No. 5,843.866, U.S. Pat. No. 6,153,632, U.S. Pat. 767, U.S. Pat. No. 6,573.287, U.S. Pat. No. 6,395,767 (com No. 6,191,105, and WO 85/05029, and primate, rodent, or pounds disclosed include BMS-477118, BMS-471211 and rabbit insulin including biologically active variants thereof BMS 538,305), WO99/38501, WO99/46272, WO99/67279, including allelic variants, more preferably human insulin WO99/67278, WO99/61431WO03/004498, WO03/004496, available in recombinant form (sources of human insulin EP1258476, WO02/083128, WO02/062764, WO03/000250, include pharmaceutically acceptable and sterile formulations WO03/002530, WO03/002531, WO03/002553, WO03/ such as those available from Eli Lilly (Indianapolis, Ind. 002593, WO03/000180, and WO03/000181; GLP-I agonists 46285) as HumulinTM (human insulin rDNA origin), also see Such as exendin-3 and exendin-4 (including the 39 aa the THE PHYSICIAN'S DESK REFERENCE,55.sup...th Ed. polypeptide synthetic exendin-4 called Exenatide(R), and (2001) Medical Economics, Thomson Healthcare (disclosing compounds disclosed in US2003087821 and NZ504256, and other Suitable human insulins); non-thiazolidinediones Such pharmaceutically acceptable salts and esters thereof; peptides as JT-501 and fargilitazar (GW-2570/GI-262579), and phar including amlintide and Symlin R (pramlintide acetate); and maceutically acceptable salts and esters thereof; PPARC/y glycokinase activators such as those disclosed in dual agonists such as AR-HO39242 (AztraZeneca), US2002103199 (fused heteroaromatic compounds) and GW-409544 (Glaxo-Wellcome), BVT-142, CLX-0940, WO02/48106 (isoindolin-1-one-substituted propionamide GW-1536, GW-1929, GW-2433, KRP-297 (Kyorin Merck; compounds). 5-(2,4-Dioxo thiazolidinyl)methylmethoxy-N-(4-(trifluo romethyl)phenylmethylbenzamide), L-796449, LR-90, MK-0767 (Merck/Kyorin/Banyu), SB 219994, muraglitazar Phosphodiesterase Inhibitors (BMS), tesaglitzar (AstraZeneca), reglitazar (JTT-501) and (0257. The GCRA peptides described herein can be used in those disclosed in WO99/16758, WO99/19313, WO99/ combination therapy with a phosphodiesterase inhibitor. PDE 20614, WO99/38850, WO00/23415, WO00/23417, WO00/ inhibitors are those compounds which slow the degradation 23445, WO00/50414, WO01/00579, WO01/79150, WO02/ of cyclic AMP (cAMP) and/or cyclic GMP (cGMP) by inhi 062799, WO03/004.458, WO03/016265, WO03/018010, bition of the phosphodiesterases, which can lead to a relative WO03/033481, WO03/033450, WO03/033453, WO03/ increase in the intracellular concentration of cAMP and/or 043985, WO 031053976, U.S. application Ser. No. 09/664, cGMP Possible PDE inhibitors are primarily those sub 598, filed Sep. 18, 2000, Murakami et al. Diabetes 47, 1841 stances which are to be numbered among the class consisting 1847 (1998), and pharmaceutically acceptable salts and of the PDE3 inhibitors, the class consisting of the PDE4 US 2016/0235807 A1 Aug. 18, 2016 46 inhibitors and/or the class consisting of the PDE5 inhibitors, MILRINONE. PDE3/4 inhibitors which may be mentioned in particular those Substances which can be designated as by way of example are BENAFENTRINE, TREQUINSIN, mixed types of PDE3/4 inhibitors or as mixed types of PDE3/ ORG-3.0029, ZARDAVERINE, L-686398, SDZ-ISQ-844, 4/5 inhibitors. By way of example, those PDE inhibitors may ORG-20241, EMD-54622, and TOLAFENTRINE. Other be mentioned such as are described and/or claimed in the PDE inhibitors include: cilomilast, pentoxifylline, roflumi following patent applications and patents: DE 1470341, last, tadalafil (Cialis(R), theophylline, and Vardenafil (Lev DE2108438, DE2123328, DE2305339, DE2305575, itra(R), Zaprinast (PDE5 specific). DE2315801, DE2402908, DE2413935, DE2451417, DE2459090, DE2646469, DE2727481, DE2825048, Anti-Uterine Contractions Agents DE2837161, DE2845220, DE2847621, DE2934747, 0258. The GCRA peptides described herein can be used in DE3021792, DE3038166, DE3044568, EP000718, combination therapy (for example, in order to decrease or EP0008.408, EP0010759, EP0059948, EP0075436, inhibit uterine contractions) with a tocolytic agent including EP00965.17, EPO1 12987, EPO1 16948, EPO150937, but not limited to beta-adrenergic agents, magnesium sulfate, EP0158380, EPO161632, EPO161918, EP0167121, prostaglandin inhibitors, and calcium channel blockers. EP0199.127, EP0220044, EP0247725, EP0258191, EP0272910, EP0272914, EP0294647, EPO300726, Anti-Neoplastic Agents EPO335386, EPO357788, EPO38.9282, EPO406958, EPO426180, EPO428302, EPO435811, EPO470805, (0259. The GCRA peptides described herein can be used in EPO482208, EPO490823, EP0506194, EP0511865, combination therapy with an antineoplastic agents including EP0527117, EP0626939, EP0664289, EP0671389, but not limited to alkylating agents, epipodophyllotoxins, EP0685474, EP0685475, EP0685479, JP92234389, nitrosoureas, antimetabolites, Vinca alkaloids, anthracycline JP94329652, JP950.10875, U.S. Pat. Nos. 4,963,561, 5,141, antibiotics, nitrogen mustard agents, and the like. Particular 931, WO9117991, WO9200968, WO9212961, WO9307146, anti-neoplastic agents may include tamoxifen, taxol, etopo WO9315044, WO9315045, WO9318024, WO9319068, side and 5-fluorouracil. WO9319720, WO931.9747, WO9319749, WO9319751, 0260 The GCRA peptides described herein can be used in WO9325517, WO9402465, WO9406423, WO9412461, combination therapy (for example as in a chemotherapeutic WO9420455, WO9422852, WO9425437, WO9427947, composition) with an antiviral and monoclonal antibody WO9500516, WO950.1980, WO9503794, WO9504045, therapies. WO9504046, WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836, WO9514667, Agents to Treat Congestive Heart Failure WO9514680, WO9514681, WO9517392, WO9517399, WO9519362, WO9522520, WO9524381, WO9527692, 0261 The GCRA peptides described herein can be used in WO9528926, WO953.5281, WO9535282, WO9600218, combination therapy (for example, in prevention/treatment of WO9601825, WO9602541, WO961 1917, DE3142982, DE1 congestive heart failure or another method described herein) 116676, DE2162096, EP0293063, EPO463756, with the partial agonist of the nociceptin receptor ORL1 EPO482208, EP0579496, EP0667345 U.S. Pat. No. 6,331, described by Dooley et al. (The Journal of Pharmacology and 543, US20050004222 (including those disclosed in formulas Experimental Therapeutics, 283 (2): 735-741, 1997). The I-XIII and paragraphs 37-39, 85-0545 and 557-577) and agonist is a hexapeptide having the amino acid sequence WO9307124, EPO163965, EPO393500, EP0510562, Ac-RYY (RK) (WI) (RK)-NH2 (“the Dooley polypeptide'), EP0553174, WO950.1338 and WO9603399. PDE5 inhibitors where the brackets show allowable variation of amino acid which may be mentioned by way of example are RX-RA-69, residue. Thus Dooley polypeptide can include but are not SCH-51866, KT-734, vesnarinone, Zaprinast, SKF-96231, limited to KYYRWR, RYYRWR, KWRYYR, RYYRWK, ER-21355, BF/GP-385, NM-702 and sildenafil (Viagra(R). RYYRWK (all-D amin acids), RYYRIK, RYYRIR, PDE4 inhibitors which may be mentioned by way of example RYYKIK, RYYKIR, RYYKWR, RYYKWK, RYYRWR, are RO-20-1724, MEM 1414 (R1533/R1500; Pharmacia RYYRWK, RYYRIK, RYYKWR, RYYKWK, RYYRWK Roche), DENBUFYLLINE, ROLIPRAM, OXAGRELATE, and KYYRWK, wherein the amino acid residues are in the NITRAQUAZONE, Y-590, DH-6471, SKF-94120, MOT L-form unless otherwise specified. The GCRA peptides APIZONE, LIXAZINONE, INDOLIDAN, OLPRINONE, described hereincan also be used in combination therapy with ATIZORAM, KS-506-G, DIPAMFYLLINE, BMY-433.51, polypeptide conjugate modifications of the Dooley polypep ATIZORAM, AROFYLLINE, FILAMINAST, PDB-093, tide described in WOO198324. UCB-29646, CDP-840, SKF-107806, PICLAMILAST, RS-17597, RS-25344-000, SB-207499, TIBENELAST, Fibrate SB-210667, SB-211572, SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, MOPIDAMOL, 0262 The GCRA peptides described herein can be used in ANAGRELIDE, IBUDILAST, AMRINONE, PIMOBEN combination therapy with a fibrate. The term “fibrate' is also DAN, CILOSTAZOL, QUAZINONE and N-(3,5-dichloro interchangeably used herein and in the art with the term pyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxyben “fibric acid derivative.” and means any of the fibric acid Zamide. PDE3 inhibitors which may be mentioned by way of derivatives useful in the methods described herein, e.g., example are SULMAZOLE, AMPIZONE, CILOSTAMIDE, fenofibrate. Fenofibrate is a fibrate compound, other CARBAZERAN, PIROXIMONE, IMAZODAN, CI-930, examples of which include, for example, bezafibrate, beclofi SIGUAZODAN, ADIBENDAN, SATERINONE, SKF brate, benzafibrate, binifibrate, ciprofibrate, clinofibrate, 95654, SDZ-MKS-492, 349-U-85, EMORADAN, EMD clofibrate, etofibrate, gemcabene, gemfibrozil, lifibrol, 53998, EMD-57033, NSP-306, NSP-307, REVIZINONE, nicofibrate, pirifibrate, ronifibrate, simfibrate, theofibrate, NM-702, WIN-62582 and WIN-63291, ENOXIMONE and etc. US 2016/0235807 A1 Aug. 18, 2016 47

