US 20150366935A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0366935 A1 COMISKEY et al. (43) Pub. Date: Dec. 24, 2015

(54) FORMULATIONS OF GUANYLATE CYCLASE (60) Provisional application No. 61/383,156, filed on Sep. CAGONSTS AND METHODS OF USE 15, 2010, provisional application No. 61/387,636, filed on Sep. 29, 2010, provisional application No. (71) Applicant: SYNERGY PHARMACEUTICALS 61/392,186, filed on Oct. 12, 2010. INC., NEW YORK, NY (US) Publication Classification (72) Inventors: Stephen COMISKEY, Doylestown, PA (US); Rong FENG, Langhorne, PA (51) Int. Cl. (US); John FOSS, Doylestown, PA A638/2 (2006.01) (US); Kunwar SHAILUBHAI, A6II 45/06 (2006.01) Audubon, PA (US) (52) U.S. C. CPC ...... A61K 38/12 (2013.01); A61K 45/06 (21) Appl. No.: 14/845,644 (2013.01)

(22) Filed: Sep. 4, 2015 (57) ABSTRACT Related U.S. Application Data The invention provides low-dose formulations of guanylate (63) Continuation of application No. 14/661,299, filed on cyclase-C (“GCC) agonist peptides and methods for their Mar. 18, 2015, which is a continuation of application use. The formulations of the invention can be administered No. 13/421,769, filed on Mar. 15, 2012, which is a either alone or in combination with one or more additional continuation-in-part of application No. PCT/US2011/ therapeutic agents, preferably an inhibitor of c(GMP-depen 051805, filed on Sep. 15, 2011. dent phosphodiesterase or a laxative. Patent Application Publication Dec. 24, 2015 Sheet 1 of 6 US 2015/0366935 A1

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FORMULATIONS OF GUANYLATE CYCLASE can also occur in other sections of the gastrointestinal tract. CAGONSTS AND METHODS OF USE Ulcerative colitis is exclusively an inflammatory disease of the colon, the large intestine. Unlike Crohn's disease, in RELATED APPLICATIONS which all layers of the intestine are involved, and in which 0001. This application is a continuation of U.S. patent there can be normal healthy bowel in between patches of application Ser. No. 14/661,299, filed Mar. 18, 2015, which is diseased bowel, ulcerative colitis affects only the innermost a continuation of U.S. patent application Ser. No. 13/421,769, lining (mucosa) of the colonina continuous manner. Depend filed Mar. 15, 2012, which is a continuation-in-part of PCT/ ing on which portion of the gastrointestinal tract is involved, US2011/051805 filed on Sep. 15, 2011, which claims the Crohn's disease may be referred to as ileitis, regional enteri benefit of priority to U.S. Provisional Application No. tis, colitis, etc. Crohn's disease and ulcerative colitis differ 61/383,156 filed on Sep. 15, 2010, U.S. Provisional Applica from spastic colon or irritable bowel syndrome, which are tion No. 61/387,636 filed on Sep. 29, 2010, and U.S. Provi motility disorders of the gastrointestinal tract. Gastrointesti sional Application No. 61/392,186 filed on Oct. 12, 2010, the nal inflammation can be a chronic condition. It is estimated that as many as 1,000,000 Americans are afflicted with contents of which are incorporated by reference in their inflammatory bowel disease, with male and female patients entireties. appearing to be equally affected. Most cases are diagnosed INCORPORATION-BY-REFERENCE OF before age 30, but the disease can occur in the sixth, seventh, SEQUENCE LISTING and later decades of life. 0007 IBS and chronic idiopathic constipation are patho 0002. The contents of the text file named “SYPA 009 logical conditions that can cause a great deal of intestinal CO2US Sequence Listing..txt, which was created on Sep.3, discomfort and distress but unlike the inflammatory bowel 2015 and is 113 KB in size, are hereby incorporated by diseases, IBS does not cause the serious inflammation or reference in their entirety. changes in bowel tissue and it is not thought to increase the risk of colorectal cancer. In the past, inflammatory bowel FIELD OF THE INVENTION disease, celiac disease and IBS were regarded as completely 0003. The present invention relates to low-dose formula separate disorders. Now, with the description of inflamma tions of guanylate cyclase C peptide agonists useful for the tion, albeit low-grade, in IBS, and of symptom overlap treatment and prevention of various diseases and disorders. between IBS and celiac disease, this contention has come under question. Acute bacterial gastroenteritis is the strongest BACKGROUND OF THE INVENTION risk factor identified to date for the subsequent development 0004 Guanylate cyclase C is a transmembrane form of of postinfective irritable bowel syndrome. Clinical risk fac guanylate cyclase that is expressed on various cells, including tors include prolonged acute illness and the absence of Vom gastrointestinal epithelial cells (reviewed in Vaandrager 2002 iting. A genetically determined Susceptibility to inflamma Mol. Cell. Biochem. 230:73-83). It was originally discovered tory stimuli may also be a risk factor for irritable bowel as the intestinal receptor for the heat-stable toxin (ST) pep syndrome. The underlying pathophysiology indicates tides secreted by enteric bacteria and which cause diarrhea. increased intestinal permeability and low-grade inflamma The ST peptides share a similar primary amino acid structure tion, as well as altered motility and visceral sensitivity. Sero with two peptides isolated from intestinal mucosa and urine, tonin (5-hydroxytryptamine 5-HT) is a key modulator of gut guanylin and uroguanylin (Currie, et al., Proc. Nat'l Acad. function and is known to play a major role in pathophysiology Sci. USA 89:947-951 (1992); Hamra, et al., Proc. Nat 'l Acad. of IBS. The activity of 5-HT is regulated by c0MP. Sci. USA 90:10464-10468 (1993); Forte, L., Reg. Pept. 81:25 0008 While the precise causes of IBS and inflammatory 39 (1999); Schulz, et al., Cell 63:941-948 (1990); Guba, et al., bowel diseases (IBD) are not known, a disruption in the Gastroenterology 1 11:1558-1568 (1996); Joo, et al., Am. J. process of continual renewal of the gastrointestinal mucosa Physiol. 274:G633-G644 (1998)). may contribute to disease pathology in IBD and aggravate 0005. In the intestines, guanylin and uroguanylin act as IBS. The renewal process of the gastrointestinal lining is an regulators of fluid and electrolyte balance. In response to high efficient and dynamic process involving the continual prolif oral salt intake, these peptides are released into the intestinal eration and replenishment of unwanted damaged cells. Pro lumen where they bind to guanylate cyclase C localized on the liferation rates of cells lining the gastrointestinal mucosa are luminal membrane of enterocytes (simple columnar epithe very high, second only to the hematopoietic system. Gas lial cells of the small intestines and colon). The binding of the trointestinal homeostasis depends on both the proliferation guanylin peptides to guanylate cyclase C induces electrolyte and programmed cellular death (apoptosis) of epithelial cells and water excretion into the intestinal lumen via a complex lining the gut mucosa. Cells are continually lost from the intracellular signaling cascade that is initiated by an increase Villus into the lumen of the gut and are replenished at a in cyclic guanosine monophosphate (cGMP). substantially equal rate by the proliferation of cells in the 0006. The coMP-mediated signaling that is initiated by crypts, followed by their upward movement to the villus. The the guanylin peptides is critical for the normal functioning of rates of cell proliferation and apoptosis in the gut epithelium the gut. Any abnormality in this process could lead to gas can be increased or decreased in a variety of circumstances, trointestinal disorders such as irritable bowel syndrome (IBS) e.g., in response to physiological stimuli such as aging, and inflammatory bowel diseases. Inflammatory bowel dis inflammatory signals, hormones, peptides, growth factors, ease is a general name given to a group of disorders that cause chemicals and dietary habits. In addition, an enhanced pro the intestines to become inflamed, characterized by red and liferation rate is frequently associated with a reduction in Swollen tissue. Examples include ulcerative colitis and turnover time and an expansion of the proliferative Zone. The Crohn's disease. Crohn's disease is a serious inflammatory proliferation index is much higher in pathological states Such disease that predominantly affects the ileum and colon, but as ulcerative colitis and other gastrointestinal disorders. US 2015/0366935 A1 Dec. 24, 2015

Intestinal hyperplasia is a major promoter of gastrointestinal 0012. The invention provides an oral dosage formulation inflammation. Apoptosis and cell proliferation together regu comprising one or more pharmaceutically acceptable excipi late cell number and determine the proliferation index. ents and at least one GCC agonist peptide, wherein the Reduced rates of apoptosis are often associated with abnor amount of GCC agonist peptide per unit dose is from 0.01 mg mal growth, inflammation, and neoplastic transformation. to 10 mg, and wherein the GCC agonist peptide is selected Thus, both increased proliferation and/or reduced cell death from the group consisting of SEQID NOs: 1-54 and 56-249. may increase the proliferation index of intestinal tissue, In one embodiment, the GCC agonist peptide has a chromato which may in turn lead to gastrointestinal inflammatory dis graphic purity of no less than 90%, no less than 90.5%, no less CaSCS. than 91%, no less than 92%, no less than 93%, no less than 0009. In addition to a role for uroguanylin and guanylinas 94%, no less than 95%, no less than 96%, no less than 97%, no modulators of intestinal fluid and ion secretion, these peptides less than 98%, or no less than 99%. The chromatographic may also be involved in the continual renewal of gastrointes purity of the GCC agonist peptide is determined as area tinal mucosa by maintaining the balance between prolifera percent by HPLC. In one embodiment, the GCC agonist tion and apoptosis. For example, uroguanylin and guanylin peptide is selected from the group consisting of SEQID NOs: peptides appear to promote apoptosis by controlling cellular 1,8,9, or 56. In one embodiment, the GCC agonist peptide is ion flux. Given the prevalence of inflammatory conditions in selected from the group consisting of SEQID NOs: 1 and 9. Western societies a need exists to improve the treatment In one embodiment, the GCC agonist peptide is selected from options for inflammatory conditions, particularly of the gas the group consisting of SEQID NOs: 8 and 9. In one embodi trointestinal tract. ment, the amount of GCC agonist peptide per unit dose is 0.1 0010 Peptide agonists of guanylate cyclase C agonists mg, 0.3 mg, 0.6 mg, 1.0 mg, 3.0 mg. 6.0 mg, 9.0 mg or 9.5 mg. (“GCC agonists”) are described in U.S. Pat. Nos. 7,041,786, 0013. In one embodiment, the GCC agonist peptide has a 7,799,897, and U.S. Patent Application Publication Nos. total impurity content of no greater than 10%, no greater than US2009/0048175, US 2010/0069306, US 2010/0120694, US 9.5%, no greater than 9%, no greater than 8%, no greater than 2010/0093635, and US 2010/0221329. However, the formu 7%, no greater than 6%, no greater than 5%, no greater than lation of peptides for pharmaceutical delivery presents a num 4%, no greater than 3%, no greater than 2%, or no greater than ber of special problems. For example, peptides are subject to 1%. The total impurity content is determined as total area structural modifications by a variety of degradation mecha percentages of impurities by HPLC. The impurities do not nisms resulting in problems of chemical and physical insta include any pharmaceutically acceptable excipient used for the formulation. In one embodiment, the formulation is Sub bility of the formulation. stantially free of inorganic acids and carboxylic acids, e.g., HCl, phosphoric acid, or acetic acid. In this context, carboxy SUMMARY OF THE INVENTION lic acids do not include amino acids or peptides. In this 0011. The present invention provides low-dose formula context “substantially’ free of acids means that the acid con tions of peptide agonists of guanylate cyclase C ("GCC) and tent of the formulation at the time of packaging is preferably methods for their use in the treatment and prevention of less than 0.2%, less than 0.1%, less than 0.05%, less than human diseases and disorders, such as a gastrointestinal 0.01%, less than 0.005%, or less than 0.001% of the total motility disorder, irritable bowel syndrome, a functional gas weight of the formulation. In one embodiment, the formula trointestinal disorder, gastroesophageal reflux disease, func tion is free of HC1. tional heartburn, dyspepsia, functional dyspepsia, nonulcer 0014. In one embodiment, the formulation is a solid for dyspepsia, gastroparesis, chronic intestinal pseudo-obstruc mulation. In one embodiment, the formulation is in the form tion, colonic pseudo-obstruction; Crohn's disease, ulcerative of a powder, granule, Sachet, troche, tablet, or capsule. In colitis, inflammatory bowel disease, colonic pseudo-obstruc another embodiment, the formulation is a liquid formulation tion, obesity, congestive heart failure, and benign prostatic and the GCC agonist peptide is in Solution or Suspension in a hyperplasia. In certain embodiments, the formulations are lipophilic liquid. In one embodiment, the liquid is a refined stabilized against chemical degradation of the peptide. The specialty oil or a medium chain triglyceride or related ester. In low-dose formulations of the invention have unexpected effi one embodiment, the refined specialty oil is selected from cacy in humans in a dosage range that was not predicted based Arachis oil, Castor oil, cottonseed oil, maize (corn) oil, olive on studies in primates. The formulations of the invention are oil, sesame oil, soybean oil, and Sunflower oil. In one embodi particularly useful for the treatment or prevention of chronic ment, the medium chain triglyceride or related ester is AKO idiopathic constipation. In certain embodiments, the GCC MED E, AKOMED R, CAPTEX 355, LABRAFAC CC, agonists are analogs of uroguanylin and bacterial ST pep LABRAFAC PG, LAUROGLYCOL FCC, MIGLYOL 810, tides. In preferred embodiments, the analogs have Superior MIGLYOL 812, MIGLYOL 829, MIGLYOL 840, and SOF properties compared to the naturally occurring or “wild-type TISAN 645. In one embodiment, the liquid is selected from peptides. Examples of such Superior properties include a high the group consisting of medium chain triglycerides, propy resistance to degradation at the N-terminus and C-terminus lene glycol dicaprylocaprate, Vitamin E, soybean oil, Crema from carboxypeptidases, aminopeptidases, and/or by other phor, PG, and PG 400. In one embodiment, the unit dose is a proteolytic enzymes present in the stimulated human intesti powder, tablet, or capsule. In one embodiment, the unit dose nal juices and human gastric juices. Examples of GCC ago is a liquid-filled capsule. In one embodiment, the capsule or nists that can be used in the formulations and methods of the tablet is in a blister pack or strip. Preferably, the blister pack invention are described in more detail below and in U.S. Pat. or strip is made of a material that is impermeable to water Nos. 7,041,786, 7,799,897, and U.S. Patent Application Pub vapor and oxygen. In one embodiment the blister pack is lication Nos. US2009/0048175, US 2010/0069306, US 2010/ comprised of a metal foil. In one embodiment the blister pack O120694, US 2010/0093635, and US 2010/0221329, each of is a FOIL/FOIL blister pack. In one embodiment, the con which is incorporated herein by reference in its entirety. tainer of the blister pack is flushed with an inert gas such as US 2015/0366935 A1 Dec. 24, 2015

nitrogen or argon. In one embodiment, the container further 0020. In one embodiment of the spray coating-drying pro includes a desiccant. In a preferred embodiment the desiccant cess above, the one or more pharmaceutically acceptable is a molecular sieve. In one embodiment, the unit dose is in a excipients comprise a divalent cation salt wherein the divalent high density polyethylene bottle having a seal. In one cation is selected from Ca", Mg", Zn", and Mn". In one embodiment, the bottle further comprises a desiccant. In one embodiment, the one or more pharmaceutically acceptable embodiment, the bottle further comprises an oxygen scaven excipients comprise an amino acid selected from leucine, ger or molecular sieve. In one embodiment, the bottle is isoleucine, and Valine. In one embodiment, the one or more nearly impermeable to oxygen and water vapor (e.g., much pharmaceutically acceptable excipients comprise a coating more impermeable than a HDPE bottle), such as an Oxy agent (such as hypromellose Methocel E5 PremLV). In one Guard bottle. embodiment, the aqueous Solution has a pH greater than 4 0015. In one embodiment, the one or more pharmaceuti (e.g., 4.5-5.5, 5-6, about 5, or greater than 5) or even greater cally acceptable excipients include an inert carrier. In one than 7. In one embodiment, the aqueous Solution is Substan embodiment, the inert carrier is a selected from mannitol, tially free of inorganic acids and carboxylic acids. In one lactose, a microcrystalline cellulose, or starch. In one embodiment, the GCC agonist peptide is selected from the embodiment, the inert carrier has a particle size of from 50 to group consisting of SEQ ID NOs: 1, 8, 9, and 56. In one 900 microns, from 50 to 800 microns, from 50 to 300 embodiment, the process further includes drying the GCC microns, from 50 to 200 microns, from 75 to 150 microns, agonist peptide-coated carrier. from 75 to 200 microns, or from 75 to 300 microns. 0021. The invention further provides an oral dosage for mulation made by the process described herein. Preferably, 0016. In one embodiment, the GCC agonist peptide is the GCC agonist peptide as made is stabilized against chemi stabilized against chemical or physical degradation for a cal or physical degradation for a period of at least 18 months period of at least 18 months at 30° C. and 65% relative at 30° C. and 65% relative humidity, or at least 18 months at humidity, or at least 18 months at 25° C. and 60% relative 25°C. and 60% relative humidity, or at least 18 months at 2-8 humidity, or at least 18 months at 2-8°C. C. 0017. In one embodiment, the one or more pharmaceuti 0022. The invention also provides a method for treating or cally acceptable excipients include a divalent cation salt Such preventing a disease or disorder in a Subject in need thereof, as calcium chloride. In one embodiment, the one or more comprising administering to the Subject an oral dosage for pharmaceutically acceptable excipients comprise an amino mulation comprising at least one GCC agonist peptide, acid, such as leucine, histidine, or arginine, or an amine Such wherein the amount of GCC agonist peptide per unit dose is TRIS or TRIS/HC1. from 0.01 mg to 10 mg, and wherein the GCC agonist peptide 0018. In one embodiment, the oral dosage formulation is selected from the group consisting of SEQID NOs: 1-54 consists of the GCC agonist peptide described herein, an inert and 56-249. Preferably, the subject is a human subject. In one carrier (e.g., Celphere SCP-100, Avicel PH 102, or Avicel PH embodiment, the GCC agonist peptide is selected from the 112), and a lubricant (e.g., magnesium Stearate). In one group consisting of SEQ ID NOs: 1, 8, 9, or 56. In one embodiment, the formulation consists of the GCC agonist embodiment, the GCC agonist peptide is selected from the peptide, an inert carrier (e.g., Avicel PH 200), a divalent group consisting of SEQID NOs: 1 and 9. In one embodi cation salt (e.g., calcium chloride or calcium ascorbate), an ment, the amount of GCC agonist peptide per unit dose is 0.1 amino acid (e.g., leucine, histidine, or arginine) or a protec mg, 0.3 mg, 0.6 mg, 1.0 mg, 3.0 mg. 6.0 mg, 9.0 mg, 9.5 mg. tive amine (e.g., TRIS), a coating agent (e.g., Methocel ES or 10 mg. Premium LV) and optionally a lubricant (e.g., magnesium 0023. In one embodiment, the disease or disorder is a Stearate) or another additive (e.g., trehalose). In one embodi gastrointestinal disease or disorder selected from the group ment, the formulation consists of the GCC agonist peptide, a consisting of irritable bowel syndrome, non-ulcer dyspepsia, binder (e.g., Provsolv SMCC 90LM), and a disintegrant (e.g., chronic intestinal pseudo-obstruction, functional dyspepsia, Explotab). In one embodiment, the formulation consists of colonic pseudo-obstruction, duodenogastric reflux, gastro the GCC agonist peptide, a diluent (e.g., Mannogem EZ), a esophageal reflux disease, constipation, gastroparesis, heart binder (e.g., Provsolv SMCC 90 LM), a disintegrant (e.g., burn, gastric cancer, and H. pylori infection. In a preferred Explotab), a lubricant (e.g., Pruv). embodiment, the gastrointestinal disease or disorder is 0019. The invention also provides a process for making chronic idiopathic constipation. the oral dosage formulations described herein, wherein the 0024. In one embodiment, the method further comprises process comprises a step of dry granulation, wet granulation, administering to the Subject an effective amount of an inhibi or spray coating followed by drying. In another embodiment, tor of a coMP-specific phosphodiesterase. In one embodi the process comprises a step of dry mixing. In a preferred ment, the coMP-dependent phosphodiesterase inhibitor is embodiment the step of dry mixing includes geometric blend selected from the group consisting of Suldinac Sulfone, Zap ing. In one embodiment, the process comprises a step of direct rinast, and motapizone, Vardenifil, and Suldenifil. compression. In one embodiment, the process for making the 0025. In one embodiment, the method further comprises oral dosage formulations described herein is a spray coating administering to the Subject an effective amount of at least drying process which includes (a) providing an aqueous solu one laxative. In one embodiment, the at least one laxative is tion comprising: a GCC agonist peptide selected from the selected from the group consisting of SENNA, MIRALAX, group consisting of SEQID NOs: 1-54 and 56-249, and one PEG, or calcium polycarbophil. or more pharmaceutically acceptable excipients, wherein the 0026. In one embodiment, the method further comprises concentration of the GCC agonist peptide ranges from 10 to administering to the Subject an effective amount of at least 60 mg/mL, and (b) applying the aqueous solution to a phar one anti-inflammatory agent. maceutically acceptable carrier to generate a GCC agonist 0027. The invention also provides pharmaceutical compo peptide-coated carrier. sitions comprising the formulations described herein. US 2015/0366935 A1 Dec. 24, 2015

0028. Other features and advantages of the invention will peroxides commonly found in many pharmaceutical excipi be apparent from and are encompassed by the following ents. The invention also discloses additives (i.e. CaCl) that detailed description and claims. may function as local desiccants in the formulation. Divalent cation salts such as calcium ascorbate, MgCl, ZnCl2, MnCl BRIEF DESCRIPTION OF THE DRAWINGS and CaCl bind plecanatide and sterically hinder reactive 0029 FIG. 1: Plecanatide (SP-304) treatment reduced species such as water or oxygen from causing plecanatide time to first BM following daily dose. degradation by molecular displacement. The invention fur 0030 FIG. 2: Effect of daily treatment with plecanatide on ther includes Scavengers of residual formaldehyde (amines spontaneous bowel movements (SBM) in chronic constipa such as TRIS or TRIS/HCl or amino acids such as leucine, tion patients. isoleucine and Valine), and discloses packaging confirma 0031 FIG.3: Effect of daily treatment with plecanatide on tions to minimize oxygen exposure and water vapor during complete spontaneous bowel movements (CSBM) in chronic storage. The invention also discloses a stable manufacturing constipation patients. process comprised of initially dissolving plecanatide in cold 0032 FIG. 4: Effect of daily treatment with plecanatide on water to minimize solution degradation, followed by spray Bristol Stool Form Scores (BSFS) in chronic constipation coating the peptide solution on particles and drying to remove patients. moisture. 0033 FIG.5: Effect of daily treatment with plecanatide on 0038. The formulations of the invention are particularly straining scores in chronic constipation patients useful for the treatment or prevention of a gastrointestinal 0034 FIG. 6: Percentage of subjects reporting improve disease or disorder selected from the group consisting of ments in abdominal discomfort scores after 14-days of daily irritable bowel syndrome, non-ulcer dyspepsia, chronic intes treatment with plecanatide. tinal pseudo-obstruction, functional dyspepsia, colonic pseudo-obstruction, duodenogastric reflux, gastro esoph DETAILED DESCRIPTION ageal reflux disease, chronic idiopathic constipation, gastro paresis, heartburn, gastric cancer, and H. pylori infection. 0035. The invention provides pharmaceutical formula tions of peptide GCC agonists. It is intended that the formu 0039. In one embodiment, the formulations of the inven lations of the invention are “pharmaceutical formulations, tion are used in a method for the treatment of constipation. meaning that they are Suitable for pharmaceutical use. Clinically accepted criteria that define constipation range Accordingly, the term “formulations” as used herein is meant from the frequency of bowel movements, the consistency of to encompass pharmaceutical formulations even if "pharma feces and the ease of bowel movement. One common defini ceutical' is not expressly stated. Pharmaceutical composi tion of constipation is less than three bowel movements per tions comprising the formulations described herein are also week. Other definitions include abnormally hard stools or provided by the invention. The formulations of the invention defecation that requires excessive straining. Constipation preferably provide stability against chemical and physical may be idiopathic (functional constipation or slow transit degradation of the peptide, e.g., plecanatide (i.e., SEQID #1). constipation) or secondary to other causes including neuro 0036. The invention is based in part upon the discovery logic, metabolic or endocrine disorders. These disorders that mannitol mixes very effectively with the GCC agonist include diabetes mellitus, hypothyroidism, hyperthyroidism, peptides described herein and provides stability against deg hypocalcaemia, Multiple Sclerosis, Parkinson's disease, Spi radation, allowing the peptides to be formulated at very low nal cord lesions, Neurofibromatosis, autonomic neuropathy, doses. The invention is also based in part on the discovery that Chagas disease, Hirschsprung disease and cystic fibrosis. very low doses of the GCC agonist peptides described herein Constipation may also be the result of Surgery or due to the are effective for the treatment of diseases and disorders in use of drugs such as analgesics (like opioids), antihyperten humans. The dosage range found to be effective was not sives, anticonvulsants, antidepressants, antispasmodics and predicted based on animal studies. The invention is also based antipsychotics. In a preferred embodiment, the constipation is in part upon the discovery that a divalent cation (e.g., Ca") chronic idiopathic constipation. and/or an amino acid (e.g., leucine or arginine) stabilize the 0040. The stabilized formulations of the invention com GCC agonist peptides described herein during a process (e.g., prise at least one GCC agonist peptide formulated with one or spray coating-drying process) of manufacturing a formula more excipients such that the peptide is stabilized against tion of the GCC agonist peptides and provides stability chemical degradation. Chemical degradation of peptides against degradation both during the manufacturing process results from a number of mechanisms including oxidation, and storage of the formulation. water-mediated degradation, and reaction with aldehydes or 0037 Plecanatide is a charged peptide due to the presence reducing Sugars. The ideal excipient or combination of of four carboxylic acids and single amine group with a cal excipients will be non-hygroscopic, have few or no reducing culated pKa of approximately 3.5. Therefore plecanatide is Sugars, and be substantially free of contaminants such as iron, likely to interact with ions in solution or in the solid state. peroxide, and formaldehyde. The formulations of the inven Plecanatide is a hygroscopic peptide requiring the control of tion are preferably substantially free of water. In this context water during manufacture and storage to promote long term “substantially’ free of water means that the water content of stability. Plecanatide is prone to degradation by oxidation in the formulation at the time of packaging is preferably less the presence of residual peroxides or formaldehyde contami than 7%, less than 5%, less than 1%, or less than 0.5% of the nants that are formed from peroxide reaction with polymeric total weight of the formulation. In one embodiment the excipients. The present invention discloses a manufacturing amount of water is between 0.1 to 5% of the total weight of the process and dry solid formulation compositions that mini formulation. In one embodiment, the amount of water in the mizes water content. The formulations are comprised of com formulation of the invention manufactured through a spray ponents to minimize levels of residual formaldehyde and coating process is less than 0.5% (e.g., about 0.47%). US 2015/0366935 A1 Dec. 24, 2015