Lipid Altering Agents US534772), glenvastatin, fluindostatin (disclosed in 0263 NF-KB has a coordinating role in inflammation and EP363934A1), velostatin (visinolin; disclosed in U.S. Pat. cellular proliferation and may be involved in early atheroscle No. 4,448,784 and U.S. Pat. No. 4,450,171), lovastatin (mevi rosis. The GCRA peptides for inhibiting NF-kB activation nolin; MEVACORR (Merck and Co.) and related compounds described herein can be used in combination therapy with a disclosed in U.S. Pat. No. 4,231.938), mevastatin (and related lipid altering agent. As used herein the term “lipid altering compound disclosed in U.S. Pat. No. 3,983,140), compactin agent” or "dyslipidemia agent” refers to compounds includ (and related compounds disclosed in U.S. Pat. No. 4,804, ing, but not limited to, bile acid sequestrants such as 770), pravastatin (also known as NK-104, itavastatin, nisvas cholestyramine (a styrene-divinylbenzene copolymer con tatin, nisbastatin disclosed in U.S. Pat. No. 5,102.888), prav taining quaternary ammonium cationic groups capable of astatin (PRAVACHOL(R) (Bristol Myers Squibb) and related binding bile acids, such as QUESTRANR) or QUESTRAN compounds disclosed in U.S. Pat. No. 4.346.227), rivastatin LIGHTR) cholestyramine which are available from Bristol (sodium 7-(4-fluorophenyl)-2,6-diisopropyl-5-methoxym Myers Squibb), colesevelam hydrochloride (such as ethylpyridin-3-yl)-3,5-dihydroxy-6-heptanoate), rosuvasta WELCHOL(R) Tablets (polyallylamine hydrochloride) cross tin (CRESTORR); also known as ZD-4522 disclosed in U.S. linked with epichlorohydrin and alkylated with 1-bromode Pat. No. 5,260.440), atavastatin, visastatin, simvastatin (ZO cane and (6-bromohexyl)-trimethylammonium bromide) CORR (Merck and Co.) and related compounds as disclosed which are available from Sankyo), colestipol (a copolymer of in U.S. Pat. No. 4,448,784 and U.S. Pat. No. 4.450,171), diethylenetriamine and 1-chloro-2,3-epoxypropane, such as simvastatin, CI-981, compounds disclosed in WO03/033481, COLESTIDR tablets which are available from Pharmacia), U.S. Pat. No. 4,231,938, U.S. Pat. No. 4,444,784, U.S. Pat. dialkylaminoalkyl derivatives of a cross-linked dextran, No. 4,647,576, U.S. Pat. No. 4,686,237, U.S. Pat. No. 4,499, LOCHOLESTR), DEAE-Sephadex (SECHOLEXR, 289, U.S. Pat. No. 4,346,227, U.S. Pat. No. 5,753,675, U.S. POLICEXIDE(R), water soluble derivatives such as 3.3- Pat. No. 4,613,610, EP0221025, and EP4.91226, and optical ioene, N-(cycloalkyl)alkylamines and poliglusam, insoluble or geometric isomers thereof, and nontoxic pharmaceutically quaternized polystyrenes, Saponins and mixtures thereof and acceptable salts, N-oxides, esters, quaternary ammonium salts, and prodrugs thereof. In HMG-CoA reductase inhibi those bile acid sequestrants disclosed in WO97/11345, tors where an open-acid form can exist, salt and ester forms WO98/57652, U.S. Pat. No. 3,692,895, and U.S. Pat. No. may preferably be formed from the open-acid, and all such 5,703,188. Suitable inorganic cholesterol sequestrants forms are included within the meaning of the term “HMG include bismuth Salicylate plus montmorillonite clay, alumi CoA reductase inhibitor” as used herein. Pharmaceutically num hydroxide and calcium carbonate antacids. acceptable salts with respect to the HMG-CoA reductase inhibitor includes non-toxic salts of the compounds which are HMG-CoA Reductase Inhibitors generally prepared by reacting the free acid with a suitable 0264. The 3-hydroxy-3-methylglutaryl coenzyme A organic or inorganic base, particularly those formed from (HMGCoA) reductase inhibitors, commonly referred to as cations such as sodium, potassium, aluminum, calcium, statins, are potent inhibitors of cholesterol biosynthesis and lithium, magnesium, Zinc and tetramethylammonium, as well widely prescribed for the treatment of hypercholesterolemia. as those salts formed from amines Such as ammonia, ethyl For example, Pitavastatin is highly potent inhibitor of HMG enediamine, N-methylglucamine, lysine, arginine, ornithine, CoA reductase, the rate-limiting enzyme in cholesterol bio choline, N,N'-dibenzylethylenediamine, chloroprocaine, synthesis. Pitavastatin shows protective action on vascular diethanolamine, procaine, N-benzylphenethylamine, 1-p- endothelial cells (ECs) by inducing the activation of eNOS, chlorobenzyl-2-pyrrolidine-1-yl-methylbenzim-idazole, thereby protecting the vascular ECs from injury due to the diethylamine, piperazine, and tris(hydroxymethyl) ami inflammatory reaction induced by TNF-C., and increasing NO nomethane. Further examples of salt forms of HMG-CoA production, which is dependent on post-transcriptional regu reductase inhibitors may include, but are not limited to, lation, and involves phosphoinositide 3-kinase and the Akt acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, pathway. bitartrate, borate, bromide, calcium edetate, camsylate, car bonate, chloride, clavulanate, citrate, dihydrochloride, ede Pitavastatin Also Inhibits NF-kB Activation and IL-6 Protein tate, edisylate, estolate, esylate, fumarate, gluceptate, glucon Production Induced by TNF-C. in Hepatocarcinoma Cells. ate, glutamate, glycolylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydrox 0265. The GCRA peptides for inhibiting NF-kB activa tion described herein can be used in combination therapy with ynapthoate, iodide, isothionate, lactate, lactobionate, laurate, a HMG-CoA reductase inhibitor. HMG-CoA reductase malate, maleate, mandelate, mesylate, methylsulfate, mucate, inhibitors are dyslipidemic agents that can be used in thera napsylate, nitrate, oleate, oxalate, pamaote, palmitate, pan peutic combinations with compounds described herein. Suit tothenate, phosphate/diphosphate, polygalacturonate, salicy able HMG-CoA reductase inhibitors for use in therapeutic late, Stearate, Subacetate, Succinate, tannate, tartrate, teoclate, combination with a compounds described herein include: tosylate, triethiodide, and Valerate. atorvastatin (LIPITORR); disclosed in U.S. Pat. No. 4,681, 0266 Other dyslipidemic agents which can be used in 893, U.S. Pat. No. 5,385,929 and U.S. Pat. No. 5,686,104), therapeutic combination with a compound described herein atorvastatin calcium (disclosed in U.S. Pat. No. 5.273.995), include: HMG-CoA synthase inhibitors such as L-659,699 dihydrocompactin, (disclosed in U.S. Pat. No. 4.450,171), ((E E)-I 1-3'R-(hydroxymethyl)-4'-oxo-2R-oxetanyl-3,5, bervastatin (disclosed in U.S. Pat. No. 5,082,859), carvasta 7R-trimethyl-2,4-undecadienoic acid) and those disclosed in tin, cerivastatin (BAYCOLOR.; disclosed in U.S. Pat. No. U.S. Pat. No. 5,120,729, U.S. Pat. No. 5,064,856, and U.S. 5,006,530, U.S. Pat. No. 5,502,199, and US5 177080), crilv Pat. No. 4,847,271; cholesterol absorption inhibitors such as astatin, dalvastatin (disclosed in EP738510A2), fluvastatin plant Sterols, plant stanols and/or fatty acid estesrs of plant (LESCOL(R); disclosed in U.S. Pat. No. 4,739,073 and stanols such as sitostanol ester used in BENECOLOR marga US 2016/0235807 A1 Aug. 18, 2016 48 rine, stanol esters, beta-sitosterol, and sterol glycosides Such fibrozil (such as 5-(2,5-dimethylphenoxy)-2,2-dimethylpen as tiqueside. Other cholesterol absorption inhibitors include tanoic acid, e.g. LopidR tablets (Parke Davis)), lifibrol, GW 1,4-Diphenylazetidin-2-ones: 4-biarylyl-1-phenylazetidin 7647, BM 170744, LY5 18674 and those fibrate and fibrate 2-ones: 4-(hydroxyphenyl)aZetidin-2-ones; 1,4-diphenyl-3- acid derivatives disclosed in WO03/033456, WO03/033481, hydroxyalkyl-2-aZetidinones: 4-biphenyl-1-phenylaZetidin WO03/043997, WO03/048116, WO03/053974, WO03/ 2-ones; 4-biarylyl-1-phenylaZetidin-2-ones; and 059864, and WO03/05875; FXR receptor modulators such as 4-biphenylylaZetidinones.acyl coenzyme A-cholesterol acyl GW4064, SR 103912, and the like; LXR receptor modulators transferase (ACAT) inhibitors such as avasimibe (Current such as GW 3965, T9013137, and XTCO179628, and those Opinion in Investigational Drugs. 3(9): 291-297 (2003)), eflu disclosed in US2003.0125357, WO03/045382, WO03/ cimibe, HL-004, lecimibe, DuP-128, KY505, SMP 797, 053352, WO03/059874, and the like; HM74 and HM74A CL-277,082 (Clin Pharmacol Ther. 48(2): 189-94 (1990)) (human HM74A is Genbank Accession No. AY148884 and and the like; and those disclosed in U.S. Pat. No. 5,510,379, rat HM74A is EMM patAR09 8624) receptor agonists such WO96/26948 and WO96/10559; CETP inhibitors such as as nicotinic acid (niacin) and derivatives thereof (e.g. com JTT 705 identified as in Nature 406, (6792):203-7 (2000), pounds comprising a pyridine-3-carboxylate structure or a torcetrapib (CP-529,414 described in US20030186952 and pyrazine-2-carboxylate structure, including acid forms, salts, WO00/017164), CP532,632, BAY63-2149, SC 591, SC 795, esters, Zwitterions and tautomers, where available) including and the like including those described in Current Opinion in but not limited to those disclosed in Wise etal (2003).J. Biol. Investigational Drugs. 4(3):291-297 (2003) and those dis Chem. 278: 98.69 (e.g. 5-methylpyrazole-3-carboxylic acid closed in J. Antibiot, 49(8): 815-816 (1996), and Bioorg. and acifran (4,5-dihydro-5-methyl-4-oxo-5-phenyl-2-furan Med. Chem. Lett, 6:1951-1954 (1996) and patent publica carboxylic acid pyradine-3-acetic acid)), as well as 5-methyl tions U.S. Pat. No. 5,512,548, U.S. Pat. No. 6,147,090, nicotinic acid, nicotinuric acid, niceritrol, nicofuranose, WO99/20302, WO99/14204, WO99/41237, WO95/04755, acipimoX (5-methylpyrazine-2-carboxylic acid 4-oxide), WO96/15141, WO96/05227, WO038721, EP796846, Niaspan R (niacin extended-release tablets; Kos) and those EP818197, EP818448, DE 19704244, DE1974 1051, which can be easily identified by one skilled in the art which DE19741399, DE197042437, DE 19709125, DE19627430, bind to and agonize the HM74A or HM74 receptor (for DE19832159, DE19741400, JP 11049743, and JP 09059155; example using the assays disclosed in Wise et al (2003) J. squalene synthetase inhibitors such as squalestatin-1, TAK Biol. Chem 278:9869 (nicotine binding and 35S-GTPyS 475, and those disclosed in U.S. Pat. No. 4,871,721, U.S. Pat. binding assays), Soga et al (2003) Biochem. Biophys. Res. No. 4,924,024, U.S. Pat. No. 5,712,396 (O-phosphono-sul Comm. 303:364 (radiolabel binding assay using the HM74 fonates), Biller et al (1988) J. Med. Chem., 31:1869 (e.g. receptor which could be adapted to the HM74A receptor), isoprenoid (phosphinyl-methyl)phosphonates), Biller et al Tunaru et al (2003) Nature Medicine 9:352 (calcium mobili (1996) Current Pharmaceutical Design, 2:1, P. Ortiz de Mon zation assay using the HM74 receptor which could be adapted tellano et al (1977) J. Med. Chem. 20:243 (terpenoid pyro to the HM74A receptor) and U.S. Pat. No. 6,420,183 (FLIPR phosphates), Corey and Volante (1976) J. Am. Chem. Soc, assays are described generally in and may be adapted to the 98: 1291 (farnesyl diphosphate analog A and presqualene HM74A or HM74 receptor); renin angiotensin system inhibi pyrophosphate (PSQ-PP) analogs), McClard etal (1987) J.A. tors; bile acid reabsorption inhibitors (bile acid reuptake C.S., 109:5544 (phosphinylphosphonates), Capson, T. L., inhibitors), such as BARI 1453, SC435, PHA384640,58921, PhD dissertation, June, 1987, Dept. Med. Chem. U of Utah, AZD7706, and the like: PPARö agonists (including partial Abstract, Table of Contents, pp 16, 17, 40-43, 48-51, Sum agonists) such as GW 501516, and GW 590735, and those mary, (cyclopropanes), Curr. Op. Ther. Patents (1993) 861, disclosed in U.S. Pat. No. 5,859,051 (acetophenols), WO03/ and patent publications EP0567026A1. EP0645378A1, 024395, WO97/28149, WO01/791.97, WO02/14291, WO02/ EP0645377A1, EP0611749A1, EP0705607A2, 46154, WO02/46176, WO02/076957, WO03/0 16291, EP0701725A1, and WO96/09827; antioxidants such as WO03/033493, WO99/20275 (quinoline phenyl com probucol (and related compounds disclosed in U.S. Pat. No. pounds), WO99/38845 (aryl compounds), WO00/63161 3,674,836), probucol derivatives such as AGI-1067 (and other (1,4-disubstituted phenyl compounds), WO01/00579 (aryl derivatives disclosed in U.S. Pat. No. 6,121,319 and U.S. Pat. compounds), WO01/12612 & WO01/12187 (benzoic acid No. 6,147.250), tocopherol, ascorbic acid, B-carotene, sele compounds), and WO97/31907 (substituted 4-hydroxy-phe nium and vitamins such as vitamin B6 or vitamin B12 and nylalconic acid compound); Sterol biosynthesis inhibitors pharmaceutically acceptable salts and esters thereof; PPARC. such as DMP-565; triglyceride synthesis inhibitors; microso agonists such as those disclosed in U.S. Pat. No. 6,028,109 mal triglyceride transport (MTTP) inhibitors, such as inpli (fluorophenyl compounds), WO00/75103 (substituted phe tapide, LAB687, and CP346086, AEGR733, implitapide and nylpropionic compounds), WO98/43.081 and fibric acid the like; HMG-CoA reductase gene expression inhibitors derivatives (fibrates) such as beclofibrate, benzafibrate, (e.g. compounds that decrease HMG-CoA reductase expres bezafibrate (C.A.S. Registry No. 41859-67-0, see U.S. Pat. sion by affecting (e.g. blocking) transcription or translation of No. 3,781.328), binifibrate (C.A.S. Registry No. 69047-39-8, HMG-CoA reductase into protein or compounds that maybe see BE884722), ciprofibrate (C.A.S. Registry No.52214-84 biotransformed into compounds that have the aforemen 3, see U.S. Pat. No. 3,948,973), clinofibrate (C.A.S. Registry tioned attributes by one or more enzymes in the cholesterol No. 30299-08-2, see U.S. Pat. No. 3,716,583), clofibrate biosynthetic cascade or may lead to the accumulation of an (such as ethyl 2-(p-chlorophenoxy)-2-methyl-propionate, isoprene metabolite that has the aforementioned activities e.g. Atromid-SR) capsules (Wyeth-Ayerst), etofibrate, fenofi (such regulation is readily determined by those skilled in the brate (such as Tricor R micronized fenofibrate ((2-4-(4-chlo art according to standard assays (Methods of Enzymology, robenzoyl)phenoxy-2-methyl-propanoic acid, 1-methyl 110:9-19 1985))) such as those disclosed in U.S. Pat. No. ethyl ester; Abbott Laboratories) or Lipanthyl(R) micronized 5,041,432 (certain 15-substituted lanosterol derivatives) and fenofibrate (Labortoire Founier, France)), gemcabene, gem E. I. Mercer (1993) Prog. Lip. Res. 32:357 (oxygenated ste US 2016/0235807 A1 Aug. 18, 2016 49 rols that suppress the biosynthesis of HMG-CoA reductase); will be made using methods that are routine in the art and will squalene epoxidase inhibitors such as NB-598 ((E)-N-ethyl be based upon the particular composition being used and N-(6,6-dimethyl-2-hepten-4-y-nyl)-3-(3,3'-bithiophen-5- clinical considerations. yl)methoxybenzene-methanamine hydrochloride); low den sity lipoprotein (LDL) receptor inducers such as HOE-402 0270. The guanylate cyclase receptor agonists used in the (an imidazolidinyl-pyrimidine derivative that directly stimu methods described above may be administered orally, sys lates LDL receptor activity, see Huettinger et al (1993) Arte temically or locally. Dosage forms include preparations for rioscler. Thromb. 13:1005); platelet aggregation inhibitors: inhalation or injection, Solutions, Suspensions, emulsions, 5-LO or FLAP inhibitors; PPAR modulators (including com tablets, capsules, topical salves and lotions, transdermal com pounds that may have multiple functionality for activating positions, other known peptide formulations and pegylated various combinations of PPARC, PPARy, and PPAR8) such peptide analogs. Agonists may be administered as either the as those disclosed in U.S. Pat. No. 6,008,237, U.S. Pat. No. sole active agent or in combination with other drugs, e.g., an 6,248,781, U.S. Pat. No. 6,166,049, WOOO/12491, WOOO/ inhibitor of c(GMP-dependent phosphodiesterase and anti 218355, WO00/23415, WO00/23416, WO00/23425, WO00/ inflammatory agent. In all cases, additional drugs should be 23442, WO00/23445, WO00/23451, WO00/23.6331, WO00/ administered at a dosage that is therapeutically effective 236332, WO00/238553, WO00/50392, WO00/53563, using the existing art as a guide. Drugs may be administered WO00/63153, WO00/63.190, WO00/63196, WO00/63209, in a single composition or sequentially. WO00/78312, WO00/78313, WO01/04351, WO01/14349, WO01/14350, WO01/16120, WO01/17994, WO01/21181, 0271 Dosage levels of the GCR agonist for use in methods WO01/21578, WO01/25 181, WO01/25225, WO01/25226, of this invention typically are from about 0.001 mg to about WO01/401.92, WO01/79150, WO02/081428, WO02/ 10,000 mg daily, preferably from about 0.005 mg to about 100403, WO02/102780, WO02/79162, WO03/016265, 1,000 mg daily. For example, an effective dosage of the WO03/033453, WO03/042194, WO03/043997, WO03/ GCRA peptide for use in methods of this invention is 0.1, 0.2. 066581, WO97/25042, WO99/07357, WO99/11255, WO99/ 0.3, 0.4. 0.5,0.6,0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 12534, WO99/15520, WO99/46232, and WO98/05331 (in 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, cluding GW233 1 or (2-(4-difluorophenyl-1 heptylureido) 14, 15, 16, 17, 18, 19, or 20 mg per day, or optionally twice a ethylphenoxy)-2-methylbutyric)); niacin-bound chromium, day. Preferably the GCRA peptide is given after a meal (i.e. as disclosed in WO03/039535; substituted acid derivatives 30 minutes). In some embodiments a second agent useful for disclosed in WO03/040114; apolipoprotein B inhibitors such treating a lipid metabolism disorder, a billary disorder, a as those disclosed in WO02/090347, WOO2/28835, WOO3/ cardiovascular disease, obesity or an an endocrine disorder is 045921, WO03/047575: Factor Xa modulators such as those administered. Suitable second agents are described herein. In disclosed in WO03/047517, WO03/047520, WO03/048081; Some aspects the secondagent is administered at less than the ileal bile acid transport (“IBAT) inhibitors (or apical sodium co-dependent bile acid transport (ASBT) inhibitors) such standard does for treating the particular disorder because the as benzothiepines (including 1,2-benzothiazepines; 1,4-ben GCRA peptide acts Synergistically with the secondagent. For Zodiazepines; 1.5-benzothiazepines; 1.2, 5-benzothiadiaz example, 2.5, 5. 7.5 or 10 mg of Liptor is given twice a day epines); PPARö activators such as disclosed in WO01/00603 after a meal (i.e., 30 minutes). On the basis of mg/kg daily (thiazole and oxazole derivates (e.g. C.A.S. Registry No. dose, either given in single or divided doses, dosages typically 317318-32-4), WO97/28149 (fluoro, chloro and thio phe range from about 0.001775 mg/kg to about 10,000/75 mg/kg, noxy phenylacetic), U.S. Pat. No. 5,093.365 (non-1-oxidiz preferably from about 0.005/75 mg/kg to about 1,000/75 able fatty acid analogues), and WO99/04815. Tests showing mg/kg. the efficacy of the therapy and the rationale for the combina 0272. The total daily dose of each inhibitor can be admin tion therapy with a dyslipidemic agent are presented in istered to the patient in a single dose, or in multiple Subdoses. US2003 0069221 (where the dyslipidemic agents are called Typically, Subdoses can be administered two to six times per “cardiovascular agents”) day, preferably two to four times per day, and even more 0267 Dosage preferably two to three times per day. Doses can be in imme 0268 Dosage levels of active ingredients in a pharmaceu diate release form or sustained release form sufficiently effec tical composition can also be varied so as to achieve a tran tive to obtain the desired control over the medical condition. sient or Sustained concentration of the compound in a subject, 0273. The dosage regimen to prevent, treat, give relief especially in and around the site of inflammation or disease from, or ameliorate a medical condition or disorder, or to area, and to result in the desired response. It is well within the otherwise protect against or treata medical condition with the skill of the art to start doses of the compound at levels lower combinations and compositions of the present invention is than required to achieve the desired effect and to gradually selected in accordance with a variety of factors. These factors increase the dosage until the desired effect is achieved. It will include, but are not limited to, the type, age, weight, sex, diet, be understood that the specific dose level for any particular and medical condition of the subject, the severity of the dis Subject will depend on a variety of factors, including body ease, the route of administration, pharmacological consider weight, general health, diet, natural history of disease, route ations such as the activity, efficacy, pharmacokinetics and and scheduling of administration, combination with one or toxicology profiles of the particular inhibitors employed, more other drugs, and severity of disease. whether a drug delivery system is utilized, and whether the 0269. An effective dosage of the composition will typi inhibitors are administered with other active ingredients. cally be between about 1 lug and about 10 mg per kilogram Thus, the dosage regimen actually employed may vary body weight, preferably between about 10 ug to 5 mg of the widely and therefore deviate from the preferred dosage regi compound per kilogram body weight. Adjustments in dosage men set forth above. US 2016/0235807 A1 Aug. 18, 2016 50