0041. In the context of the present formulations, the term 0046 Carriers having small particle sizes, and/or spheri “stable' or “stabilized’ refers to the resistance of the peptide cal shape, and narrow size distribution are preferred. Particles to chemical or physical degradation over time. Preferably, a of less than 20 microns have a relatively high surface area to stable formulation of the invention retains an amount of the Volume ratio causing inter-particle attractive forces to domi peptide in the formulation over a period of time that is at least nate and resist bulk flow. Larger particles (greater than 100 90%, preferably at least 95%, and most preferably at least microns) tend to roll or slide over one another and exhibit 99% the amount of peptide initially present in the formula superior bulk flow properties compared with small particles. tion. In one embodiment, a stable formulation of the inven A narrow particle-size distribution reduces particle packing tion, over a period of time (e.g., 18 month), has an increase in and increases flow. In one embodiment, the particles are the total impurity content not greater than 8%, not greater between 20 and 500 microns in size (as measured across the than 7%, not greater than 6%, not greater than 5%, not greater largest diameter of the particle, on average). In one embodi than 4%, not greater than 3%, not greater than 2%, or not ment, a small particle size and a narrow particle size range greater than 1%. In one embodiment, the peptide is chemi refers to particles having a size range of from 20-300 microns, cally stable in the formulation for a period of time that is at 50-200 microns, or 75-150 microns. In certain embodiments, least 18 months, at least 20 months, or at least 24 months the carrier has a Substantially spherical shape Such as can be when stored at 25 degrees Celsius (25 C) and 60% relative obtained with a spray drying process. humidity. In one embodiment, the peptide is chemically 0047. In one embodiment, the low-dose formulation is a stable in the formulation for a period of time that is at least 18 solid formulation and the unit dose is in the form of a tabletor months, at least 20 months, or at least 24 months when stored capsule. In one embodiment, the low-dose formulation is a at 2-8 degrees Celsius (2-8 C). In one embodiment, the pep liquid formulation and the unit dosage form is a liquid-filled tide is chemically stable in the formulation for a period of capsule. In one embodiment, the liquid formulation in the time that is at least 3 months, 12 months, 18 months and form of a solution or Suspension of the GCC agonist peptide preferably 24 months when stored at 25 degrees Celsius (25 in an lipophilic liquid. Examples of Suitable liquids include C) and 60% relative humidity. In one embodiment, the pep medium chain triglycerides (e.g., LABRAFAC Lipophile), tide is chemically stable in the formulation for a period of propylene glycol dicaprylocaprate (e.g., LABRAFAC PG), time that is at least 3 months, 18 months and preferably 24 vitamin E (e.g., a tocopherol), PEG 400 (e.g., Polyethylene months when stored at 30 degrees Celsius (30 C). glycol low M.W. (liquid)), propylene glycol, soybean oil, and 0042. The low-dose formulations of the invention com Castor oil. In one embodiment, the liquid is selected from the prise an amount of at least one GCC agonist peptide per unit group consisting of medium chain triglycerides, propylene dose that is less than 10 mg. It is especially advantageous to glycol dicaprylocaprate, vitamin E, and soybean oil. In one formulate oral compositions in unit dosage form for ease of embodiment, the refined specialty oil is selected from Arachis administration and uniformity of dosage. The term “unit dos oil, Castor oil, cottonseed oil, maize (corn) oil, olive oil, age form' as used herein refers to physically discrete units sesame oil, soybean oil, and Sunflower oil. In one embodi Suited as unitary dosages for the Subject to be treated; each ment, the medium chain triglyceride or related ester is AKO unit containing a predetermined quantity of active compound MED E, AKOMED R, CAPTEX 355, LABRAFAC CC, calculated to produce the desired therapeutic effect in asso LABRAFAC PG, LAUROGLYCOL FCC, MIGLYOL 810, ciation with the required pharmaceutical carrier. The specifi MIGLYOL 812, MIGLYOL 829, MIGLYOL 840, and SOF cation for the dosage unit forms of the invention are dictated TISAN 645. by and directly dependent on the unique characteristics of the 0048. A formulation according to the invention may be active compound and the particular therapeutic effect to be contained in a blister pack. In a particular embodiment, the achieved. In one embodiment, the unit dosage form is a tablet powder, tablet, or capsule comprising the formulation is con or a capsule. tained in ablister pack. Preferably, the blister pack is made of 0043. In one embodiment of the low-dose formulations of a material that allows only minimal permeation by water the invention, the amount of GCC agonist peptide per unit vapor and oxygen. In one embodiment the blister pack is dose is from 0.01 mg to 10 mg. In one embodiment, the comprised of a metal foil. In one embodiment, the blister pack amount of GCC agonist peptide per unit dose is 0.1 mg, 0.3 is comprised of ACLAR. In one embodiment, the container of mg, 0.6 mg, 1.0 mg, 3.0 mg., 6.0 mg, 9.0 mg, 9.5 mg, or 10 mg. the blister pack is flushed with an inert gas Such as nitrogen or argon. In one embodiment, the container further includes a 0044. In one embodiment, the low-dose formulation con desiccant. In one embodiment, the desiccant is calcium chlo tains a carrier that is non-hygroscopic. In one embodiment, ride. In one embodiment the desiccant is a molecular sieve. the carrier is selected from mannitol and maltose (e.g., 0049. While any GCC agonist known in the art can be ADVANTOSE 100). formulated according to the present invention, analogs of 0045. In one embodiment, the carrier is cellulose, prefer uroguanylin and bacterial ST peptides are preferred. In cer ably microcrystalline cellulose (e.g., Avicel PH 102, low tain embodiments, the uroguanylin and bacterial ST peptide moisture Avicel PH 112, Avicel PH 200, or Celphere SCP analogs have Superior properties compared to naturally 100). In one embodiment, the carrier is calcium phosphate or occurring, or “wild-type' peptides. For example, the urogua calcium Sulphate. In another embodiment, the carrier is a nylin and bacterial ST peptides for use in the present inven saccharide. The term "saccharide' as used herein also refers tion are preferably modified to increase their resistance to to polysaccharides. Thus, the term saccharide is meant to degradation at the N-terminus and C-terminus from carbox include polysaccharides. In one embodiment, the saccharide ypeptidases, aminopeptidases, and/or by other proteolytic is selected from mannitol, trehalose, lactose, Sucrose, Sorbi enzymes present in the stimulated human intestinal juices and tol, and maltose. In a preferred embodiment, the Saccharide is human gastric juices. In certain embodiments, the GCC ago mannitol. Preferably the saccharide has a low water content, nist formulation comprises a peptide consisting essentially of a small particle size and a narrow particle-size distribution. an amino acid sequence selected from SEQID NOs: 1-249. In US 2015/0366935 A1 Dec. 24, 2015 a preferred embodiment, the peptide consists essentially of an Suldinac Sulfone, Zaprinast, motapizone, Vardenifil, and amino acid sequence selected from SEQID NOs: 1, 8, 9, 55 sildenafil. In another embodiment, the GCC agonist formu and 56. The term “consists essentially of refers to a peptide lation is administered in combination with inhibitors of cyclic that is identical to the reference peptide in its amino acid nucleotide transporters. sequence or to a peptide that does not differ Substantially in terms of either structure or function from the reference pep 1.1 Formulations tide. A peptide differs substantially from the reference pep 0052. The formulations of the invention contain one or tide if its primary amino acid sequence varies by more than more GCC agonist peptides described herein, in combination three amino acids from the reference peptide or if its activa with one or more pharmaceutically acceptable carriers (also tion of cellular cGMP production is reduced by more than referred to as diluents) and/or excipients. In a preferred 50% compared to the reference peptide. Preferably, substan embodiment, the formulations of the invention include an tially similar peptides differ by no more than two amino acids inert carrier. The inert carrier is preferably non-hygroscopic. and not by more than about 25% with respect to activating In one embodiment, the carrier in the formulation contains cGMP production. In preferred embodiments, the GCC ago few or no reducing Sugars and is Substantially free of con nist is a peptide comprising at least 12 amino acid residues, taminants including, but not limited to, iron, peroxide, and and most preferably comprising between 12 and 26 amino formaldehyde. In one embodiment, the carrier is selected acids. Non-limiting examples of such analogs of uroguanylin from the group consisting of sorbitol, mannitol, EMDEX, and and bacterial ST peptides are described in Section 1.2 below. starch. In one embodiment, the carrier is mannitol (e.g., 0050. The invention provides methods for treating or pre MANNOGEM) or microcrystalline cellulose (e.g. PRO venting certain diseases and disorders and methods for SOLV, CELPHERE, CELPHERE beads). increasing gastrointestinal motility in a Subject in need 0053. The low-dose formulations of the invention contain thereof by administering an effective amount of a GCC ago no greater than 10 mg per unit dose of a GCC agonist peptide. nist formulation to the subject. The term “treating as used The remainder of the formulation is comprised of the carrier herein refers to a reduction, a partial improvement, amelio and one or more optional excipients. In one embodiment, the ration, or a mitigation of at least one clinical symptom asso amount of carrier is at least 90% of the total weight of the ciated with the gastrointestinal disorders being treated. The formulation. In another embodiment, the amount of carrier is term “preventing refers to an inhibition or delay in the onset at least 95% or at least 98% of the total weight of the formu or progression of at least one clinical symptom associated lation. In one embodiment, the amount of carrier is between with the gastrointestinal disorders to be prevented. The term 90 and 99.9% of the total weight of the formulation. In one “effective amount’ as used herein refers to an amount that embodiment, the one or more optional excipients comprise a provides some improvement or benefit to the subject. In cer disintegrant which is present at 1 to 5% of the total weight of tain embodiments, an effective amount is an amount that the formulation. In one embodiment, the one or more optional provides some alleviation, mitigation, and/or decrease in at excipients comprise a lubricant which is present at 0.02 to 5% least one clinical symptom of the gastrointestinal disorder to of the total weight of the formulation. In one embodiment, the be treated. In other embodiments, the effective amount is the one or more optional excipients comprise an amino acid Such amount that provides some inhibition or delay in the onset or as arginine, leucine, isoleucine, Valine, histidine, phenylala progression of at least one clinical symptom associated with nine, alanine, glutamic acid, aspartic acid, glutamine, the gastrointestinal disorder to be prevented. The therapeutic methionine, asparagine, tyrosine, threonine, tryptophan, or effects need not be complete or curative, as long as some glycine, which is present at 0.1 to 4% (e.g., 0.1-1%) of the benefit is provided to the subject. The term “subject’ prefer total weight of the formulation. In one embodiment, the molar ably refers to a human Subject but may also refer to a non ratio between the amino acid and the GCC agonist peptide is human primate or other mammal preferably selected from from about 2:1 to about 30:1 or about 2:1 to about 20:1 (e.g., among a mouse, a rat, a dog, a cat, a cow, a horse, or a pig. 5:1). In one embodiment, the one or more optional excipients 0051. In accordance with the methods of the present comprise a stabilizer Such as a divalent cation salt, more invention, the GCC agonist formulation can be administered specifically, a water-soluble divalent cation salt (e.g., calcium alone or in combination with one or more additional thera chloride, magnesium chloride, Zinc chloride, manganese peutic agents to prevent or treat inflammation, cancer and chloride, or calcium ascorbate), which is present at 0.1 to 12% other disorders, particularly of the gastrointestinal tract. In a (e.g., 0.1-4%) of the total weight of the formulation. In one preferred embodiment, the GCC agonist formulation is embodiment, the molar ratio between the salt and the GCC administered for the treatment of chronic constipation. In one agonist peptide is from about 5:1 to about 20:1 (e.g., 10:1). embodiment, the GCC agonist formulation is administered in 0054 The formulations may contain other additives as combination with one or more additional therapeutic agents needed, including for example lactose, glucose, fructose, selected from the group consisting of phosphodiesterase galactose, trehalose, Sucrose, maltose, raffnose, maltitol, inhibitors, cyclic nucleotides (such as cCMP and cAMP), a melezitose, stachyose, lactitol, palatinite, starch, Xylitol, laxative (such as SENNA, METAMUCIL, MIRALAX, PEG, mannitol, myoinositol, and the like, and hydrates thereof, and or calcium polycarbophil), a stool softener, an anti-tumor amino acids, for example alanine, glycine and betaine, and necrosis factor alpha therapy for IBD (such as REMICADE, polypeptides and proteins, for example albumen. ENBREL, or HUMAIRA), and anti-inflammatory drugs 0055. Further examples of pharmaceutically acceptable (such as COX-2 inhibitors, sulfasalazine, 5-ASA derivatives carriers and excipients include, but are not limited to binders, and NSAIDS). In certain embodiments, the GCC agonist fillers, disintegrants, lubricants, anti-microbial agents, anti formulation is administered in combination with an effective oxidant, and coating agents such as: BINDERS: corn starch, dose of an inhibitor of c(GMP-specific phosphodiesterase potato starch, other starches, gelatin, natural and synthetic (cGMP-PDE) either concurrently or sequentially with said gums such as acacia, Xanthan, Sodium alginate, alginic acid, GCC agonist. c6MP-PDE inhibitors include, for example, other alginates, powdered tragacanth, guar gum, cellulose US 2015/0366935 A1 Dec. 24, 2015