EXAMPLES was used in all cell culture experiments to inhibit cOMP degradation. Treatment of T84 cells with Zaprinast (500 uM) Example 1 alone did not increase cQMP levels and it did not inhibit NF-kB activation. The control cells (without LPS) were pro GC-C Agonists Ameliorate Colitis in Mice Via a cessed using identical procedures. Cyclic GMP Mediated Mechanism to Downregulate 0282 Western Blot analysis: This was performed essen NF-KB and Pro-Inflammatory Cytokines tially as described employing either 50 g of cytoplasmic or 25ug proteins of the nuclear fractions. The blots were probed Materials and Methods overnight with primary antibodies of interest (anti-p65 anti 0274. Materials: body, 1:200 dilution; anti-IKB antibody 1:500 dilution; anti 0275 T84 cells were obtained from Leonard Forte, Uni phosphorylated p65 antibody, 1:250 dilution; anti-phospho versity of Missouri, Columbia, Mo. UG and its analogs were rylated IKB antibody 1:250 dilution; all were from Santa Cruz chemically synthesized using the procedure as described and Biotechnology (Santa Cruz, Calif.). Anti-IKK-f and phos purified by BACHEMBioSciences, PA. Plecanatide, SP-333 phorylated IKK-C/B (Ser 176/180) were purchased from Cell and UG were chemically synthesized by procedures as Signaling Technology, Boston, Mass. and used at 1:500 dilu described previously (Shailubhai and Jacob, 2009). All other tion; anti-mouse or anti-rabbit antibodies conjugated to chemicals, cytokines ELISA kits, and antibodies were horseradish peroxide were used for chemiluminescence obtained from commercially available vendors. detection as recommended by manufacturer (GE Healthcare, (0276 Cyclic GMP Stimulation Assay: Piscataway, N.J.). (0277. The potency of test peptides to stimulate coMP (0283 Quantitative RT-PCR for Transcripts: synthesis in T84 cells was assayed by a published procedure (0284 T-84 cells (-107 cells) were treated with 1 uM (Shailubhai et al. 2000). Briefly, confluent monolayers of SP-333 for 5h. Subsequently, cDNA was prepared from total T-84 cells in 24-well plates were washed twice with 250 ul of RNA using High Capacity RNA-to-cDNA Kit (Applied Bio DMEM containing 50 mMHEPES (pH 7.4), pre-incubated at systems, Carlsbad, Calif.) according to manufacturers 37° C. for 10 min with 250 ul of DMEM containing 50 mM instructions. Quantitative Real-Time PCR amplification and HEPES (pH 7.4) and 1 mM isobutylmethylxanthine (IBMX), analysis were carried out using Lightcycler 480 (Roche, followed by incubation with test peptide for 30 min. The Basel Switzerland), p65 and IKK-13 specific Taqman gene medium was aspirated, and the reaction was terminated by the expression assays and Lightcycler 480 Probe master. All addition of 3% perchloric acid. Following centrifugation, and amplification assays were performed in duplicate employing neutralization with 0.1 N NaOH, the resulting supernatant 20ng cDNA (based on input RNA). The total reaction volume was used directly for measurements of intracellular cGMP was adjusted to 20 Jull with appropriate amounts of TaqMan using an ELISA kit (Caymen Chemical, Ann Arbor, Mich.). gene expression assays, Lightcycler 480 probe master and Results are expressed as pmol of c(GMP/mg of protein in the distilled water, then subjected to 45 PCR cycles using default cell extracts. cycling parameters. GAPDH transcript was used as endog (0278 Inhibition of NF-kB Signaling by SP-333: enous control. The data generated were analyzed and (0279. Approximately 1.5x10° T84 cells were seeded into expressed as target gene expression relative to endogenous 100 mm dishes and cultured essentially as described control using comparative Cp method and 2^Cp formula (Shailubhai et al. 2000). At 85-90% confluence, cells were (comparative Cp values and advanced quantitative relative washed and treated with 10 ug/ml lipopolysaccharides (LPS) quantification method). TaqMan reagents were obtained from from Escherichia coli (Sigma-Aldrich, St. Louis, Mo.) for 4 Applied Biosystems (Carlsbad, Calif.). h. Subsequent to LPS treatment, cells were washed and (0285) Studies in Experimental Models of Murine Colitis: replenished with fresh media containing 8-Bromo-cyclic 0286 BALB/c and TCRC-/- mice were obtained from GMP (8-Br-cGMP) at 0, 0.1, 1, 10, 100 and 1000 uM (Enzo Jackson Laboratories and handled as per the IACUC-ap Life Science, Farmingdale, N.Y.), or SP-333 at 0, 0.01, 0.1, 1 proved protocols of the University of Pittsburgh School of and 10LM together with 500 uMofphosphodiesterase inhibi Medicine, PA. BDF1 mice used for dextran sulfate sodium tor Zaprinast (Sigma-Aldrich, St. Louis, Mo.) for 16 h before (DSS)- and 2, 4, 6 trinitrobenzenesulfonic acid (TNBS)-in harvest. For analyzing phosphorylation status of IKK-C/B. duced colitis studies conducted by Epistem Co. (Manchester, T84 cells were stimulated with LPS (10 ug/ml) for 4 h fol UK) were obtained from Harlan Laboratories, UK. All ani lowed by treatment with 0.1 and 1.0 M of SP-333 in the mals were handled and euthanized in compliance with the presence of 500 uM Zaprinast, for 2 h. The control cells institutional and national animal welfare regulations. (without LPS treatment) were washed and refilled with fresh 0287 Cytokines Expression Analysis: media and harvested simultaneously with treated cells. 0288 To evaluate the effect of different concentrations of Nuclear and cytosolic extracts were prepared by the previ SP-333 on secretion of cytokines (IL-8 and TNF), 12-well ously described method (Aiamkitsumrit et al. 2007). Protein plates seeded with 10 cells/well were used. At 85-90% con concentration was measured with a Bio-Rad protein assay kit fluence, cells were washed with DPBS and treated with or (Bio-Rad, Hercules, Calif.). The extracts were stored at -80° without LPS (10 ug/ml) for 4 h, followed by washing with C. until use. phosphate buffered saline (PBS). SP-333 at 0,0.01, 0.1, 1 and 0280 Inhibition of NF-kB Signaling by SP-333: 10 uM) together with 500 uM of Zaprinast was added to the 0281. T84 cells at 85-90% confluence were washed and treated cells and incubated further for 16h. The levels of IL-8 treated with 10 g/ml Escherichia coli LPS (Sigma-Aldrich, and TNF were quantified using human IL-8 and TNF ELISA St. Louis, Mo.) for 4 h. Subsequently, cells were washed, development kits (Peprotech, Rocky Hill, N.J.) Each ELISA replenished with fresh media containing either SP-333 (0 to was performed in triplicate with cell-free supernatants from 10 uM) or 8-Bromo-cyclic GMP (8-Br-cGMP (0-1 mM) for two independent experiments. The protein concentration of 16 h. Zaprinast (500 uM), a coMP-specific PDE inhibitor, each well was assessed by Bio-Rad protein dye detection kit. US 2016/0235807 A1 Aug. 18, 2016