and its derivatives (e.g., ethyl cellulose, cellulose acetate, trin, maltose, mannitol, microcrystalline cellulose and guar carboxymethyl cellulose calcium, sodium carboxymethyl gum, Sorbitol crystalline); parenterals (like mannitol and cellulose), polyvinyl pyrrolidone (e.g., povidone, crospovi povidone); plasticizers (like dibutyl sebacate, plasticizers for done, copovidone, etc), methyl cellulose, Methocel, pre-ge coatings, polyvinylacetate phthalate); powderlubricants (like latinized starch (e.g., STARCH 15000 and STARCH 1500 glyceryl behenate); softgelatin capsules (like Sorbitol special LM(R), sold by Colorcon, Ltd.), hydroxypropyl methyl cellu Solution); spheres for coating (like Sugar spheres); spheroni lose, microcrystalline cellulose (FMC Corporation, Marcus Zation agents (like glyceryl behenate and microcrystalline Hook, Pa., USA), Emdex, Plasdone, or mixtures thereof, cellulose); Suspending/gelling agents (like carrageenan, gel FILLERS: talc, calcium carbonate (e.g., granules or powder), lan gum, mannitol, microcrystalline cellulose, povidone, dibasic calcium phosphate, tribasic calcium phosphate, cal Sodium starch glycolate, Xanthan gum); Sweeteners (like cium sulfate (e.g., granules or powder), microcrystalline cel aspartame, aspartame and lactose, dextrose, fructose, honey, lulose, powdered cellulose, dextrates, kaolin, mannitol, maltodextrin, maltose, mannitol, molasses, Sorbitol crystal silicic acid, Sorbitol, starch, pre-gelatinized starch, dextrose, line, Sorbitol special Solution, Sucrose); wet granulation fructose, honey, lactose anhydrate, lactose monohydrate, lac agents (like calcium carbonate, lactose anhydrous, lactose tose and aspartame, lactose and cellulose, lactose and micro monohydrate, maltodextrin, mannitol, microcrystalline cel crystalline cellulose, maltodextrin, maltose, mannitol, micro lulose, povidone, starch), caramel, carboxymethylcellulose crystalline cellulose & guar gum, molasses, sucrose, or Sodium, cherry cream flavor and cherry flavor, citric acid mixtures thereof, DISINTEGRANTS: agar-agar, alginic anhydrous, citric acid, confectioner's sugar, D&C Red No. acid, calcium carbonate, microcrystalline cellulose, croscar 33, D&C Yellow #10 Aluminum Lake, disodium edetate, mellose Sodium, crospovidone, polacrilin potassium, sodium ethyl alcohol 15%, FD&C Yellow No. 6 aluminum lake, starch glycolate (such as Explotab), potato or tapioca starch, FD&C Blue #1 Aluminum Lake, FD&C Blue No. 1, FD&C other starches, pre-gelatinized starch, clays, other algins, blue no. 2 aluminum lake, FD&C Green No. 3, FD&C Red other celluloses, gums (like gellan), low-substituted hydrox No. 40, FD&C Yellow No. 6 Aluminum Lake, FD&C Yellow ypropyl cellulose, ploy plasdone, or mixtures thereof, No. 6, FD&C Yellow No. 10, glycerol palmitostearate, glyc LUBRICANTS: calcium stearate, magnesium stearate, min eryl monostearate, indigo carmine, lecithin, manitol, methyl eral oil, light mineral oil, glycerin, Sorbitol, mannitol, poly and propyl parabens, mono ammonium glycyrrhizinate, natu ethylene glycol, other glycols, compritol, Stearic acid, sodium ral and artificial orange flavor, pharmaceutical glaze, poloX lauryl Sulfate, sodium Stearyl fumarate (such as Pruv), Veg amer 188, Polydextrose, polysorbate 20, polysorbate 80, etable based fatty acids lubricant, talc, hydrogenated veg polyvidone, pregelatinized corn starch, pregelatinized starch, etable oil (e.g., peanut oil, cottonseed oil, Sunflower oil, red iron oxide, Saccharin sodium, Sodium carboxymethyl sesame oil, olive oil, corn oil and Soybean oil), Zinc Stearate, ether, sodium chloride, sodium citrate, sodium phosphate, ethyl oleate, ethyl laurate, agar, syloid silica gel (AEROSIL strawberry flavor, synthetic black iron oxide, synthetic red 200, W.R. Grace Co., Baltimore, Md. USA), a coagulated iron oxide, titanium dioxide, and white wax. aerosol of synthetic silica (Deaussa Co., Piano, Tex. USA), a 0057 Solid oral dosage forms may optionally be treated pyrogenic silicon dioxide (CAB O-SIL, Cabot Co., Boston, with coating systems (e.g. Opadry(R) fix film coating system, Mass. USA), or mixtures thereof, ANTI-CAKINGAGENTS: for example Opadry(R) blue (OY-LS-20921), Opadry(R) white calcium silicate, magnesium silicate, silicon dioxide, colloi (YS-2-7063), Opadry(R) white (YS-1-7040), and black ink dal silicon dioxide, talc, or mixtures thereof, ANTIMICRO (S-1-8 106). BIAL AGENTS: benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butyl paraben, 0058. The agents either in their free form or as a salt can be cetylpyridinium chloride, cresol, chlorobutanol, dehydroace combined with a polymer Such as polylactic-glycoloic acid tic acid, ethylparaben, methylparaben, phenol, phenylethyl (PLGA), poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) alcohol, phenoxyethanol, phenylmercuric acetate, phenylm (WO 01/12233), polyglycolic acid (U.S. Pat. No. 3,773.919), ercuric nitrate, potassium Sorbate, propylparaben, Sodium polylactic acid (U.S. Pat. No. 4,767,628), poly(e-caprolac benzoate, sodium dehydroacetate, Sodium propionate, Sorbic tone) and poly(alkylene oxide) (U.S. 2003.0068384) to create acid, thimersol, thymo, or mixtures thereof, ANTOXI a sustained release formulation. Other sustained release for DANTS: ascorbic acid, BHA, BHT, EDTA, or mixture mulations and polymers for use in the compositions and thereof, and COATING AGENTS: sodium carboxymethyl methods of the invention are described in EP 0 467389 A2, cellulose, cellulose acetate phthalate, ethylcellulose, gelatin, WO 93/24150, U.S. Pat. No. 5,612,052, WO 97/40085, WO pharmaceutical glaze, hydroxypropyl cellulose, hydroxypro 03/075887, WO 01/01964A2, U.S. Pat. No. 5,922,356, WO pyl methylcellulose (hypromellose), hydroxypropyl methyl 94/155587, WO 02/074247A2, WO 98/25642, U.S. Pat. No. cellulose phthalate, methylcellulose, polyethylene glycol, 5,968,895, U.S. Pat. No. 6,180,608, U.S. 2003.0171296, U.S. 20020176841, U.S. Pat. No. 5,672,659, U.S. Pat. No. 5,893, polyvinyl acetate phthalate, shellac, Sucrose, titanium diox 985, U.S. Pat. No. 5,134,122, U.S. Pat. No. 5,192,741, U.S. ide, carnauba wax, microcrystalline wax, gellan gum, malto Pat. No. 5,192,741, U.S. Pat. No. 4,668,506, U.S. Pat. No. dextrin, methacrylates, microcrystalline cellulose and carra 4,713,244, U.S. Pat. No. 5,445,832 U.S. Pat. No. 4,931,279, geenan or mixtures thereof. U.S. Pat. No. 5,980,945, WO 02/058672, WO 97/26015, WO 0056. The formulation can also include other excipients 97/04744, and US20020019446. In such sustained release and categories thereof including but not limited to Pluronic R, formulations microparticles (Delie and Blanco-Prieto 2005 Poloxamers (such as Lutrol(R) and Poloxamer 188), ascorbic Molecule 10:65-80) of polypeptide are combined with micro acid, glutathione, protease inhibitors (e.g. soybean trypsin particles of polymer. U.S. 6,011.0 1 and WO 94/06452 inhibitor, organic acids), pH lowering agents, creams and describe a Sustained release formulation providing either lotions (like maltodextrin and carrageenans); materials for polyethylene glycols (i.e. PEG 300 and PEG 400) or triacetin. chewable tablets (like dextrose, fructose, lactose monohy WO 03/053401 describes a formulation which may both drate, lactose and aspartame, lactose and cellulose, maltodex enhance bioavailability and provide controlled release of the US 2015/0366935 A1 Dec. 24, 2015

agent within the GI tract. Additional controlled release for is placed in a drug capsule for oral administration. The cap mulations are described in WO 02/38129, EP 326151, U.S. sule shell can be a HPMC capsule shell or Gelatin capsule Pat. No. 5,236,704, WO 02/30398, WO 98/13029; U.S. shell. Upon dissolution of the capsule, a gas-generating sys 20030064105, U.S. 20030138488A1, U.S. 20030216307A1, tem inflates the pouch in the stomach where it is retained for U.S. Pat. No. 6,667,060, WO 01/49249, WO 01/49311, WO 16-24 hours, all the time releasing agents described herein. O1/49249, WO 01/49311, and U.S. Pat. No. 5,877,224 mate rials which may include those described in WOO4041195 0061 The GCC peptides described herein can also be (including the seal and enteric coating described therein) and formulated using the multi matrix system technology pH-sensitive coatings that achieve delivery in the colon (MMX). including those described in U.S. Pat. No. 4,910,021 and 0062. The GCC peptides described herein can be formu WO9001329. U.S. Pat. No. 4,910,021 describes using a pH lated in an osmotic device including the ones disclosed in sensitive material to coat a capsule. WO9001329 describes U.S. Pat. No. 4,503,030, U.S. Pat. No. 5,609,590 and U.S. using pH-sensitive coatings on beads containing acid, where Pat. No. 5,358,502. U.S. Pat. No. 4,503,030 discloses an the acid in the bead core prolongs dissolution of the pH osmotic device for dispensing a drug to certain pH regions of sensitive coating. U.S. Pat. No. 5,175,003 discloses a dual the gastrointestinal tract. More particularly, the invention mechanism polymer mixture composed of pH-sensitive relates to an osmotic device comprising a wall formed of a enteric materials and film-forming plasticizers capable of semi-permeable pH sensitive composition that Surrounds a conferring permeability to the enteric material, for use in compartment containing a drug, with a passageway through drug-delivery systems; a matrix pellet composed of a dual the wall connecting the exterior of the device with the com mechanism polymer mixture permeated with a drug and partment. The device delivers the drug at a controlled rate in Sometimes covering a pharmaceutically neutral nucleus; a the region of the gastrointestinal tract having a pH of less than membrane-coated pellet comprising a matrix pellet coated 3.5, and the device self-destructs and releases all its drug in with a dual mechanism polymer mixture envelope of the same the region of the gastrointestinal tract having a pH greater or different composition; and a pharmaceutical dosage form than 3.5, thereby providing total availability for drug absorp containing matrix pellets. The matrix pellet releases acid tion. U.S. Pat. Nos. 5,609,590 and 5,358,502 disclose an soluble drugs by diffusion in acid pH and by disintegration at osmotic bursting device for dispensing a beneficial agent to pH levels of nominally about 5.0 or higher. an aqueous environment. The device comprises a beneficial 0059. The GCC peptides described herein may be formu agent and osmagent Surrounded at least in part by a semi lated in the pH triggered targeted control release systems permeable membrane. The beneficial agent may also function described in WOO4052339. The agents described herein may as the osmagent. The semi-permeable membrane is perme beformulated according to the methodology described in any able to water and substantially impermeable to the beneficial of WO03105812 (extruded hyrdratable polymers): agent and osmagent. A trigger means is attached to the semi WO0243767 (enzyme cleavable membrane translocators); permeable membrane (e.g., joins two capsule halves). The WO03007913 and WO03086297 (mucoadhesive systems); trigger means is activated by a pH of from 3 to 9 and triggers WO02072075 (bilayer laminated formulation comprising pH the eventual, but sudden, delivery of the beneficial agent. lowering agent and absorption enhancer); WOO4064769 These devices enable the pH-triggered release of the benefi (amidated polypeptides); WO05063156 (solid lipid suspen cial agent core as a bolus by osmotic bursting. sion with pseudotropic and/or thixotropic properties upon 0063. In one embodiment the formulation contains a GCC melting); WO03035029 and WO03035041 (erodible, gastric agonist peptide, mannitol, silicified microcrystalline cellu retentive dosage forms); U.S. Pat. No. 5,007,790 and U.S. lose, sodicum starch glycolate, and Sodium Stearyl fumarate. Pat. No. 5,972,389 (sustained release dosage forms); WOO41 The GCC agonist is at a concentration of less than 5% w/w, 12711 (oral extended release compositions); WO05027878, less than 4%, less than 3% w/w, less than 2% w/w, less than WO02072033, and WO02072034 (delayed release composi 1% w/w, less than 0.5% w/w, or less than 0.25% w/w. In some tions with natural or synthetic gum); WO05030182 (con embodiments the GCC peptide is at a concentration of about trolled release formulations with an ascending rate of 0.23% w/w. The GCC peptide is preferably SEQ NO: 1 or release); WO05048998 (microencapsulation system); U.S. SEQNO: 9. The mannitol is at a concentration of at least 60% Pat. No. 5,952,314 (biopolymer); U.S. Pat. No. 5,108,758 w/w, at least 65% w/w, at least 70% w/w, at least 75% w/w, or (glassy amylose matrix delivery); U.S. Pat. No. 5,840.860 at least 80% w/w. In some embodiments the mannitol is (modified starch based delivery). JP10324642 (delivery sys present at about 79% w/w (e.g., 79.77%). The mannitol is tem comprising chitosanand gastric resistant material Such as preferably Mannogem EZ. The silicified microcrystalline wheat gliadin or zein): U.S. Pat. No. 5,866.619 and U.S. Pat. cellulose is at a concentration of at least 5% w/w, at least 10% No. 6,368,629 (saccharide containing polymer); U.S. Pat. No. wfw, or at least 15% w/w. In some embodiments the concen 6.531,152 (describes a drug delivery system containing a tration of the silicified microcrystalline cellulose is about water soluble core (Ca pectinate or other water-insoluble 15% w/w. The silicified microcrystalline cellulose is prefer polymers) and outer coat which bursts (e.g. hydrophobic ably Prosolv SMCC 90 LM. The sodicum starch glycolate is polymer-Eudragrit)); U.S. Pat. No. 6,234,464; U.S. Pat. No. at a concentration of at least 1% w/w, at least 2% w/w, at least 6,403,130 (coating with polymer containing casein and high 3% w/w, or at least 4% w/w. In some embodiments the con methoxy pectin; WOO174 175 (Maillard reaction product): centration of the sodicum starch glycolate is about 4% w/w. WO05063206 (solubility increasing formulation); WO040 The sodicum starch glycolate is preferably Explotab. The 19872 (transferring fusion proteins). Sodium Stearyl fumarate is at a concentration of at least 0.2% 0060. The GCC peptides described herein may be formu w/w, at least 0.5% w/w, at least 0.7% w/w, at least 0.8% w/w, lated using gastrointestinal retention system technology at least 0.9, or at least 1% w/w. In some embodiments the (GIRES: Merrion Pharmaceuticals). GIRES comprises a con concentration of the sodium stearyl fumarate is about 1% trolled-release dosage form inside an inflatable pouch, which w/w. The sodium stearyl fumarate is preferably Pruv. US 2015/0366935 A1 Dec. 24, 2015