0289 TNBS-Induced Colitis in Mice: H&E stained tissue sections (Hegazi et al., 2005; Dave et al., 0290 Mouse strains BDF1 and BALB/c were used to 2009) employing the following criteria: (score 0) normal examine the efficacy of plecanatide in TNBS induced colitis. appearing crypts; (score 1) abnormal crypt pathology without The procedures used were as previously described (HegaZi et ulceration; (score 2) depleted crypts with ulceration; (score 3) al., 2005; Dave et al., 2009). Briefly, to induce colitis in 20-50% depleted crypts and increased ulceration; (score 4) specific pathogen-free 2-4 months old female BALB/c mice, >50% depleted crypts with substantial ulceration and (score 0.5 mg of TNBS in 50% ethanol was slowly administered into 5) totally ulcerated/inflamed colon with no crypts remaining. the lumen of the colon via a 3.5 F catheter fitted onto a 1-ml All slides were scored in a blinded manner. Colitis severity in syringe (total injection volume 100 ul). Plecanatide formu TCRO model was scored as previously described. All lated in PBS was administered by oral gavage for a total of 7 slides were scored in a blinded manner. Disease activity index days, with the first dose given one day prior to TNBS chal (DAI) was calculated by assessing body weight, stool consis lenge. Mice were observed daily for body weight, food con tency, and the presence of overt blood in stools or around the Sumption and for signs of any apparent toxicity, rectal bleed anus, essentially similar to that reported earlier (Hamamoto et ing or prolapse. After 7 days of treatment, GI tissue al., 1999). collection, fixation and histopathological scoring were per 0299| Explant Cultures to Measure Cytokine Expression: formed essentially as described previously (Sheikh et al., 0300 Colonic tissue fragments (0.5g dry weight) from the 2011). GI tissues were also collected and immediately pro TNBS-induced colitis mice and TCRC, knockout mice cessed for explant culture and cytokine expression as were processed as previously described (Hegazi et al., 2005). described below. Tissue fragment supernatants were collected after 24 h for 0291 TNBS-Induced Colitis in BDF-1 Mice: cytokine ELISAs. Murine immunoassay kits (R&D System) 0292 To induce colitis in BDF1 mice, a total of 90, 10-12 were used according to the manufacturers instructions for week old, H. pylori free male mice were used. Animals were IL-12p40, IL-23 and TNF. Linco Cytokine-16 plex Mouse randomized into 9 groups. TNBS was rectally administered ELISA was performed for MIP-1C, RANTES, IL-10 and as described above in a total injection volume of 100 ul. Oral IL-17 (Millipore) as per manufacturer's instructions. administration of PBS formulated plecanatide (0.0005-50 0301 Cytokine Analysis: mg/kg), animal monitoring and collection of GI tissues was (0302 Murine IL-12 p40, IL-1 B, TNF, IFN-Y, immunoas performed as described above. Daily administration of ple say kits (R&D System, MN, USA) were used according to the canatide was continued through day 6, when the mice were manufacturers instructions. Linco Cytokine-16 plex Mouse euthanized. Colon tissues were removed and weighed. Distal ELISA was performed for MIP-1C, RANTES, IL-10, TNF sections were fixed, stained with H&E, and evaluated for and IL-17 (Millipore, Billerica, Mass.) as per manufacturers histopathology and visual severity scores (HegaZiet al., 2005; instructions. Dave et al., 2009). Scoring criteria were similar to those described for DSS-induced colitis studies. 0303 Selection of Samples for Ki-67 Labeling and 0293 TCRO. Knockout Mice: Myeloperoxidase (MPO) Assay: 0294 TCRO mice were matched for age and sex in all 0304. A total of 40 samples were chosen for analysis of experiments. 16 week old TCRO. mice were treated with MPO activity and Ki-67 labeling from the treatment groups plecanatide at 0.5 or 2.5 mg/kg/day or PBS vehicle control by that received SP-333, its vehicle control (0.1M phosphate oral gavage for 14 days (6 mice per group). Mice were sacri buffer, pH 7) and the reference compound (5-ASA). A ran ficed 12 h after the final dose, and GI tissues were collected dom number generator (www.random.org) was employed to for experimental analyses. Colitis severity in TCRO model select samples from these groups (7 from each of the SP-333 was scored as described (Berg et al., 1996). recipient groups and 6 from each of the vehicle and 5-ASA 0295. DSS-Induced Colitis in Mice: groups). 0296. To evaluate the efficacy of plecanatide in DSS 0305 Ki-67 Labeling in Large Bowel Cross Sections: induced colitis model, 48 BDF-1 mice were divided into 8 0306 Employing standard procedures, two non-serial sec treatment groups (6 mice/group). One group was not exposed tions from each animal, were de-waxed in Xylene and rehy to DSS (untreated control) and groups 2-8 were treated with drated in graded alcohols to PBS (pH 7.4). Endogenous per 5% DSS in the drinking water on day 0. Plecanatide or SP-333 oxidase was blocked by incubation in 0.3% hydrogen solutions formulated in 0.1M phosphate buffer (pH 7) was peroxidase for 30 min. Antigen retrieval was performed by administered by oral gavage, once a day at 0.005, 0.05, 0.5, placing the slides in 1 L of citrate buffer (pH 6) and micro 2.5 and 5 mg/kg, from study day -1 (i.e. prior to initiation of waving at high power for 20 minutes. After cooling for 15 DSS treatment) through day 6 for SP-333 or day 7 for ple minutes, sections were incubated with 10% normal goat canatide. DSS treatment was initiated on day 0 for groups 2-8 serum for 30 minutes to block non-specific binding Primary until the end of the study. Sulfasalazine (80 mg/kg) or 5-ASA antibody (mouse monoclonal IgG, clone MIB-1, Dako) at (100 mg/kg) was used as positive control and was adminis 1/50 dilution was added and incubated for 1 hour at room tered daily. Mice were sacrificed on day 7. The large intestine temperature. Sections were washed and incubated with bioti was removed and weighed. Distal section of the large intes nylated secondary IgG antibody (1/200 dilution: Vector Labs tine was collected and fixed as described above. Inc.), for 45 minutes at room temperature. Subsequently, 0297 Evaluation of Colitis: slides were washed and incubated with ABC Elite reagents 0298 Disease activity index (DAI) was calculated by scor (Vector Labs Inc.), for 30 minutes at room temperature. Pres ing in-life parameters starting from day-1 until the end of the ence of bound antibody was demonstrated with 3,3'-diami study. Once daily, all mice were weighed and assessed for nobenzidine (DAB: 0.5 mg/ml in PBS). Sections were coun stool consistency, and the presence of overt blood in the stool terstained with thionine, dehydrated and mounted. or around the anus essentially similar to that reported earlier. (0307 MPO Activity in Lysates Prepared from Snap-Fro Colitis severity was assessed by histopathological analyses of Zen, Mid-Colon Tissues: US 2016/0235807 A1 Aug. 18, 2016 52