0064. In one embodiment the formulation contains a GCC w/w, at least 0.1% w/w, at least 0.12% w/w, or at least 0.15% agonist peptide, silicified microcrystalline cellulose, and W/w. In some embodiments the concentration of leucine is Sodium starch glycolate. The GCC agonist is at a concentra about 0.13% w/w. The hypromellose is at a concentration of tion of less than 5% w/w, less than 4% w/w, less than 3% w/w, at least 0.1% w/w, at least 0.15% w/w, at least 0.2% w/w, or at less than 2% w/w, less than 1% w/w, less than 0.5% w/w, or least 0.25% w/w. In some embodiments the concentration of less than 0.25% w/w. In some embodiments the GCC peptide the hypromellose is about 0.22% w/w. The hypromellose is is at a concentration of about 0.3% w/w. The GCC peptide is preferably Methocel E5 PremLV. The magnesium stearate is preferably SEQ NO: 1 or SEQ NO: 9. The silicified microc at a concentration of at least 0.1% w/w, at least 0.15% w/w, at rystalline cellulose is at a concentration of at least 10% w/w, least 0.2% w/w, or at least 0.25% w/w. In some embodiments at least 20% w/w, at least 30% w/w, at least 40% w/w, at least the concentration of the magnesium stearate is about 0.25% 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% wfw. wfw, at least 90% w/w, or at least 95% w/w. In some embodi 0067. In one embodiment the formulation contains a GCC ments the concentration of the silicified microcrystalline cel agonist peptide, microcrystalline cellulose, and magnesium lulose is about 95.7% w/w. The silicified microcrystalline Stearate. The GCC agonist is at a concentration of less than cellulose is preferably Prosolv SMCC 90 HD. The sodicum 5% w/w, less than 4% w/w, less than 3% w/w, less than 2% starch glycolate is at a concentration of at least 1% w/w, at wfw, less than 1% w/w, less than 0.5% w/w, or less than least 2% w/w, at least 3% w/w, or at least 4% w/w. In some 0.25% w/w. In some embodiments the GCC peptide is at a embodiments the concentration of the sodicum starch glyco concentration of about 0.32% w/w. The GCC peptide is pref late is 4% w/w. The sodicum starch glycolate is preferably erably SEQ NO: 1 or SEQ NO: 9. The microcrystalline cel Explotab. lulose is at a concentration of at least 50% w/w, at least 60% 0065. In one embodiment the formulation contains a GCC w/w, at least 70% w/w, at least 80% w/w, at least 90% w/w, at agonist peptide, microcrystalline cellulose, calcium chloride least 95% w/w, or at least 99% w/w. In some embodiments the dihydrate, leucine, and hyrpomellose. The GCC agonist is at concentration of the microcrystalline cellulose is about a concentration of less than 5% w/w, less than 4% w/w, less 99.43% w/w. The microcrystalline cellulose is preferably than 3% w/w, less than 2% w/w, less than 1% w/w, less than Avicel PH 102. The magnesium stearate is at a concentration 0.5% w/w, or less than 0.25% w/w. In some embodiments the of at least 0.1% w/w, at least 0.15% w/w, at least 0.2% w/w, or GCC peptide is at a concentration of about 0.324.6% w/w. The at least 0.25% w/w. In some embodiments the concentration GCC peptide is preferably SEQ NO: 1 or SEQ NO: 9. The of the magnesium stearate is about 0.25% w/w. microcrystalline cellulose is at a concentration of at least 50% 0068. In one embodiment the formulation contains a GCC w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w, at agonist peptide, microcrystalline cellulose, and magnesium least 90% w/w, at least 95% w/w, or at least 99% w/w. In some Stearate. The GCC agonist is at a concentration of less than embodiments the concentration of the microcrystalline cel 5% w/w, less than 4% w/w, less than 3% w/w, less than 2% lulose is about 99.10% w/w. The microcrystalline cellulose is wfw, less than 1% w/w, less than 0.5% w/w, or less than preferably Celphere SCP-100. The calcium chloride dihy 0.25% w/w. In some embodiments the GCC peptide is at a drate is at a concentration of at least 0.1% w/w, at least 0.15% concentration of about 0.32% w/w, about 1.18% w/w. The wfw, at least 0.2% w/w, or at least 0.25% w/w. In some GCC peptide is preferably SEQ NO: 1 or SEQ NO: 9. The embodiments the concentration of the calcium chloride dihy microcrystalline cellulose is at a concentration of at least 30% drate is about 0.2622% w/w. The leucine is at a concentration wfw, at least 40% w/w, at least 50% w/w, at least 60% w/w, at of at least 0.05% w/w, at least 0.1% w/w, at least 0.12% w/w, least 70% w/w, at least 80% w/w, at least 90% w/w, at least or at least 0.15% w/w. In some embodiments the concentra 95% w/w, or at least 99% w/w. In some embodiments the tion of leucine is about 0.12% w/w. The hypromellose is at a concentration of the microcrystalline cellulose is about concentration of at least 0.1% w/w, at least 0.15% w/w, at 98.57% w/w. The microcrystalline cellulose is preferably least 0.2% w/w, or at least 0.25% w/w. In some embodiments Avicel PH 102. The magnesium stearate is at a concentration the concentration of the hypromellose is about 0.2% w/w. The of at least 0.1% w/w, at least 0.15% w/w, at least 0.2% w/w, or hypromellose is preferably Methocel E5 PremLV. at least 0.25% w/w. In some embodiments the concentration 0.066. In one embodiment the formulation contains a GCC of the magnesium stearate is about 0.25% w/w. agonist peptide, microcrystalline cellulose, calcium chloride 0069. In one embodiment the formulation contains a GCC dihydrate, leucine, hypromellose, and magnesium Stearate. agonist peptide, microcrystalline cellulose, and magnesium The GCC agonist is at a concentration of less than 5% w/w, Stearate. The GCC agonist is at a concentration of less than less than 4% w/w, less than 3% w/w, less than 2% w/w, less 5% w/w, less than 4% w/w, less than 3% w/w, less than 2% than 1% w/w, less than 0.5% w/w, or less than 0.25% w/w. In wfw, less than 1% w/w, less than 0.5% w/w, or less than some embodiments the GCC peptide is at a concentration of 0.25% w/w. In some embodiments the GCC peptide is at a about 0.36% w/w. The GCC peptide is preferably SEQNO: 1 concentration of about 1.18% w/w. The GCC peptide is pref or SEQ NO: 9. The microcrystalline cellulose is at a concen erably SEQ NO: 1 or SEQ NO: 9. The microcrystalline cel tration of at least 50% w/w, at least 60% w/w, at least 70% lulose is at a concentration of at least 30% w/w, at least 40% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w, or w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, at at least 99% w/w. In some embodiments the concentration of least 80% w/w, at least 90% w/w, at least 95% w/w, or at least the microcrystalline cellulose is about 98.75% w/w. The 99% w/w. In some embodiments the concentration of the microcrystalline cellulose is preferably Avicel PH 102. The microcrystalline cellulose is about 97.09% w/w. The micro calcium chloride dihydrate is at a concentration of at least crystalline cellulose is preferably Avicel PH 112. The mag 0.1% w/w, at least 0.15% w/w, at least 0.2% w/w, at least nesium Stearate is at a concentration of at least 0.1% w/w, at 0.25% w/w, or at least 0.3% w/w. In some embodiments the least 0.15% w/w, at least 0.2% w/w, or at least 0.25% w/w. In concentration of the calcium chloride dihydrate is about Some embodiments the concentration of the magnesium 0.29% w/w. The leucine is at a concentration of at least 0.05% stearate is about 0.25% w/w. US 2015/0366935 A1 Dec. 24, 2015

0070. In one embodiment the formulation contains a GCC concentration of the hypromellose is 0.2-2% w/w. In a par agonist peptide, trehalose granules, hypromellose, histidine, ticular embodiment the concentration of the hypromellose calcium ascorbate, trehalose powder, microcrystalline cellu about 0.5% w/w. The hypromellose is preferably Methocel lose, and magnesium Stearate. The GCC agonist is at a con ES Premium LV. The arginine is a concentration of at least centration of less than 5% w/w, less than 4% w/w, less than 0.5% w/w, at least 1% w/w, at least 1.5% w/w, or at least 2% 3% w/w, less than 2% w/w, less than 1% w/w, less than 0.5% W/w. In some embodiments the concentration of the arginine wfw, or less than 0.25% w/w. In some embodiments the GCC is 1-6% w/w. In a particular embodiment, the concentration of peptide is at a concentration of about 1.18% w/w. The GCC the arginine is 1.66% w/w. The calcium ascorbate is at a peptide is preferably SEQNO: 1 or SEQNO:9. The trehalose concentration of at least 0.05% w/w, at least 0.07% w/w, at granules are at a concentration of at least 50% w/w, at least least 0.09% w/w, or at least 0.1% w/w. In some embodiments 55% w/w, at least 60% w/w, at least 65% w/w, at least 70% the concentration of the calcium ascorbate is 0.05-10% w/w. wfw, or at least 75% w/w. In some embodiments the concen In a particular embodiment, the concentration of the calcium tration of the trehalose granules is 55-75% w/w. In a particu ascorbate is about 0.1% w/w. The trehalose powder is at a lar embodiment, the concentration of the trehalose granules is concentration of at least 0.5% w/w, at least 0.7% w/w, at least 70.48% w/w. The hypromellose is at a concentration of at 0.8% w/w, at least 0.9% w/w, at least 1% w/w, or at least 1.2% least 0.1% w/w, at least 0.2% w/w, at least 0.3% w/w, at least wfw. In some embodiments the concentration of the trehalose 0.4% w/w, or at least 0.5% w/w. In some embodiments the powder is 0.5-4% w/w. In a particular embodiment, the con concentration of the hypromellose is 0.2-2% w/w. In a par centration of the trehalose powder is 1.02% w/w. The micro ticular embodiment the concentration of the hypromellose crystalline cellulose is at a concentration of at least 10% w/w, about 0.5% w/w. The hypromellose is preferably Methocel at least 20% w/w, or at least 25% w/w. In some embodiments ES Premium LV. The histine is a concentration of at least the concentration of the microcrystalline cellulose is 20-40% 0.6% w/w, at least 0.8% w/w, at least 0.9% w/w, at least 1% w/w. In a particular embodiment, the concentration of the wfw, at least 3% w/w, or at least 5% w/w. In some embodi microcrystalline cellulose is 25% w/w. The microcrystalline ments the concentration of the histidine is 1-6% w/w. In a cellulose is preferably Avicel PH 200. The magnesium stear particular embodiment, the concentration of the arginine is ate is at a concentration of at least 0.1% w/w, at least 0.15% 1.48% w/w. The calcium ascorbate is at a concentration of at wfw, at least 0.2% w/w, or at least 0.25% w/w. In some least 0.05% w/w, at least 0.07% w/w, at least 0.09% w/w, or at embodiments the concentration of the magnesium Stearate is least 0.1% w/w. In some embodiments the concentration of 0.2-1% w/w. In a particular embodiment the concentration of the calcium ascorbate is 0.05-10% w/w. In a particular the magnesium stearate is about 0.25% w/w. embodiment, the concentration of the calcium ascorbate is 0072. In one embodiment the formulation contains a GCC about 0.1% w/w. The trehalose powder is at a concentration of agonist peptide, trehalose granules, hypromellose, TRIS, cal at least 0.5% w/w, at least 0.7% w/w, at least 0.8% w/w, at cium ascorbate, trehalose powder, microcrystalline cellulose, least 0.9% w/w, at least 1% w/w, or at least 1.2% w/w. In some and magnesium Stearate. The GCC agonist is at a concentra embodiments the concentration of the trehalose powder is tion of less than 5% w/w, less than 4% w/w, less than 3% w/w, 0.5-4% w/w. In a particular embodiment, the concentration of less than 2% w/w, less than 1% w/w, less than 0.5% w/w, or the trehalose powder is 1.02% w/w. The microcrystalline less than 0.25% w/w. In some embodiments the GCC peptide cellulose is at a concentration of at least 10% w/w, at least is at a concentration of about 1.17% w/w. The GCC peptide is 20% w/w, or at least 25% w/w. In some embodiments the preferably SEQ NO: 1 or SEQNO: 9. The trehalose granules concentration of the microcrystalline cellulose is 20-40% are at a concentration of at least 50% w/w, at least 55% w/w, w/w. In a particular embodiment, the concentration of the at least 60% w/w, at least 65% w/w, at least 70% w/w, or at microcrystalline cellulose is 25% w/w. The microcrystalline least 75% w/w. In some embodiments the concentration of the cellulose is preferably Avicel PH 200. The magnesium stear trehalose granules is 55-75% w/w. In a particular embodi ate is at a concentration of at least 0.1% w/w, at least 0.15% ment, the concentration of the trehalose granules is 70.81% wfw, at least 0.2% w/w, or at least 0.25% w/w. In some w/w. The hypromellose is at a concentration of at least 0.1% embodiments the concentration of the magnesium Stearate is wfw, at least 0.2% w/w, at least 0.3% w/w, at least 0.4% w/w, 0.2-1% w/w. In a particular embodiment the concentration of or at least 0.5% w/w. In some embodiments the concentration the magnesium stearate is about 0.25% w/w. of the hypromellose is 0.2-2% w/w. In a particular embodi 0071. In one embodiment the formulation contains a GCC ment the concentration of the hypromellose about 0.5% w/w. agonist peptide, trehalose granules, hypromellose, arginine, The hypromellose is preferably Methocel ES Premium LV. calcium ascorbate, trehalose powder, microcrystalline cellu The TRIS is a concentration of at least 0.6% w/w, at least lose, and magnesium Stearate. The GCC agonist is at a con 0.8% w/w, at least 0.9% w/w, or at least 1% w/w. In some centration of less than 5% w/w, less than 4% w/w, less than embodiments the concentration of the TRIS is 0.5-6% w/w. In 3% w/w, less than 2% w/w, less than 1% w/w, less than 0.5% a particular embodiment, the concentration of the arginine is wfw, or less than 0.25% w/w. In some embodiments the GCC 1.15% w/w. The calcium ascorbate is at a concentration of at peptide is at a concentration of about 1.17% w/w. The GCC least 0.05% w/w, at least 0.07% w/w, at least 0.1% w/w, or at peptide is preferably SEQNO: 1 or SEQNO:9. The trehalose least 1% w/w. In some embodiments the concentration of the granules are at a concentration of at least 50% w/w, at least calcium ascorbate is 0.05-10% w/w. In a particular embodi 55% w/w, at least 60% w/w, at least 65% w/w, at least 70% ment, the concentration of the calcium ascorbate is about wfw, or at least 75% w/w. In some embodiments the concen 0.1% w/w. The trehalose powder is at a concentration of at tration of the trehalose granules is 55-75% w/w. In a particu least 0.5% w/w, at least 0.7% w/w, at least 0.8% w/w, at least lar embodiment, the concentration of the trehalose granules is 0.9% w/w, at least 1% w/w, or at least 1.2% w/w. In some 70.31% w/w. The hypromellose is at a concentration of at embodiments the concentration of the trehalose powder is least 0.1% w/w, at least 0.2% w/w, at least 0.3% w/w, at least 0.5-4% w/w. In a particular embodiment, the concentration of 0.4% w/w, or at least 0.5% w/w. In some embodiments the the trehalose powder is 1.02% w/w. The microcrystalline US 2015/0366935 A1 Dec. 24, 2015

cellulose is at a concentration of at least 10% w/w, at least (0076. The term “GCC agonist” refers to both peptides and 20% w/w, or at least 25% w/w. In some embodiments the non-peptide compounds Such as that bind to an intestinal concentration of the microcrystalline cellulose is 20-40% guanylate cyclase C and stimulate the intracellular produc w/w. In a particular embodiment, the concentration of the tion of c(GMP. Where the GCC agonist is a peptide, the term microcrystalline cellulose is 25% w/w. The microcrystalline encompasses biologically active fragments of Such peptides cellulose is preferably Avicel PH 200. The magnesium stear and pro-peptides that bind to guanylate cyclase C and stimu ate is at a concentration of at least 0.1% w/w, at least 0.15% late the intracellular production of c(GMP. wfw, at least 0.2% w/w, or at least 0.25% w/w. In some (0077. Preferably, the GCC agonists for use in the formu embodiments the concentration of the magnesium Stearate is lations and methods of the invention stimulate intracellular 0.2-1% w/w. In a particular embodiment the concentration of cGMP production at higher levels than naturally occurring the magnesium stearate is about 0.25% w/w. GCC agonists such as uroguanylin, guanylin, and ST pep 0073. In one embodiment the formulation contains a GCC tides. In some embodiments, the GCC agonists stimulate agonist peptide, microcrystalline cellulose, and magnesium intracellular ccjMP production at higher levels than the pep Stearate. The GCC agonist is at a concentration of less than tide designated SP-304 (SEQID NO:1). In specific embodi 5% w/w, less than 4% w/w, less than 3% w/w, less than 2% ments, a GCC agonist for use in the formulations and methods wfw, less than 1% w/w, less than 0.5% w/w, or less than of the invention stimulates 5%, 10%. 20%, 30%, 40%, 50%, 0.25% w/w. In some embodiments the GCC peptide is at a 75%.90% or more intracellular cGMP compared to urogua concentration of about 1.10% w/w. The GCC peptide is pref nylin, guanylin, lymphoguanylin, linaclotide, ST peptides, or erably SEQ NO: 1 or SEQ NO: 9. The microcrystalline cel SP-304. The terms “induce” and “stimulate” are used inter lulose is at a concentration of at least 30% w/w, at least 40% changeably throughout the specification. w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, at (0078 Preferably, the GCC agonists for use in the formu least 80% w/w, at least 90% w/w, at least 95% w/w, or at least lations and methods of the invention are more stable than 99% w/w. In some embodiments the concentration of the naturally occurring GCC agonists Such as uroguanylin, gua microcrystalline cellulose is about 98.64% w/w. The micro nylin, and ST peptides. In some embodiments, the GCC ago crystalline cellulose is preferably Avicel PH 102. The mag nists are more stable than the peptide designated SP-304. nesium Stearate is at a concentration of at least 0.1% w/w, at "Stability” in this context refers to resistance to degradation least 0.15% w/w, at least 0.2% w/w, or at least 0.25% w/w. In in gastrointestinal fluid and/or intestinal fluid (or simulated Some embodiments the concentration of the magnesium gastrointestinal or intestinal fluids) compared to the reference stearate is about 0.25% w/w. peptide. For example, the GCC agonists for use in the formu 0.074. In one embodiment the formulation contains a GCC lations and methods of the invention preferably degrade 2%, agonist peptide, microcrystalline cellulose, and magnesium 3%. 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 90% or less Stearate. The GCC agonist is at a concentration of less than compared to naturally occurring GCC angonists and/or 5% w/w, less than 4% w/w, less than 3% w/w, less than 2% SP-3O4. wfw, less than 1% w/w, less than 0.5% w/w, or less than 007.9 The GCC agonists for use in the formulations and 0.25% w/w. In some embodiments the GCC peptide is at a methods of the invention are preferably peptides. In some concentration of about 3.32% w/w. The GCC peptide is pref embodiments, the GCC agonist peptide is less than 30 amino erably SEQ NO: 1 or SEQ NO: 9. The microcrystalline cel acids in length. In particular embodiments, the GCC agonist lulose is at a concentration of at least 30% w/w, at least 40% peptide is less than or equal to 30, 25, 20, 15, 14, 13, 12, 11, w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, at 10, or 5 amino acids in length. Examples of GCC agonist least 80% w/w, at least 90% w/w, at least 95% w/w, or at least peptides for use in the formulations and methods of the inven 99% w/w. In some embodiments the concentration of the tion include those described in U.S. Ser. Nos.: 12/133,344, microcrystalline cellulose is about 96.43% w/w. The micro filed Jun. 4, 2008, Ser. No. 12/478,505, filed Jun. 4, 2009; Ser. crystalline cellulose is preferably Avicel PH 102. The mag No. 12/478,511, filed Jun. 4, 2009: Ser. No. 12/504,288, filed nesium Stearate is at a concentration of at least 0.1% w/w, at Jul. 16, 2009; and U.S. Provisional Application Ser. Nos.: least 0.15% w/w, at least 0.2% w/w, or at least 0.25% w/w. In 60/933,194, filed Jun. 4, 2007: 61/058,888, filed Jun. 4, 2008: Some embodiments the concentration of the magnesium 61/058,892, filed Jun. 4, 2008; and 61/081,289, filed Jul. 16, stearate is about 0.25% w/w. 2008, each of which is incorporated by reference herein in its entirety. 1.2 GCC Agonists 0080 Specific examples of GCC agonist peptides for use 0075. The GCC agonists for use in the formulations and in the formulations and methods of the invention include methods of the invention bind to guanylate cyclase C and those described in Tables I-VII below. As used Tables I-VII, stimulate intracellular production of c(GMP. Optionally, the the terms “PEG3' or “3PEG” refer to a polyethylene glycol GCC agonists induce apoptosis and inhibit proliferation of Such as aminoethyloxy-ethyloxy-acetic acid (AeeA), and epithelial cells. The term, “guanylate cyclase C refers to a polymers thereof. The term “X” refers to any natural or transmembrane form of guanylate cyclase that acts as the unnatural amino acid or amino acid analogue. The term intestinal receptor for the heat-stable toxin (ST) peptides “M” refers to a cysteine (Cys), penicillamine (Pen) secreted by enteric bacteria. Guanylate cyclase C is also the homocysteine, or 3-mercaptoproline. The term "Xaa, is receptor for the naturally occurring peptides guanylin and meant to denote an amino acid sequence of any natural or uroguanylin. The possibility that there may be different unnatural amino acid or amino acid analogue that is one, two receptors for each of these peptides has not been excluded. or three residues in length; Xaa, is meant to denote an amino Hence, the term “guanylate cyclase C may also encompass a acid sequence that is Zero or one residue in length; and Xaa, class of transmembrane guanylate cyclase receptors is meant to denote an amino acid sequence Zero, one, two, expressed on epithelial cells lining the gastrointestinal three, four, five or six residues in length. Additionally, any COSa. amino acid represented by Xaa, Xaa, Xaa, or Xaa, may US 2015/0366935 A1 Dec. 24, 2015

bean L-amino acid, a D-amino acid, a methylated amino acid programmed cell death, or activate the cystic fibrosis trans or any combination of thereof. Optionally, any GCC agonist membrane conductance regulator (CFTR). peptide represented by Formulas I to XX in the tables may I0087. In some embodiments, the GCC agonist peptides for contain on or more polyethylene glycol residues at the N-ter use in the formulations and methods of the invention are more minus, C-terminus or both. stable than naturally occurring GCC agonists and/or SP-304 0081. In certain embodiments, a GCC agonist formulation (SEQ ID NO:1), SP-339 (linaclotide) (SEQ ID NO: 55) or of the invention comprises a peptide selected from SEQ ID SP-340 (SEQ ID NO: 56). For example, the GCC agonist NOs: 1-249, the sequences of which are set forth below in peptide degrades 2%. 3%. 5%, 10%, 15%, 20%, 30%, 40%, Tables I to VII below. In one embodiment, a GCC agonist 50%, 75%, 90% or less compared to naturally occurring GCC formulation comprises the peptide designated by SEQ ID agonists and/or SP-304, SP-339 (linaclotide) or SP-340. In NOs: 1, 8, 9,55, or 56. certain embodiments, the GCC agonist peptides for use in the 0082 In certain embodiments, a GCC agonist formulation formulations and methods of the invention are more stable to of the invention comprises a peptide that is Substantially proteolytic digestion than naturally occurring GCC agonists equivalent to a peptide selected from SEQ ID NOs: 1-249. and/or SP-304 (SEQID NO:1), SP-339 (linaclotide) (SEQID The term “substantially equivalent” refers to a peptide that NO: 55) or SP-340 (SEQID NO:56). In one embodiment, a has an amino acid sequence equivalent to that of the binding GCC agonist peptide is pegylated in order to render the pep domain where certain residues may be deleted or replaced tides more resistant towards protealysis by enzymes of the with otheramino acids without impairing the peptide’s ability gastrointestinal tract. In a preferred embodiment, the GCC to bind to an intestinal guanylate cyclase receptor and stimu agonist peptide is pegylated with the aminoethyloxy-ethy late fluid and electrolyte transport. loxy-acetic acid (Aeea) group at its C-terminal end, at its I0083) 1.2.1 GCC Agonist Peptides N-terminal end, or at both termini. 0084. In a preferred embodiment, the GCC agonists for I0088 Specific examples of GCC agonist peptides that can use in the formulations and methods of the invention are GCC be used in the methods and formulations of the invention agonist peptides. In certain embodiments, the GCC agonist include a peptide selected from the group designated by SEQ peptides are analogues of uroguanylin or a bacterial ST pep ID NOS: 1-249. tide. Uroguanylin is a circulating peptide hormone with natri I0089. In one embodiment, the GCC agonist peptide is a uretic activity. An ST peptide is a member of a family of heat peptide having the amino acid sequence of any one of For stable enterotoxins (ST peptides) secreted by pathogenic mulas X-XVII (e.g. SEQID NO:87–98). strains of E. coli and other enteric bacteria that activate gua nylate cyclase receptor and cause secretory diarrhea. Unlike 0090. In some embodiments, GCC agonist peptides bacterial ST peptides, the binding of uroguanylinto guanylate include peptides having the amino acid sequence of Formula cyclase receptor is dependent on the physiological pH of the I, wherein at least one amino acid of Formula I is a D-amino gut. Therefore, uroguanylin is expected to regulate fluid and acid or a methylated amino acid and/or the amino acid at electrolyte transport in a pH dependent manner and without position 16 is a serine. Preferably, the amino acid at position causing severe diarrhea. 16 of Formula I is a D-amino acid or a methylated amino acid. 0085. The GCC agonist peptides for use in the formula For example, the amino acid at position 16 of Formula I is a tions and methods of the invention can be polymers of d-leucine or a d-serine. Optionally, one or more of the amino L-amino acids, D-amino acids, or a combination of both. For acids at positions 1-3 of Formula I are D-amino acids or example, in various embodiments, the peptides are D retro methylated amino acids or a combination of D-amino acids or inverso peptides. The term “retro-inverso isomer refers to an methylated amino acids. For example, Asn', Asp or Glu (or isomer of a linear peptide in which the direction of the a combination thereof) of Formula I is a D-amino acid or a sequence is reversed and the chirality of each amino acid methylated amino acid. Preferably, the amino acid at position residue is inverted. See, e.g., Jameson et al., Nature, 368, Xaa' of Formula I is a leucine, serine or tyrosine. 744-746 (1994); Brady et al., Nature, 368, 692-693 (1994). 0091. In alternative embodiments, GCC agonist peptides The net result of combining D-enantiomers and reverse Syn include peptides having the amino acid sequence of Formula thesis is that the positions of carbonyl and amino groups in II, wherein at least one amino acid of Formula II is a D-amino each amide bond are exchanged, while the position of the acid or a methylated amino acid. Preferably, the amino acid side-chain groups at each alpha carbon is preserved. Unless denoted by Xaa, of Formula II is a D-amino acid or a methy specifically stated otherwise, it is presumed that any given lated amino acid. In some embodiments, the amino acid L-amino acid sequence of the invention may be made into a D denoted by Xaa, of Formula II is a leucine, a d-leucine, a retro-inverso peptide by Synthesizing a reverse of the serine, or ad-serine. Preferably, the one or more amino acids sequence for the corresponding native L-amino acid denoted by Xaa, of Formula II is a D-amino acid or a methy Sequence. lated amino acid. Preferably, the amino acid at positionXaa I0086. The GCC agonist peptides for use in the formula of Formula II is a leucine, a serine, or a tyrosine. tions and methods of the invention are able to induce intrac 0092. In some embodiments, GCC agonist peptides ellular ccjMP production in cells and tissues expressing gua include peptides having the amino acid sequence of Formula nylate cyclase C. In certain embodiments, the GCC agonist III, wherein at least one amino acid of Formula III is a peptide stimulates 5%, 10%, 20%, 30%, 40%, 50%, 75%, D-amino acid or a methylated amino acid and/or Maa is not a 90% or more intracellular ccjMP compared to naturally cysteine. Preferably, the amino acid denoted by Xaa, of occurring GCC agonists such as uroguanylin, guanylin, or ST Formula III is a D-amino acid or a methylated amino acid. In peptides. Optionally, the GCC agonist peptide stimulates 5%, Some embodiments the amino acid denoted by Xaa, of For 10%, 20%, 30%, 40%, 50%, 75%, 90% or more intracellular mula III is a leucine, a d-leucine, a serine, or a d-serine. cGMP compared SP-304 (SEQID NO:1). In further embodi Preferably, the one or more amino acids denoted by Xaa, of ments, the GCC agonist peptide stimulates apoptosis, e.g., Formula III is a D-amino acid or a methylated amino acid. US 2015/0366935 A1 Dec. 24, 2015