0308 Myeloperoxidase activity in colonic tissue samples (lane2, FIG.3C). Treatment with SP-333 (1 uM) completely was performed according to the method described (Krawisc reversed the LPS-mediated increase in phosphorylation of et al., 1984). Briefly, the rate of change in absorbance at 450 IKKB (lane 4, FIG. 3C). The total levels of IKK-B and actin nm was recorded when 20 ul of tissue extract was incubated remained unchanged, indicating the same level of protein with 150 ml of reaction buffer containing 0.26 mg/ml 0-dia loading in each lane (FIG. 3C). Taken together, these results nisidine and 0.6 ul/ml H.O. Each reaction was performed in suggested that SP-333-mediated inhibition of NF-kB activa triplicate. The rate of reaction was determined (initial slope) tion might occur through a coMP-mediated mechanism. and normalized to protein concentration of sample. 0313. It was also of interest to examine if SP-333 treat 0309 Statistical Analysis: ment had any on expression of IKKB and p65 transcripts. 0310. Where mentioned, statistical comparisons of group Treatment with SP-333 reduced transcript levels of IKK-13 data were performed using 2-way, unpaired T tests, assuming and p65 by approximately 50% as compared to those in the unequal variance, using Microsoft Excel. Statistically signifi unstimulated T84 cells (FIG. 3D). These in vitro results cant differences (p<0.05) and borderline non-significant dif prompted us to further examine the anti-inflammatory actions ferences (p<0.1) are indicated on the figures. of plecanatide and SP-333 in experimental models of murine colitis. Results 0314 NF-kB is a central regulator of pro-inflammatory cytokine expression in multiple cell types. T84 cells were Plecanatide and SP-333 are Potent Analogs of Human stimulated with LPS (10 ug/ml) for 4 h and subsequently Natriuretic Peptide UG treated with indicated amounts of SP-333 for 16 hr. Secretion of IL-8, and TNF in the extracellular medium were deter 0311 Plecanatide is structurally similar to UG except for mined by ELISA. Results presented in FIGS. 4A and B dem the substitution of aspartic acid with glutamate at the 3rd onstrated that LPS treatment caused a significant increase in position from N-terminal. SP-333 is similar to plecanatide in the levels of IL-8 and TNF as compared to those in untreated structure except that L-stereoisomeramino acids at the N- and controls. SP-333 treatment caused concentration dependent C-termini are replaced with the corresponding D Stereoiso reversal of LPS stimulated production of IL-8 and TNF mers Based on three-dimensional structures and energy cal (FIGS. 4A and B). culations of UG isomers, substitution of the third amino acid from the N-terminus (D->E) in the parent UG peptide was Plecanatide Ameliorates GI Inflammation in Several Murine predicted to stabilize the peptide structure predominantly in Colitis Models an active form. Plecanatide and SP-333 represent two potent synthetic analogs of human UG (FIG. 1). These analogs were 0315. Plecanatide ameliorates DSS- and TNBS-induced stable during Solid-phase synthesis and folding, and produced colitis in Mice: negligible amounts of the inactive isomer during purification. 0316. In a preliminary study, oral treatment with ple SP-333 was designed with D-stereoisomeramino acids at the canatide at 0.5 and 2.5 mg/kg, given once daily dose for 7 N- and C-terminus (FIG. 1) to make the peptide more resis days, effectively ameliorated TNBS-induced colitis in tant to proteolytic degradation. Both analogs possess disul BALB/c mice (FIG. 6A). Colon tissues from these mice were fide-linked bridges between amino acid position 4-12 and used for explant cultures to examine secretion of certain 7-15 as observed in UG peptide (schematically shown FIG. pro-inflammatory cytokines. Plecanatide treatment Sup 1). Potencies of plecanatide and SP-333 were evaluated in a pressed secretion of IL-12p40 (37%), IL-23 (84%), and TNF bioassay employing the human T84 colon cell line that exhib (67%) in colonic explants as compared to the vehicle-treated its robust cOMP responses. The peptides are equipotent caus explants (FIG. 6B). ing a dose dependent increase incCMP (FIG.2) with an ECso 0317 Effectiveness of oral treatment with plecanatide to ameliorate colitis was also examined in TCRC. -/- mice, of 1.889x107M and 2.826x107M, respectively. which are known to spontaneously develop chronic colitis. SP-333. Inhibits NF-kB Activation Daily treatment with plecanatide for two weeks reduced coli tis scores as compared to those in the vehicle treated mice 0312 Phosphorylated-p65 (phospho-p65) protein expres (FIG. 7A). Colon tissues from these mice were used in Sion, a measure of NF-KB activation, was markedly enhanced explant cultures to measure secretion of chemokines and in the cytosolic fraction of T84 cells following 4 h treatment cytokines in the culture media. Plecanatide treatment consid with LPS (10 ug/mL) (FIG. 2A). Treatment of LPS-activated erably suppressed production of RANTES and MIP-1C., and T84 cells, either with 8-Br-cGMP (1 mM) or with SP-333 (1.0 IL-17 (FIG. 7B). Interestingly, the treatment also increased and 10 uM), reduced levels of phospho-p65 in a dose-depen levels of the secreted IL-10, a cytokine known to be reduced dent manner (FIGS. 3 A&B). In addition, SP-333 also during GI inflammation (FIG. 7C). Taken together, these reduced levels of phospho-IKBC. with concomitant increase preliminary results prompted a further evaluation of ple in levels of IKBC, an endogenous inhibitor of NF-KB, which canatide in other murine models of experimental colitis. is phosphorylated primarily by I-K-kinase (IKKB). In FIG. 0318. The effectiveness of plecanatide to ameliorate coli 3C, the immunoblot, developed using an anti-IKKB antibody tis was furtherevaluated in DSS- and TNBS-induced colitis in recognizing both phosphorylated and unphosphorylated BDF-1 mice. Consistent with the results from the preliminary IKKB, showed that the level of total IKKB remained studies described above, oral treatment with plecanatide, unchanged following treatment with SP-333. However, when even at a dose as low as 0.005 mg/kg, was as effective as the immunoblot was developed using an antibody specific for Sulfasalazine (80 mg/kg) in ameliorating DSS-induced colitis phosphorylated IKKB, low levels of phosphorylated IKKB in BDF-1 mice (FIG. 5A). However, doses higher than 0.005 (phospho-IKKB) were detected in untreated control T84 cells mg/day did not produce incremental effect on amelioration of (lane 1, FIG.3C), but stimulation of cells with LPS resulted in colitis. Surprisingly, the colitis severity Scores in the group of a significant increase in the levels of phosphorylated IKKB mice treated with 2.5 mg/kg showed an unusually high level US 2016/0235807 A1 Aug. 18, 2016 of experimental variability. Nevertheless, the anti-inflamma major metabolite appears to retain biological activity, as only tory activity of plecanatide, at a broader range between ~35% of biological activity was lost within 2.5 h (FIG. 8A). 0.0005 to 50 mg/kg, was further examined in the TNBS HPLC analyses reveled that as expected, SP-333 was rela induced colitis in BDF1 mice. As shown in FIG. 5B, ple tively more stable in SIF (FIG. 8F) losing ~20% biological canatide treatment at doses under 0.005 mg/kg was ineffec activity after a 24h incubation in SIF (FIG.8D). These results tive but 0.05 mg/kg and higher doses produced statistically Suggested that plecanatide is sensitive to proteolysis, which is significant reduction in colitis severity. Again, there was no problematic especially when considering administration to further reduction in colitis severity with incremental doses species with far longer GI transit times than mice. Hence, we was observed, Suggesting that the dose above 0.05 mg/kg chose to advance SP-333 in further animal studies. might be Saturating. The effective dose-range of plecanatide 0322 Mice, randomly selected from treatment groups, was found to be in range between 0.05-0.5 mg/kg in this were used to determine the proportion of normal Ki-67-im model (FIG. 5B). mune reactive colonic epithelial cells (as a marker of regen Oral Treatment with SP-333 Ameliorates DSS-Induced Coli erative capability) in large bowel cross-sections (FIG. 11A) tis in Mice and levels of MPO activity in lysates prepared from large 0319 BDF-1 mice were administered SP-333 (0.005 bowel samples (as a marker for neutrophil infiltration and mg/kg to 5 mg/kg) by oral gavage to determine efficacy in acute inflammation severity) (FIG. 11B). Strong immune acute DSS-induced colitis. Mice that received either SP-333 reactivity demonstrating nuclear localization was observed in or reference compound 5-ASA (100 mg/kg) had lower mean large bowel cross-sections incubated with anti-Ki-67 IgG. As DAI scores than mice treated with vehicle control or DSS shown in FIG. 11A SP-333 treated sections exhibited larger alone (FIG. 9A). Effect of oral treatment with SP-333 was proportions of crypts with Ki-67 labeling than in DSS (35. evaluated on DSS-induced colitis in BDF-1 mice to further 3+4.86%) treated control sections. The proportion of Ki-67 confirm anti-inflammatory activity of GC-C agonists. As positive crypts observed in animals administered 0.005 and shown in FIG.9, a daily dose of SP-333 of between a 0.005 to 0.05 mg/kg/day of SP-333 was 60.4+6.96% and 58.7+7.3% 5 mg/kg dose range or of 5-ASA (100 mg/kg, as positive respectively. The corresponding value in 5-ASA treated mice control) consistently showed lower mean scores of colitis was 53.5+12.33%. Spearman rank correlation coefficients severity (FIG.9B) and DAI (FIG.9A) as compared to those in showed a statistically significant inverse correlation between DSS alone or DSS plus vehicle-treated mice. The effect of colitis severity and the percentage of epithelial crypts dem SP-333 treatment on levels of MPO activity in colon tissues onstrating normal Ki-67 labeling (correlation coefficient of was measured as an indirect way to assess severity of GI -0.449, p=0.003). Thus, oral treatment with SP-333 amelio inflammation. As expected, the colon tissues from DSS rated DSS-induced changes in the Ki-67 labeling of crypt treated mice exhibited the highest levels of MPO (0.048+0. epithelial cells. 004 units/min). The MPO activity was considerably reduced 0323 Lysates prepared from snap-frozen colon were ana following treatment either with 5-ASA (100 mg/kg) or with lyzed for myeloperoxidase (MPO) activity (FIG. 11B). The SP-333 at all doses (FIG. 11B). Mice administered 0.05 DSS treated group exhibited the highest MPO activity mg/kg of SP-333 exhibited the lowest (-50% reduction) level (0.048+0.004 units/minute). MPO activity was much lower in of MPO activity (0.024+0.003 units/minute; p=0.001). lysates derived from SP-333 treated groups compared to DSS 0320. Histological evaluation of colon tissues from DSS treated animals. The 0.05 mg/kg SP-333 treatment group had treated mice showed substantial loss of crypts, changes in the lowest initial MPO activity (0.024+0.003 units/minute: crypt architecture, ulceration, and localized infiltration of p=0.001 compared to DSS group). Statistical significance inflammatory cells (FIG. 10B; score=4) as compared to those was achieved for all other SP-333 treated groups except the in the naive tissues (FIG. 10A; score 0). Substantial loss of 0.5 mg/kg group. Treatment with the reference compound, crypts and ulceration can be seen upon DSS treatment (FIGS. 5-ASA, was also associated with a low initial MPO activity 10B and H score=/>3). As expected, colon tissues from the (0.029+0.004; p=0.010). Calculation of the Spearman rank 5-ASA-treated mice group exhibited only patchy loss of correlation coefficient revealed a significant, positive rela crypts and the distortion of crypt morphology was limited tionship between MPO activity and colitis severity scores (FIG. 10F, score=2). The colon tissues from mice in SP-333 (rs=0.277, p=0.05). treatment groups (0.005 mg/kg and 0.05 mg/kg) exhibited with minimal loss of crypts with very low distortions in crypt Discussion morphology (FIGS. 10C, D and E: scores 1, 2 and 2, respec 0324. This is the first study reporting that plecanatide and tively). Stability of plecanatide and SP-333 against proteoly SP-333, Superior analogs of UG. ameliorated GI inflamma S1S tion in acute as well as chronic models of murine colitis. Oral 0321 Plecanatide and SP-333 were incubated in simu treatment, once daily, with plecanatide or SP-333 at a dose lated intestinal fluid (SIF) for 6-24 h. Inactivated SIF was range between 0.005 to 2.5 mg/kg was observed to be as used as a control. Following the treatment, samples were effective as once daily treatment with 5-ASA (100 mg/kg) or analyzed by HPLC by c0MP production in T84 cells. HPLC sulfasalazine (80 mg/kg). However, doses of either of the analysis revealed that intact plecanatide incubated in heat GC-C agonists higher than 2.5 mg/kg did not show incremen inactivated SIF was stable up to 6 h, eluting at 13.8 min (FIG. tal dose response in amelioration of colitis, which may be 8B). Treatment with active SIF resulted in complete degra attributed to the saturation of the available GC-C receptors on dation of plecanatide within 2 h to form an active metabolite the epithelial cells lining the GI mucosa. The lowest effective that eluted at 9.4 min (indicated by an asterisk in FIG. 8C). dose of plecanatide varied from 0.005 to 2.5 mg/day in dif The active metabolite, SP-338 was characterized by MS and ferent models used at three different sites, which may be due MS/MS and identified to be formed via removal of carboxyl to external factors such as diet and animal husbandry condi terminal leucine possibly due to cleavage by carboxypepti tions of contract research organizations in US and UK which dases. Although, plecanatide is degraded rapidly in SIF, the are known to impact the composition of gut microflora influ US 2016/0235807 A1 Aug. 18, 2016 54 encing the severity of colitis in different species mice FCCC mice that were treated with DSS to induce GI inflam (Friswell et al., 2010; Gill and Finlay, 2011). mation (Shailubhai et al., 2012: Chang et al., 2014). 0325 The GI immune system is controlled primarily by a 0328. Although the potency of plecanatide to stimulate milieu of pro-inflammatory and anti-inflammatory cytokines cGMP in T84 cells and to ameliorate colitis in animal studies and growth factors promoting mucosal defense and integrity was comparable to that of SP-333, the latter drug candidate of intestinal tissue. Following the loss of barrier function, this was advanced further for clinical development because of its regulatory balance is disrupted by the massive recruitment of enhanced stability against proteolysis in SIF. The non-clinical leucocytes and macrophages, producing increased amounts safety and toxicology studies conducted in rodents and mon of destructive inflammatory cytokines (Neuman, 2007). Our keys suggest that SP-333 is an orally-safe drug candidate. results demonstrate that plecanatide treatment significantly Example 2 reduced production of IL-12 p40, IL-23 and TNF in colon explants from TNBS-treated BALB/c mice. Similarly, ple canatide treatment reduced production of RANTES, IL-17 SP-333, A Guanylate Cyclase-C Agonist, Inhibits and MIP1C. with a concomitant increase in IL-10 in colon NF-KB Signaling and Modulates Related Genes and explants from TCRC mice. Furthermore, our in vitro miRNAs Implicated in GI Inflammation and results further show that SP-333 inhibits NF-KB activation via Carcinogenesis acciMP-mediated mechanism in T84 cells. We also recently 0329 MicroRNAs are small non-coding RNA that are reported that oral treatment with plecanatide reduced forma post-transcriptional negative regulators of gene expression tion of colon dysplasia in DSS treated Apc" through and play important roles in biological processes such as cell downregulation of a number of pro-inflammatory cytokines cycle differentiation, metabolic pathways, and immune and growth factors (Chang et al., 2014). Taken together, these responses. MiRS are known to regulate expression of genes results suggest that orally-administered plecanatide or involved in the processes leading to inflammation and car SP-333 ameliorate colitis, though inhibition of NF-kB and cinogenesis. Aberrant expression of miR-21, let-7 family, downstream signaling resulting in Suppressed production of miR-155, and miR-133a is linked to several human diseases pro-inflammatory cytokines. Such as ulcerative colitis, Crohn's disease, and colitis-in 0326. During the GI renewal process, the epithelium goes duced colorectal cancer. SP-333, a proteolytically resistant through a cycle of proliferation, migration, differentiation, analog of uroguanylin, is currently under clinical develop apoptosis and eventual loss of epithelial cells into the lumen ment for the treatment of ulcerative colitis. Studies have (Lipkin, 1972: Eastwood, 1992). This process is crucial for shown that SP-333 activated guanylate cyclase-C (GC-C) maintaining the integrity of the intestinal mucosa and barrier expressed on the epithelial cells lining the gastrointestinal function. Any disruption in this renewal process, impairing (GI) mucosa to stimulate production of cyclic GMP (cGMP), epithelial cell homeostasis and causing loss of intestinal bar a second messenger known to mediate anti-inflammatory rier function, could potentially lead to GI inflammation and effects on NF-kB and downstream signaling. Previous studies colon carcinogenesis (Lipkin, 1972: Eastwood, 1992). In this have revealed that oral treatment with SP-333 ameliorates regard, deletion of GC-C gene in mice disrupts barrier func colitis in DSS- and TNBS-induced colitis animal models. In tion, resulting in increased permeation of luminal antigens human T84 cells, SP-333 treatment results in down regulation that promote inflammation, damaging the intestinal mucosal of NF-KB signaling and production of pro-inflammatory architecture (Liet al., 2007; Han et al., 2011; Harmel-Laws et cytokines. Previously, we reported that SP-333 ameliorated al., 2013). Disruption in GC-C signaling, reflecting early colitis through inhibition of NF-kB signaling in mice. The down regulation of UG and GN in colon polyps, tumors and objective of this study was to examine the effect of SP-333 in inflamed tissues from UC and CD patients, could contrib treatment on expression of genes and miRS that are implica ute to loss of barrier function and chromosomal instability tion in processes underlying GI inflammation and cancer. underlying the colonic inflammation. 0330 FIG. 1 shows the relationship of Uroguanylin (UG) and SP-333. SP-333 is a 16-mer of the endogenous BC-C 0327 Oral treatment with analogs of the endogenous ligand UG. The aspartic acid (D) at position 3 from the NH2 GC-C ligands has a unique mechanism of action, enabling terminus of UG is substituted with glutamic acid (E). D-ste restoration of homeostatic signaling responsible for mainte reoisomers of asparagine (N) and leucine (L) at position 1 and nance of colonic mucosa integrity. This study has important 16 respectively enhance proteolytic stability. This enhanced implications for treatment and maintenance of IBD in proteolytic stability results in SP-333 being stable in simu humans. First, oral treatment with locally-acting GC-C ago lated gastric and intestinal fluids, making this a highly potent nists eliminates toxicity concerns associated with the existing peptide that can be administered orally. systemic therapies of IBD. Second, the evolving paradigm demonstrates that the reduced production of UG and GN is Methods associated with the pathologies of UC and CD in humans. Accordingly, oral therapy with homologues of UG may be a 0331. The T84 cell line was used to evaluate the ability f replacement therapy to overcome the UG deficiency under SP-333 to stimulate coMP production. Total RNA and miRs lying the etiology of IBD. Finally, it is now well established were isolated from SP-333 treated T84 cells and levels of that patients suffering with chronic UC and CD are at higher gene transcripts were determined using the TaqMan Open risk of developing colon cancer (Ullman and Itzkowitz, Array platform. Extracted total miRs were analyzed by MiR 2011). Chronic treatment with orally safe drug candidates array. Statistical analyses were performed using Students such plecanatide or SP-333 are useful as maintenance therapy t-teSt. to delay the onset of IBD in to colon carcinogenesis. In this 0332 Cell Culture: Human colon carcinoma cells T84 regard, we recently reported that oral treatment with ple (ATCC Number CCL-248) were provided by Dr. Leonard canatide considerably reduced colonic dysplasia in Apc" Forte, University of Missouri at Columbia, Mo. Cells were US 2016/0235807 A1 Aug. 18, 2016