Preferably, the amino acid at positionXaa' of Formula III is a amino acid at position 9 of Formula XVIII is a an asparagine. leucine, a serine, or a tyrosine. Preferably, the amino acid at position 10 of Formula XVIII is 0093. In other embodiments, GCC agonist peptides a valine oran methionine. Preferably, the amino acid at posi include peptides having the amino acid sequence of Formula tion 11 of Formula XVIII is an alanine. Preferably, the amino IV, wherein at least one amino acid of Formula IV is a acid at position 13 of Formula XVIII is a threonine. Prefer D-amino acid or a methylated amino acid, and/or Maa is not ably, the amino acid at position 14 of Formula XVIII is a a cysteine. Preferably, the Xaa, of Formula IV is a D-amino glycine. Preferably, the amino acid at position 16 of Formula acid or a methylated amino acid. In some embodiments, the XVIII is a leucine, serine or threonine amino acid denoted by Xaa, of Formula IV is a leucine, a (0099. In alternative embodiments, GCRA peptides d-leucine, a serine, or ad-serine. Preferably, the one or more include peptides containing the amino acid sequence of For of the amino acids denoted by Xaa, of Formula IV is a mula XIX. Preferably, the amino acid at position 1 of Formula D-amino acid or a methylated amino acid. Preferably, the XIX is a serine or asparagine. Preferably, the amino acid at amino acid denoted Xaa' of Formula IV is a leucine, a serine, position 2 of Formula XIX is a histidine or an aspartic acid. or a tyrosine. Preferably, the amino acid at position 3 of Formula XIX is a 0094. In further embodiments, GCC agonist peptides threonine or a glutamic acid. Preferably, the amino acid at include peptides having the amino acid sequence of Formula position 5 of Formula XIX is a glutamic acid. Preferably, the V, wherein at least one amino acid of Formula V is a D-amino amino acid at position 6 of Formula XIX is an isoleucine, acid or a methylated amino acid. Preferably, the amino acid at leucine, Valine or tyrosine. Preferably, the amino acid at posi position 16 of Formula V is a D-amino acid or a methylated tion 8, 10, 11, or 13 of Formula XIX is a alanine. Preferably, amino acid. For example, the amino acid at position 16 (i.e., the amino acid at position 9 of Formula XIX is an asparagine Xaa') of Formula V is a d-leucine or a d-serine. Optionally, or a phenylalanine. Preferably, the amino acid at position 14 one or more of the amino acids at position 1-3 of Formula V of Formula XIX is a glycine. are D-amino acids or methylated amino acids or a combina 0100. In further embodiments, GCRA peptides include tion of D-amino acids or methylated amino acids. For peptides containing the amino acid sequence of Formula XX. example, Asn', Asp or Glu (or a combination thereof) of Preferably, the amino acid at position 1 of Formula XX is a Formula V is a D-amino acids or a methylated amino acid. glutamine Preferably, the amino acid at position 2 or 3 of Preferably, the amino acid denoted at Xaa' of Formula V is a Formula XX is a glutamic acid or a aspartic acid. Preferably, leucine, a serine, or a tyrosine. the amino acid at position 5 of Formula XX is a glutamic acid. 0095. In additional embodiments, GCC agonist peptides Preferably, the amino acid at position 6 of Formula XX is include peptides having the amino acid sequence of Formula threonine, glutamine, tyrosine, isoleucine, or leucine. Prefer VI,VII, VIII, or IX. Preferably, the amino acid at position 6 of ably, the amino acid at position 8 of Formula XX is isoleucine Formula VI, VII, VIII, or IX is a leucine, a serine, or a or valine. Preferably, the amino acid at position 9 of Formula tyrosine. In some aspects the amino acid at position 16 of XX is asparagine. Preferably, the amino acid at position 10 of Formula VI, VII, VIII, or IX is a leucine or a serine. Prefer Formula XX is methionine or valine. Preferably, the amino ably, the amino acid at position 16 of Formula V is a D-amino acid at position 11 of Formula XX is alanine. Preferably, the acid or a methylated amino acid. amino acid at position 13 of Formula XX is a threonione. 0096. In additional embodiments, GCC agonist peptides Preferably, the amino acid at position 1 of Formula XX is a include peptides having the amino acid sequence of Formula glycine. Preferably, the amino acid at position 15 of Formula X, XI, XII, XIII, XIV. XV, XVI or XVII. Optionally, one or XX is a tyrosine. Optionally, the amino acid at position 15 of more amino acids of Formulas X, XI, XII, XIII, XIV, XV, XVI Formula XX is two amino acid in length and is Cysteine or XVII is a D-amino acid or a methylated amino acid. Pref (CyS), Penicillamine (Pen) homocysteine, or 3-mercaptopro erably, the amino acid at the carboxy terminus of the peptides line and serine, leucine or threonine. according to Formulas X, XI, XII, XIII, XIV. XV, XVI or 0101. In certain embodiments, one or more amino acids of XVII is a D-amino acid or a methylated amino acid. For the GCC agonist peptides are replaced by a non-naturally example the amino acid at the carboxy terminus of the pep occurring amino acid or a naturally or non-naturally occur tides according to Formulas X, XI, XII, XIII, XIV,XV,XVI or ring amino acid analog. Such amino acids and amino acid XVII is a D-tyrosine. analogs are known in the art. See, for example, Hunt, “The 0097. Preferably, the amino acid denoted by Xaa' of For Non-Protein Amino Acids.” in Chemistry and Biochemistry mula XIV is a tyrosine, phenyalanine or a serine. Most pref of the Amino Acids, Barrett, Chapman and Hall, 1985. In erably the amino acid denoted by Xaa' of Formula XIV is a Some embodiments, an amino acid is replaced by a naturally phenyalanine or a serine. Preferably, the amino acid denoted occurring, non-essential amino acid, e.g., taurine. Non-limit by Xaa' of Formula XV. XVI or XVII is a tyrosine, a phen ing examples of naturally occurring amino acids that can be yalanine, or a serine. Most preferably, the amino acid position replaced by non-protein amino acids include the following: Xaa' of Formula V. XVI or XVII is a phenyalanine or a serine. (1) an aromatic amino acid can be replaced by 3,4-dihydroxy 0098. In some embodiments, GCRA peptides include pep L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine, L-thy tides containing the amino acid sequence of Formula XVIII. roXine, phenylglycine (Phg) or nor-tyrosine (norTyr); (2) Phg Preferably, the amino acid at position 1 of Formula XVIII is a and norTyr and other amino acids including Phe and Tyrcan glutamic acid, aspartic acid, glutamine or lysine. Preferably, be substituted by, e.g., a halogen, —CH3, —OH, the amino acid at position 2 and 3 of Formula XVIII is a CH2NH3, —C(O)H, CH2CH3, CN, glutamic acid, or an aspartic acid. Preferably, the amino acid —CH2CH2CH3, SH, or another group; (3) glutamine resi at position 5 a glutamic acid. Preferably, the amino acid at dues can be substituted with gamma-Hydroxy-Glu or position 6 of Formula XVIII is an isoleucine, valine, serine, gamma-Carboxy-Glu; (4) tyrosine residues can be substi threonine or tyrosine. Preferably, the amino acid at position 8 tuted with an alpha Substituted amino acid Such as L-alpha of Formula XVIII is a valine or isoleucine. Preferably, the methylphenylalanine or by analogues such as: 3-Amino-Tyr; US 2015/0366935 A1 Dec. 24, 2015

Tyr(CH3); Tyr(PO3(CH3)2); Tyr(SO3H); beta-Cyclohexyl derivative; aminobutyric acid (Abu); citrulline; aminohex Ala; beta-(1-Cyclopentenyl)-Ala; beta-Cyclopentyl-Ala; anoic acid; aminoisobutyric acid (AIB); cyclohexylalanine; beta-Cyclopropyl-Ala; beta-Quinolyl-Ala; beta-(2-Thiaz d-cyclohexylalanine, hydroxyproline; nitro-arginine; nitro olyl)-Ala; beta-(Triazole-1-yl)-Ala; beta-(2-Pyridyl)-Ala; phenylalanine; nitro-tyrosine; norvaline; octahydroindole beta-(3-Pyridyl)-Ala; Amino-Phe: Fluoro-Phe, Cyclohexyl carboxylate; ornithine (Orn); penicillamine (PEN); tetrahy Gly; thBu-Gly; beta-(3-benzothienyl)-Ala; beta-(2-thienyl)- droisoquinoline; acetamidomethyl protected amino acids and Ala; 5-Methyl-Trp.; and A-Methyl-Trp.; (5) proline residues pegylated amino acids. Further examples of unnatural amino can be substituted with homopro (L-pipecolic acid); hydroxy acids and amino acid analogs can be found in U.S. Pro; 3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro or 20030108885, U.S. 20030082575, US20060019347 (para an N(alpha)-C(alpha) cyclized amino acid analogues with the graphs 410-418) and the references cited therein. The structure: n=0, 1, 2, 3; and (6) alanine residues can be substi polypeptides of the invention can include further modifica tuted with alpha-substituted or N-methylated amino acid such tions including those described in US20060019347, para as alpha-amino isobutyric acid (aib), L/D-alpha-ethylalanine graph 589. Exemplary GCC agonist peptides which include a (L/D-isovaline), L/D-methylvaline, or L/D-alpha-methyleu non-naturally occurring amino acid include for example cine or a non-natural amino acid Such as beta-fluoro-Ala. SP-368 and SP-369. Alanine can also be substituted with: n=0, 1, 2, 3 Glycine 0103) In some embodiments, the GCC agonist peptides residues can be substituted with alpha-amino isobutyric acid are cyclic peptides. GCC agonist cyclic peptides can be pre (aib) or L/D-alpha-ethylalanine (L/D-isovaline). pared by methods known in the art. For example, macrocy 0102. Further examples of non-natural amino acids clization is often accomplished by forming an amide bond include: an unnatural analog of tyrosine; an unnatural ana between the peptide N- and C-termini, between a side chain logue of glutamine; an unnatural analogue of phenylalanine; and the N- or C-terminus (e.g., with KFe(CN) at pH 8.5 an unnatural analogue of serine; an unnatural analogue of (Samson et al., Endocrinology, 137: 5182-5185 (1996)), or threonine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, between two amino acid side chains, such as cysteine. See, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, Sulfonyl, e.g., DeGrado, Adv. Protein Chem, 39: 51-124 (1988). In Seleno, ester, thioacid, borate, boronate, phospho, various embodiments, the GCC agonist peptides are 4,12; phosphono, phosphine, heterocyclic, enone, imine, aldehyde, 7.15 bicycles. hydroxylamine, keto, or amino Substituted amino acid, or any 0104. In certain embodiments, one or both Cys residues combination thereof, an amino acid with a photoactivatable which normally form a disulfide bond in a GCC agonist cross-linker; a spin-labeled amino acid; a fluorescent amino peptide are replaced with homocysteine, penicillamine, acid; an amino acid with a novel functional group; an amino 3-mercaptoproline (Kolodziejet al. 1996 Int. J. Pept. Protein acid that covalently or noncovalently interacts with another Res. 48:274), B.B dimethylcysteine (Hunt et al. 1993 Int. J. molecule; a metal binding amino acid; an amino acid that is Pept. Protein Res. 42:249), or diaminopropionic acid (Smith amidated at a site that is not naturally amidated, a metal et al. 1978.J. Med. Chem. 21:117) to form alternative internal containing amino acid; a radioactive amino acid; a photo cross-links at the positions of the normal disulfide bonds. caged and/or photoisomerizable amino acid; a biotin or 0105. In certain embodiments, one or more disulfide biotin-analogue containing amino acid; a glycosylated or car bonds in a GCC agonist peptide are replaced by alternative bohydrate modified amino acid; a keto containing amino covalent cross-links, e.g., an amide linkage (-CH2CH(O) acid; amino acids comprising polyethylene glycol or poly NHCH- or —CH-NHCH(O)CH ), an ester linkage, a ether; a heavy atom Substituted amino acid (e.g., an amino thioester linkage, a lactambridge, a carbamoyl linkage, aurea acid containing deuterium, tritium, C, N, or 'O); a linkage, a thiourea linkage, a phosphonate ester linkage, an chemically cleavable orphotocleavable amino acid; an amino alkyl linkage (-CH2CH2CHCH ), an alkenyl linkage acid with an elongated side chain; an amino acid containing a ( CH-CH=CHCH ), al ether linkage toxic group; a Sugar Substituted amino acid, e.g., a Sugar (—CHCHOCH or —CHOCHCH ), a thioether Substituted serine or the like; a carbon-linked Sugar-contain linkage ( CHCH-SCH or CH-SCHCH ), an ing amino acid; a redox-active amino acid; an O-hydroxy amine linkage ( CHCH-NHCH- O containing acid; an amino thio acid containing amino acid; an —CH-NHCHCH ) or a thioamide linkage (—CHCH(S) C.C. disubstituted amino acid; a B-amino acid; a cyclic amino HNHCH-or-CHNHCH(S)CH, ). For example, Ledu acid other than proline; an O-methyl-L-tyrosine; an L-3-(2- et al. (Proc. Natl. Acad. Sci. 100:11263-78, 2003) describe naphthyl)alanine; a 3-methyl-phenylalanine; a p-acetyl-L- methods for preparing lactam and amide cross-links. Exem phenylalanine; an O-4-allyl-L-tyrosine; a 4-propyl-L-ty plary GCC agonist peptides which include a lactam bridge rosine; a tri-O-acetyl-GlcNAc B -serine; an L-Dopa; a include, for example, SP-370. fluorinated phenylalanine; an isopropyl-L-phenylalanine; a 0106. In certain embodiments, the GCC agonist peptides p-azido-L-phenylalanine; a p-acyl-L-phenylalanine; a p-ben have one or more conventional polypeptide bonds replaced by Zoyl-L-phenylalanine; an L-phosphoserine; a phospho an alternative bond. Such replacements can increase the sta noSerine; a phosphonotyrosine; a p-iodo-phenylalanine; a bility of the polypeptide. For example, replacement of the 4-fluorophenylglycine; a p-bromophenylalanine; a p-amino polypeptide bond between a residue amino terminal to an L-phenylalanine; an isopropyl-L-phenylalanine; L-3-(2- aromatic residue (e.g. Tyr, Phe, Trp) with an alternative bond naphthyl)alanine: D-3-(2-naphthyl)alanine (dNal); an can reduce cleavage by carboxypeptidases and may increase amino-, isopropyl-, or O-allyl-containing phenylalanine ana half-life in the digestive tract. Bonds that can replace logue; a dopa, O-methyl-L-tyrosine; a glycosylated amino polypeptide bonds include: a retro-inverso bond (C(O) NH acid; a p-(propargyloxy)phenylalanine; dimethyl-Lysine; instead of NH C(O); a reduced amide bond (NH-CH-); a hydroxy-proline; mercaptopropionic acid; methyl-lysine; thiomethylene bond (S CH or CH S); an oxomethylene 3-nitro-tyrosine; norleucine, pyro-glutamic acid; Z (Car bond (O CH or CH O); an ethylene bond (CH, CH): bobenzoxyl); e-Acetyl-Lysine; B-alanine; aminobenzoyl a thioamide bond (C(S)- NH); a trans-olefine bond US 2015/0366935 A1 Dec. 24, 2015