cultured in Dulbeco's Modified Eagle Medium (DMEM) and down-regulate IKKC. and inhibit proliferation, and induce Ham's F-12 medium (1:1) containing 10% fetal bovine apoptosis. Down regulation of miR-15b promotes production serum, 1% Glutamax, 1% penicillin, and 1% streptomycin at of TNFC. Let-7f and miR-936 levels inversely correlate with 37° C. in a 5% C)2 controlled incubator. IL-23R and IL-17. Treatment with SP-333 upregulated miRs 0333 c6MP Stimulation Assay: such as miR-15a (p<0.05), miR-16 (p<0.01), let-7i (p<0. 0334 Confluent monolayers of T84 cells in 24-well plates 005), miR-125b (p<0.001) and the family of miR-29 (p<0. were pre-incubated with indicated amounts of SP-333 in the 05), all of which are negative regulators of NF-kB signaling, presence of Zaprinast for 30 minutes at 37° C. Subsequently, which is known to augment production of pro-inflammatory the reaction was terminated by 3% perchloric acid and neu cytokines during GI inflammation. tralized with 0.5N NaOH. Intracellular cGMP levels were 0342 SP-333 also significantly increased levels of miR determined in lysates using a ELISA kit (Cayman Chemical). 41-3p (p<0.01), which is a negative regulator of c-Src. Acti All Samples were analyzed in duplicates. Total protein was Vation of c-Src is known to activate proto-oncogene c-Myc quantitated using Pierce BCA protein assay (Thermo Fisher and Suppress GC-C signaling. Activation of c-Myc transcrip Scientific). tionally suppresses miR-29b resulting in enhanced prolifera 0335 MicroRNA Microarray Analysis: tion and resistance to apoptosis in malignant cells. In addi 0336 T84 cells were grown to confluence in 100 mm tion, Sp-333 treatment reduced transcripts of several putative tissue culture dishes. Cells were treated with pre-determined c-Myc target genes such as cell division cycle 25a, cyclin D1, concentration of SP-333 (1 uM) in the presence of phosphodi and p53. Consistent with these findings, SP-333 treatment esterase inhibitor, Zaprinast (500 LM: Sigma Aldrich) for 5 increased levels of anti-carcinogenic miR-126 (p<0.01), hours. Following treatment, cells were trypsinized and the miR-133a (p<0.01), miR-30c (p<0.05), and reduced levels of sample was divided into 3 aliquots. One aliquot was used to pro-carcinogenic miR-21 (p<0.005), miR-155 (p<0.005), determine total c(3MP. Another was used for extracting total miR-92a (p<0.05), miR-200c (p<0.01), and miR-494 (p<0. RNA. The last aliquot was used to extract and purify miRNAs 005). Table 8 shows that treatment with SP-333 modulates using Norgen’s Kit (Norgen Biotek). Briefly, cells were lysed expression of miRNAs implicated in IBD and colon cancer. using lysis buffer, passed through a column to remove large RNA, followed by capturing miRNA using microRNA TABLE 8 enrichment column. Purified samples were stored at -20°C. MicroRNAs were profiled on a LC Sciences microarray chip miRNA SP-333 and TaqMan Open Array Human miRNA panel. miRNA expression SP-333 Treatment Expression Treatment 0337 Quantitative RT-PCR for Transcripts: IBD NF-kB 0338 T-84 cells were treated with 1 uM SP-333 for 5 Upregulated Upregulated hours. Subsequently, cDNA was prepared from total RNA miR-21 decrease miR-21 decrease and subjected to quantitative Real-Time PCR using TaqMan miR-362-3p decrease miR-27b. decrease reagents from Applied BioSystems. miR-532-3p decrease miR-3Ob decrease miR-28-5p decrease miR-10a decrease miR-99a decrease miR-199-3p decrease Results miR-203 decrease miR-301 decrease miR-26b decrease 0339 SP-333 stimulated coMP synthesis in T84 cells miR-98 decrease with an ECs of 2.83x107 M. As shown in FIG. 14, SP-333 Downregulated Downregulated treatment stimulated coMP synthesis in a dose-dependent manner in T84 cells, and approached a plateau at a concen miR-192 increase miR-218 increase miR-223 increase tration of 1 uM. Further, treatment with SP-333 enhanced miR-16 increase cGMP production and Expression of Protein Kinase GI and mir-29b. increase II Transcripts (FIG. 15). miR-15a increase 0340. As shown in FIG. 16, as compared to vehicle, treat CRC cMyc ment with SP-333 downregulated NF-kB subunits, IKK-B, Upregulated Upregulated c-Src, and p65 as judged by reduction in their transcript and miR-21 decrease miR-21 decrease protein levels. After treatment with SP-333, a 59% decrease miR-92 decrease miR-192 decrease in IKKB expression, a 55% decrease in p65 expression, and a miR-155 decrease miR-199-3p decrease 52% decrease in c-Src expression compared to untreated miR-224 decrease cells. FIG. 17 shows that SP-333 downregulates c-Myc and Downregulated Downregulated transcripts of genes related to cell-cycle in T84 cells. Treat miR-133a increase ment with SP-333 results in a 92% decrease in c-Myc expres miR-126 increase miR-101 increase sion a 58% decrease in Cyclin D1 expression, and a 50% miR-133b increase decrease in Survivin expression. Treatment with SP-333 miR-139-5p increase appears to have no effect on the expression of B-Catenin. 0341 SP-333 treatment modulates miRNAs known to be dysregulated in inflammation and cancer. As shown in FIG. 18, shows that in IBD and colon cancer, treatment with Conclusions SP-333 upregulates miR-21 and MiR-155 levels, while treat (0343 SP-333 binds and activates GC-C receptors ment with SP-333 downregulates levels of miR-126 and miR expressed on T84 cells to stimulate coMP production. 101 in colon cancer. Further, FIG. 19 shows that SP-333 SP-333 treatment attenuates LPS-mediated activation of NF upregulates expression of miRNAs that are known to be KB at transcriptional and post-translation levels in T-84 cells, expressed following NF-kB activation. NF-kB activation and down-regulates transcripts of proto-oncogenes c-Src and down-regulates miR-29 family and let-7i. MiR-15a/miR-16 c-Myc in T84 cells. Key miRNAs that are known to be dys US 2016/0235807 A1 Aug. 18, 2016 56 regulated in inflammation and cancer are inversely modulated responses in guanylyl cyclase/natriuretic peptide recep by SP-333 treatment. Further SP-333-mediated activation of tor-A gene-knockout mice. Physiological genomics GC-C signaling may contribute to its anti-inflammatory 44:43.0-442. effects through modulation of miRNAs and genes implicated 0353 Dave SH, Tilstra J S. Matsuoka K, Li F. DeMarco R in activation of NF-kB, C Src, and c-Myc signaling. This is A, Beer-Stolz D. Sepulveda AR, Fink M. P. Lotze MT and the first report demonstrating that SP-333 mediated activation Plevy S E (2009) Ethyl pyruvate decreases HMGB1 of GC-C signaling may contribute to its anti-inflammatory release and ameliorates murine colitis. J Leukoc Biol effects through suppression of c-Src, c-Myc, IKK-B, and 86:633-643. NF-kB signaling, possibly through modulation of related 0354 Eastwood G L (1992) Epithelial renewal in prema miRs implicated in GI inflammation and cancer. FIG. 20 lignant conditions of the gastrointestinal tract: a review. J shows the putative mechanism by which SP-333 modulates Clin Gastroenterol 14 Suppl 1:S29-33. expression of genes and miRNAS implicated in inflammation 0355 Forte L. R. Eber SL, Fan X, London RM, Wang Y. and cancer. These data will facilitate evaluation of the select Rowland LM, Chin DT, Freeman R H and Krause W J miRNAS and corresponding target genes in IBD tissues. (1999) Lymphoguanylin: cloning and characterization of a unique member of the guanylin peptide family. Endocri Other Embodiments nology 140:1800-1806. 0344) Those skilled in the art will recognize, or be able to 0356. Friswell MK, Gika H. Stratford IJ. Theodoridis G, ascertain using no more than routine experimentation, many Telfer B, Wilson I D and McBain A J (2010) Site and equivalents to the specific embodiments described herein. strain-specific variation in gut microbiota profiles and Such equivalents are intended to be encompassed by the metabolism in experimental mice. PLoS One 5:e8584. claims provided herein. 0357 Gill N and Finlay B B (2011) The gut microbiota: challenging immunology. Nature reviews Immunology INCORPORATION BY REFERENCE 11636-637. 0358 Hamamoto N. Maemura K, Hirata I, Murano M, 0345. This application incorporates by reference all pub Sasaki Sand Katsu K (1999) Inhibition of dextran sulphate lications or references disclosed herein for all purposes in sodium (DSS)-induced colitis in mice by intracolonically their entireties. administered antibodies against adhesion molecules (en dothelial leucocyte adhesion molecule-1 (ELAM-1) or REFERENCES intercellular adhesion molecule-1 (ICAM-1)). Clin Exp Immunol 1 17:462-468. 0346 Aiamkitsumrit B, Zhang X, Block TM, Norton P. 0359 Han X, Mann E, Gilbert S, GuanY, Steinbrecher K Fraser NW and SuYH (2007) Herpes simplex virus type A, Montrose M H and Cohen MB (2011) Loss of guanylyl 1 ICP4 deletion mutant virus dil 20 infection failed to cyclase C (GCC) signaling leads to dysfunctional intestinal induce apoptosis in nerve growth factor-differentiated barrier. PLoS One 6:e 16139. PC12 cells. J NeuroVirol 13:305-314. 0360 Harmel-Laws E. Mann EA, Cohen M B and Stein (0347 Berg DJ, DavidsonN, Kuhn R. Muller W. Menon S, brecher KA (2013) Guanylate cyclase C deficiency causes Holland G, Thompson-Snipes L. Leach MW and Rennick severe inflammation in a murine model of spontaneous D (1996) Enterocolitis and colon cancer in interleukin-10 colitis. PLoS One 8:e79180. deficient mice are associated with aberrant cytokine pro 0361 Hegazi RA, Rao K N, Mayle A, Sepulveda A R, duction and CD4(+) TH1-like responses. J Clin Invest Otterbein L E and Plevy S E (2005) Carbon monoxide 98:1010-102O. ameliorates chronic murine colitis through a heme oxyge 0348 Blomain E. S. Lin J. E. Kraft CL, Trela UT, Rock J nase 1-dependent pathway. J Exp Med 202: 1703-1713. M, Aing AS, Snook A E and Waldman SA (2013) Trans 0362 Krawisz, J. E. Sharon P and Stenson W F (1984) lating colorectal cancer prevention through the guanylyl Quantitative assay for acute intestinal inflammation based cyclase C signaling axis. Expert review of clinical pharma on myeloperoxidase activity. Assessment of inflammation cology 6:557-564. in rat and hamster models. Gastroenterology 87: 1344 (0349 Chang W, Masih S. Cooper H. Paleiwala V. Clapper 1350. M L and Shailubhai K (2014) Enhancement of Cyclic 0363 Ladetzki-Baehs K, Keller M. Kiemer AK. Koch E, GMP Production through Activation of Guanylate Zahler S, Wendel A and Vollmar AM (2007) Atrial natri Cyclase-C Inhibits Colorectal Adenomas in Apc--/Min uretic peptide, a regulator of nuclear factor-kappaB activa FCCC Mice with Dextran Sulfate Sodium-induced Inflam tion in vivo. Endocrinology 148:332-336. mation. Mol Cancer Ther Communicated. 0364 Li P. Lin J. E. Chervoneva I, Schulz S, Waldman SA 0350 Choudhari S R. Khan MA, Harris G. Picker D, and Pitari G. M. (2007) Homeostatic control of the crypt Jacob G. S. Block Tand Shailubhai K (2007) Deactivation Villus axis by the bacterial enterotoxin receptor guanylyl of Akt and STAT3 signaling promotes apoptosis, inhibits cyclase C restricts the proliferating compartment in intes proliferation, and enhances the sensitivity of hepatocellu tine. Am J Pathol 171:1847-1858. lar carcinoma cells to an anticancer agent, atiprimod. 0365 Lin J. E. Snook A E, Li P. Stoecker BA, Kim G. W. Molecular Cancer Therapeutics 6:112-121. Magee MS, Garcia A.V. Valentino MA, Hyslop T. Schulz 0351 Cohen M. B. Hawkins J A and Witte DP (1998) S and Waldman SA (2012) GUCY2C opposes systemic Guanylin mRNA expression in human intestine and col genotoxic tumorigenesis by regulating AKT-dependent orectal adenocarcinoma. Lab Invest 78: 101-108. intestinal barrier integrity. PLoS One 7:e31686. 0352 Das S. Periyasamy Rand Pandey K N (2012) Acti 0366 Lipkin M (1972) Gastric cell regeneration. Arch Fr vation of IKK/NF-kappaB provokes renal inflammatory Mal App Dig 61:691-693. US 2016/0235807 A1 Aug. 18, 2016 57