(CH=CH); a fluoro substituted trans-olefime bond 0112 1.2.2 Preparation of GCC Agonist Peptides (CF–CH); a ketomethylene bond (C(O) CHR or CHR 0113 GCC agonist peptides can be prepared using art C(O) wherein R is H or CH; and a fluoro-ketomethylene recognized techniques such as molecular cloning, peptide bond (C(O) CFR or CFR C(O) wherein R is H or For synthesis, or site-directed mutagenesis. CH. 0114 Peptide synthesis can be performed using standard 0107. In certain embodiments, the GCC agonist peptides Solution phase or solid phase peptide synthesis techniques or are modified using standard modifications. Modifications a combination of both process where segments are synthe may occur at the amino (N-), carboxy (C-) terminus, inter sized by Solid phase and condensed in Solution phase, in nally or a combination of any of the preceeding. In one aspect which a peptide linkage occurs through the direct condensa described herein, there may be more than one type of modi tion of the amino group of one amino acid with the carboxy fication on the polypeptide. Modifications include but are not group of the other amino acid with the elimination of a water limited to: acetylation, amidation, biotinylation, cinnamoyla molecule. Peptide bond synthesis by direct condensation, as tion, farnesylation, formylation, myristoylation, palmitoyla formulated above, requires Suppression of the reactive char tion, phosphorylation (Ser, Tyr or Thr), Stearoylation, Succi acter of the amino group of the first and of the carboxyl group nylation, Sulfurylation and cyclisation (via disulfide bridges of the second amino acid. The masking Substituents must oramide cyclisation), and modification by Cys3 or Cys5. The permit their ready removal, without inducing breakdown of GCC agonist peptides described herein may also be modified the labile peptide molecule. by 2,4-dinitrophenyl (DNP). DNP-lysine, modification by 0.115. In solution phase synthesis, a wide variety of cou 7-Amino-4-methyl-coumarin (AMC), flourescein, NBD pling methods and protecting groups may be used (See, Gross (7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide, and Meienhofer, eds., “The Peptides: Analysis, Synthesis, rhodamine B, EDANS (5-((2-aminoethyl)amino)naphtha Biology. Vol. 1-4 (Academic Press, 1979); Bodansky and lene-1-sulfonic acid), dabcyl, dabsyl, dansyl, texas red, Bodansky, “The Practice of Peptide Synthesis. 2d ed. FMOC, and Tamra (Tetramethylrhodamine). The GCC ago (Springer Verlag, 1994)). In addition, intermediate purifica nist peptides described herein may also be conjugated to, for tion and linear scale up are possible. Those of ordinary skill in example, polyethylene glycol (PEG); alkyl groups (e.g., the art will appreciate that Solution synthesis requires consid C1-C20 straight or branched alkyl groups); fatty acid radi eration of main chain and side chain protecting groups and cals; combinations of PEG, alkyl groups and fatty acid radi activation method. In addition, careful segment selection is cals (See, U.S. Pat. No. 6,309,633; Soltero et al., 2001 Inno necessary to minimize racemization during segment conden vations in Pharmaceutical Technology 106-110); BSA and sation. Solubility considerations are also a factor. Solid phase KLH (Keyhole Limpet Hemocyanin). The addition of PEG peptide synthesis uses an insoluble polymer for Support dur and other polymers which can be used to modify polypeptides ing organic synthesis. The polymer-supported peptide chain of the invention is described in US20060 19347 section IX. permits the use of simple washing and filtration steps instead 0108. A GCC agonist peptide can also be a derivatives of of laborious purifications at intermediate steps. Solid-phase a GCC agonist peptide described herein. For example, a peptide synthesis may generally be performed according to derivative includes hybrid and modified forms of GCC ago the method of Merrifield et al., J. Am. Chem. Soc., 1963, nist peptides in which certain amino acids have been deleted 85:21.49, which involves assembling a linear peptide chain on or replaced. A modification may also include glycosylation. a resin Support using protected amino acids. Solid phase Preferrably, where the modification is an amino acid substi peptide synthesis typically utilizes either the Boc or Fmoc tution, it is a conservative Substitution at one or more posi strategy, which are well known in the art. tions that are predicted to be non-essential amino acid resi 0116. Those of ordinary skill in the art will recognize that, dues for the biological activity of the peptide. A “conservative in Solid phase synthesis, deprotection and coupling reactions substitution' is one in which the amino acid residue is must go to completion and the side-chain blocking groups replaced with an amino acid residue having a similar side must be stable throughout the synthesis. In addition, solid chain. Families of amino acid residues having similar side phase synthesis is generally most Suitable when peptides are chains have been defined in the art. These families include to be made on a small scale. amino acids with basic side chains (e.g., lysine, arginine, 0117 Acetylation of the N-terminal can be accomplished histidine), acidic side chains (e.g., aspartic acid, glutamic by reacting the final peptide with acetic anhydride before acid), uncharged polar side chains (e.g., glycine, asparagine, cleavage from the resin. C-amidation is accomplished using glutamine, serine, threonine, tyrosine, cysteine), nonpolar an appropriate resin Such as methylbenzhydrylamine resin side chains (e.g., alanine, Valine, leucine, isoleucine, proline, using the Boc technology. phenylalanine, methionine, tryptophan), beta-branched side 0118. Alternatively the GCC agonist peptides are pro chains (e.g., threonine, Valine, isoleucine) and aromatic side duced by modern cloning techniques For example, the GCC chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). agonist peptides are produced either in bacteria including, 0109. In one embodiment, a GCC agonist peptide without limitation, E. coli, or in other existing systems for described herein is Subjected to random mutagenesis in order polypeptide or protein production (e.g., Bacillus subtilis, to identify mutants having biological activity. baculovirus expression systems using Drosophila Sf9 cells, 0110. In one embodiment, the GCC agonist peptide is yeast or filamentous fungal expression systems, mammalian Substantially homologous is a GCC agonist peptide described cell expression systems), or they can be chemically synthe herein. Such substantially homologous peptides can be iso sized. If the GCC agonist peptide or variant peptide is to be lated by virtue of cross-reactivity with antibodies to a GCC produced in bacteria, e.g., E. coli, the nucleic acid molecule agonist peptide described herein. encoding the polypeptide may also encode a leader sequence 0111. Further examples of GCC agonist peptides that can that permits the secretion of the mature polypeptide from the be used in the methods and formulations of the invention are cell. Thus, the sequence encoding the polypeptide can include found in Tables I-VII below. the pre sequence and the pro sequence of for example, a US 2015/0366935 A1 Dec. 24, 2015 naturally-occurring bacterial ST polypeptide. The secreted, I0121 The protein coding sequence that includes a GCC mature polypeptide can be purified from the culture medium. agonist peptide described hereincan also be fused to a nucleic 0119 The sequence encoding a GCC agonist peptide acid encoding a polypeptide affinity tag, e.g., glutathione described herein can be inserted into a vector capable of S-transferase (GST), maltose E binding protein, protein A, delivering and maintaining the nucleic acid molecule in a FLAG tag, hexa-histidine, myc tag or the influenza HA tag, in bacterial cell. The DNA molecule may be inserted into an order to facilitate purification. The affinity tag or reporter autonomously replicating vector (Suitable vectors include, for fusion joins the reading frame of the polypeptide of interest to example, pGEM37 and pcDNA3, and derivatives thereof). the reading frame of the gene encoding the affinity tag. Such The vector nucleic acid may be a bacterial or bacteriophage that a translational fusion is generated. Expression of the DNA such as bacteriophage lambda or M13 and derivatives fusion gene results in translation of a single polypeptide that thereof. Construction of a vector containing a nucleic acid includes both the polypeptide of interest and the affinity tag. described herein can be followed by transformation of a host In some instances where affinity tags are utilized, DNA cell such as a bacterium. Suitable bacterial hosts include but sequence encoding a protease recognition site will be fused are not limited to, E. coli, B subtilis, Pseudomonas, Salmo between the reading frames for the affinity tag and the nella. The genetic construct also includes, in addition to the polypeptide of interest. encoding nucleic acid molecule, elements that allow expres Sion, such as a promoter and regulatory sequences. The 0.122 Genetic constructs and methods suitable for produc expression vectors may contain transcriptional control tion of immature and mature forms of the GCC agonist pep sequences that control transcriptional initiation, Such as pro tides and variants described herein in protein expression sys moter, enhancer, operator, and repressor sequences. tems other than bacteria, and well known to those skilled in 0120 A variety of transcriptional control sequences are the art, can also be used to produce polypeptides in a biologi well known to those in the art. The expression vector can also cal system. include a translation regulatory sequence (e.g., an untrans I0123. The peptides disclosed herein may be modified by lated 5' sequence, an untranslated 3' sequence, or an internal attachment of a second molecule that confers a desired prop ribosome entry site). The vector can be capable of autono erty upon the peptide. Such as increased half-life in the body, mous replication or it can integrate into host DNA to ensure for example, pegylation. Such modifications also fall within stability during polypeptide production. the scope of the term “variant” as used herein. US 2015/0366935 A1 Dec. 24, 2015 17

US 2015/0366935 A1 Dec. 24, 2015 18

penu?quoo–IETTEVIL US 2015/0366935 A1 Dec. 24, 2015 19

penu?quoo–IETTEVIL US 2015/0366935 A1 Dec. 24, 2015 20 US 2015/0366935 A1 Dec. 24, 2015 21

penU?quoo–IIETTEVIL US 2015/0366935 A1 Dec. 24, 2015 22

penU?quoo–IIETTEVIL

US 2015/0366935 A1 Dec. 24, 2015 24

US 2015/0366935 A1 Dec. 24, 2015 25

penu??Uoo–AIETI@HVIL

US 2015/0366935 A1 Dec. 24, 2015 32

TABLE VI - continued Lymphoguanylin and Analogs

Position of Disulfide SEQ Name bonds Structure ID NO

N160 C4: C12, Gln-Glu-Asp-Cys'-Glu-Ile-Cys-Ile-Asn-Met-Ala- 241 C7 : C1s cys 2-Thr-Gly 14-cysis-serie

TABLE WII ST Peptide and Analogues

Position SEQ ID Name of Disulfide bonds Structure NO ST C3: C8, C4: C12, Asn-Ser-Ser-Asn-Ser-Ser-Asn-Tyr-Cys- Cys-Glu-Lys - Cys- 242 Peptide C7: 15 Cys-Asn' -Pro-Ala- cysis -Thr 19 -Gly29- Cys?'- Tyr22 N161 C3: C8, C4: C12, PEG3-Asn-Phe-Cys- Cys-Glu-Thr- Cys 7-Cys-Asn-Pro-Ala- 243 C7 : 1s Cys'2-Thr-Gly-Cys-Tyr-PEG3 N162 C3: C8, C4: C12, PEG3-Asn-Phe-Cys- Cys-Glu-Thr- Cys 7-Cys-Asn-Pro-Ala- 244 C7 : 1s cys 2-Thr-G y 14-cysis-Tyr16 N163 C3: C8, C4: C12, Asn-Phe-Cys-Cys'-Glu-Thr-Cys"- Cys-Asn-Pro-Alla'- 245 C7 : 1s cys 2-Thr-Gly 14- Cys-Tyr16-PEG3 N164 C3: C8, C4: C12, Asn-Phe-Cys-Cys'-Glu-Tyr-Cys"- Cys-Asn-Pro-Alla'- 246 C7 : 1s Cys'2-Thr-G y 4-cys-Tyr16 N165 C3: C8, C4: C12, dAsn-Phe-Cys-Cys'-Glu-Tyr- Cys 7-Cys-Asn-Pro-Ala- 247 C7 : 1s Cys'2-Thr-G y-Cys-dTyr N166 C3: C8, C4: C12, Asn-Phe-Cys- Cys-Glu-Tyr- Cys 7- Cys-Asn-Pro-Ala- 248 C7 : 1s cys 2-Thr-Gly 14- Cys-dTyr16 N167 C3: C8, C4: C12, dAsn-Phe-Cys-Cys'-Glu-Tyr- Cys 7-Cys-Asn-Pro-Ala- 249 C7 : 1s cys 2-Thr-G y 14-cysis-Tyr16

1.3 Methods of Use I0126. In one embodiment, the invention provides methods for treating or preventing constipation and/or increasing gas 0.124. The invention provides methods for treating or pre- trointestinal motility in a subject in need thereof by adminis venting gastrointestinal disorders and increasing gastrointes- tering an effective amount of a GCC agonist formulation to tinal motility in a Subject in need thereof by administering an the Subject. Clinically accepted criteria that define constipa effective amount of a GCC agonist formulation to the subject. tion range from the frequency of bowel movements, the con Non-limiting examples of gastrointestinal disorders that can sistency of feces and the ease of bowel movement. One com be treated or prevented according to the methods of the inven- mon definition of constipation is less than three bowel tion include irritable bowel syndrome (IBS), non-ulcer dys- movements per week. Other definitions include abnormally pepsia, chronic intestinal pseudo-obstruction, functional dys- hard stools or defecation that requires excessive straining pepsia, colonic pseudo-obstruction, duodenogastric reflux, (Schiller 2001 Aliment Pharmacol Ther 15:749-763). Consti gastroesophageal reflux disease (GERD), ileus (e.g., post- pation may be idiopathic (functional constipation or slow operative ileus), gastroparesis, heartburn (high acidity in the transit constipation) or secondary to other causes including GI tract), constipation (e.g., constipation associated with use neurologic, metabolic or endocrine disorders. These disor of medications such as opioids, osteoarthritis drugs, or ders include diabetes mellitus, hypothyroidism, hyperthy osteoporosis drugs); post Surgical constipation, constipation roidism, hypocalcaemia, Multiple Sclerosis, Parkinson's dis associated with neuropathic disorders, Crohn's disease