0367 Loftus EV, Jr. and Sandborn W J (2002) Epidemi Apc(Min/+) mouse and induces apoptosis in human colon ology of inflammatory bowel disease. Gastroenterol Clin adenocarcinoma cells via cyclic GMP. Cancer Res North Am 31:1-20. 60:5151-5157. 0368 London RM, Krause WJ, Fan X, Eber SL and Forte (0380 Sheikh SZ, Hegazi RA, Kobayashi T, Onyiah J C, L R (1997) Signal transduction pathways via guanylin and Russo S M, Matsuoka K. Sepulveda AR, Li F. Otterbein L uroguanylin in stomach and intestine. Am J Physiol 273: E and Plevy S E (2011) An anti-inflammatory role for G93-105. carbon monoxide and heme oxygenase-1 in chronic Th2 mediated murine colitis. J Immunol 186:5506-5513. 0369 Neuman M G (2007) Immune dysfunction in (0381 Tak PP and Firestein GS (2001) NF-kappaB: a key inflammatory bowel disease. Translational research: the role in inflammatory diseases. J Clin Invest 107:7-11. journal of laboratory and clinical medicine 149:173-186. (0382 Targan SR, Hanauer S. B. van Deventer SJ, Mayer 0370 Papachristou GI and Plevy S (2004) Novel biolog L., Present D H, Braakman T. DeWoody K L. Schaible TF ics in inflammatory bowel disease. Gastroenterol Clin and Rutgeerts P J (1997) A short-term study of chimeric North Am 33:251-269, ix. monoclonal antibody cA2 to tumor necrosis factor alpha 0371 Rogler G, Brand K, Vogl D, Page S. Hofmeister R, for Crohn's disease. Crohn's Disease cA2 Study Group. N Andus T, Knuechel R. Baeuerle PA, Scholmerich J and Engl J Med 337: 1029-1035. Gross V (1998) Nuclear factor kappaB is activated in mac 0383 Tsukagoshi H, Shimizu Y. Kawata T. Hisada T. rophages and epithelial cells of inflamed intestinal mucosa. Shimizu Y. Iwamae S, IshizukaT, Iizuka K, Dobashi Kand Gastroenterology 115:357-369. Mori M. (2001) Atrial natriuretic peptide inhibits tumor 0372 Rutgeerts P. Sandborn W. J. Feagan B G, Reinisch necrosis factor-alpha production by interferon-gamma-ac W. Olson A, Johanns J. Travers S, Rachmilewitz, D, tivated macrophages via Suppression of p38 mitogen-acti Hanauer SB, Lichtenstein GR, de Villiers WJ, Present D, vated protein kinase and nuclear factor-kappa Bactivation. Sands B E and Colombel J F (2005) Infliximab for induc Regul Pept 99:21-29. tion and maintenance therapy for ulcerative colitis. NEngl (0384 Ullman T A and Itzkowitz S H (2011) Intestinal J Med 353:2462-2476. inflammation and cancer. Gastroenterology 140: 1807 0373 Sandborn W J and Targan S R (2002) Biologic 1816. therapy of inflammatory bowel disease. Gastroenterology (0385 Vaandrager A B, Bot A G, Ruth P. Pfeifer A, Hof mann F and DeJonge HR (2000) Differential role of cyclic 122:1592-1608. GMP-dependent protein kinase II in ion transport in 0374 Schreiber S, Nikolaus S and Hampe J (1998) Acti murine Small intestine and colon. Gastroenterology 118: Vation of nuclear factor kappa B inflammatory bowel dis 108-114. ease. Gut 42:477-484. 0386 Vaandrager AB and de Jonge HR (1996) Signalling 0375 Shailubhai K (2002) Therapeutic applications of by c0MP-dependent protein kinases. Mol Cell Biochem guanylate cyclase-C receptor agonists. Curr Opin Drug 157:23-30. Discov Devel 5:261-268. (0387 Vellaichamy E. Sommana N K and Pandey KN 0376 Shailubhai K, Chang W-C, Masih S. Cooper HS and (2005) Reduced coMP signaling activates NF-kappaB in Clapper M L (2012) Enhancement of cyclic GMP produc hypertrophied hearts of mice lacking natriuretic peptide tion by guanylate cyclase-Cagonists delays progression of receptor-A. Biochem Biophy's Res Commun327: 106-111. colitis into colon cancer though downregulation of pro (0388 Wu F, Dassopoulos T. Cope L, Maitra A, Brant SR, inflammatory cytokines. Cancer Res 72: Abstract nr 1636. Harris ML, Bayless T M. Parmigiani G and Chakravarti S 0377 Shailubhai K, Comiskey S, Foss JA, Feng R, Bar (2007) Genome-wide gene expression differences in row L. Comer G M and Jacob GS (2013) Plecanatide, an Crohn's disease and ulcerative colitis from endoscopic oral guanylate cyclase C agonist acting locally in the gas pinch biopsies: insights into distinctive pathogenesis. trointestinal tract, is safe and well-tolerated in single doses. Inflamm Bowel Dis 13:807-821. Dig Dis Sci 58:2580-2586. (0389. Xavier RJ and Podolsky DK (2007) Unravelling the 0378 Shailubhai K and Jacob G (2009) Agonists of gua pathogenesis of inflammatory bowel disease. Nature 448: nylate cyclase useful for the treatment of gastrointestinal 427-434. disorders, in (Office USPaTed), Synergy Pharmaceuticals 0390 Zhang G, Arjunan K. P. Foss J. Comiskey S and Inc. Shailubhai K (2012) SP-333, a Proteolysis-resistant Ago 0379 Shailubhai K, Yu HH, Karunanandaa K, Wang JY. nist of Guanylate Cyclase-C, Inhibits Activation of NF EberSL, WangY, Joo NS, Kim HD, Miedema B W, Abbas kappa B and Suppresses Production of Inflammatory S Z, Boddupalli S S, Currie M G and Forte L R (2000) Cytokines to Ameliorate DSS-induced Colitis in Mice. Am Uroguanylin treatment Suppresses polyp formation in the J Gastroenterol 107:S626-S626.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 257

<21 Os SEQ ID NO 1 &211s LENGTH: 16 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized US 2016/0235807 A1 Aug. 18, 2016 58

- Continued

<4 OOs, SEQUENCE: 1 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu. 1. 5 1O 15

<210s, SEQ ID NO 2 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized

<4 OOs, SEQUENCE: 2 Asp Glu. Cys Glu Lieu. Cys Val Asn Val Ala Cys Thr Gly Cys Lieu. 1. 5 1O 15

<210s, SEQ ID NO 3 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized <4 OOs, SEQUENCE: 3 Asp Glu. Cys Glu Lieu. Cys Val Asn Val Ala Cys Thr Gly Cys 1. 5 1O

<210s, SEQ ID NO 4 & 211 LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemcially Synthesized

<4 OOs, SEQUENCE: 4 Glu Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu. 1. 5 1O

<210s, SEQ ID NO 5 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized <4 OOs, SEQUENCE: 5 Glu Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys 1. 5 1O

<210s, SEQ ID NO 6 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized

<4 OOs, SEQUENCE: 6 Cys Glu Lieu. Cys Val Asn. Wall Ala Cys Thr Gly Cys Lieu 1. 5 1O

<210s, SEQ ID NO 7 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2016/0235807 A1 Aug. 18, 2016 59

- Continued <223> OTHER INFORMATION: Chemically Synthesized

<4 OO > SEQUENCE: 7 Cys Glu Lieu. Cys Val Asn. Wall Ala Cys Thr Gly Cys 1. 5 1O

<210s, SEQ ID NO 8 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 8 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 9 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222 LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 9 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 10 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein ASP is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 10 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 11 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2016/0235807 A1 Aug. 18, 2016 60

- Continued <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein ASP is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein GLU is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 1 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 12 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223 OTHER INFORMATION: wherein ASN is a D amino acid

<4 OOs, SEQUENCE: 12 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 13 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: wherein LEU is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 13 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 14 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized

<4 OOs, SEQUENCE: 14 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys US 2016/0235807 A1 Aug. 18, 2016 61

- Continued

1. 5 1O 15

<210s, SEQ ID NO 15 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU at position 16 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 15 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 16 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU at position 16 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 16 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 17 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid US 2016/0235807 A1 Aug. 18, 2016 62

- Continued

22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein ASP is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU at position 16 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 17 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 18 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU at position 16 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 18 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 19 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 19 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 2 O &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE US 2016/0235807 A1 Aug. 18, 2016 63

- Continued

<222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 2O Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 21 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU at position 16 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 21 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 22 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein ASP is a D amino acid 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein GLU is a D amino acid 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU at position 16 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 22 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu US 2016/0235807 A1 Aug. 18, 2016 64

- Continued

1. 5 1O 15

<210s, SEQ ID NO 23 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein ASP is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein GLU is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 23 ASn Asp Glu. Cys Glu Lieu. CyS Val ASn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 24 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein ASP is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein GLU is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU at position 16 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 24 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 25 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2016/0235807 A1 Aug. 18, 2016 65

- Continued

OTHER INFORMAT ON: Chemically Synthesized FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) . . (1) OTHER INFORMAT ON: wherein ASN is a D amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (2) .. (2) OTHER INFORMAT ON: wherein ASP is a D amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (16) . . (16) OTHER INFORMAT ON: where in LEU at position 16 is attached to polyethylene g ycol FEATURE: NAME/KEY: MISC FEATURE LOCATION: (16) . . (16) OTHER INFORMAT ON: wherein LEU is a D amino acid

SEQUENCE: 25 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

SEQ ID NO 26 LENGTH: 16 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMAT ON: Chemically Synthesized FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) . . (1) OTHER INFORMAT ON: where in ASN at position 1 is attached to polyethylene g ycol FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) . . (1) OTHER INFORMAT ON: wherein ASN is a D amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (2) .. (2) OTHER INFORMAT ON: wherein ASP is a D amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (16) . . (16) OTHER INFORMAT ON: wherein LEU is a D amino acid

SEQUENCE: 26 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

SEO ID NO 27 LENGTH: 16 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Chemically Synthesized FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) . . (1) OTHER INFORMATION: wherein ASN is a D amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (16) . . (16) OTHER INFORMATION: wherein x is 3-(2-naphthyl)alanine FEATURE: NAME/KEY: MISC FEATURE LOCATION: (16) . . (16) OTHER INFORMATION: wherein x is a D amino acid

SEQUENCE: 27 US 2016/0235807 A1 Aug. 18, 2016 66

- Continued Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Xaa 1. 5 1O 15

<210s, SEQ ID NO 28 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: wherein x at position 8 is alpha-amino isobutyric acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein x at position 10 is alpha-amino isobutyric acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 28 Asn Asp Glu. Cys Glu Lieu. Cys Xaa Asn. Xaa Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 29 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein ASP at position 7 is attached to a Lactam bridge 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: wherein x at position 15 is ornithine 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 29 Asn Asp Glu. Cys Glu Lieu. Asp Val Asn Val Ala Cys Thr Gly Xaa Lieu 1. 5 1O 15

<210s, SEQ ID NO 3 O &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: US 2016/0235807 A1 Aug. 18, 2016 67

- Continued <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 30 Asn Asp Glu. Cys Glu Tyr Cys Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 31 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 31 Asn Asp Glu. Cys Glu Ser Cys Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 32 &211s LENGTH: 16 212. TYPE PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU at position 16 is attached to polyethylene glycol <4 OOs, SEQUENCE: 32 Asn Asp Glu. Cys Glu Tyr Cys Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 33 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid US 2016/0235807 A1 Aug. 18, 2016 68

- Continued

22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 33 Asn Asp Glu. Cys Glu Tyr Cys Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 34 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU at position 16 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid

<4 OOs, SEQUENCE: 34 ASn Asp Glu. Cys Glu Tyr CyS Val ASn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 35 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein LEU at position 16 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 35 Asn Asp Glu. Cys Glu Ser Cys Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 36 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE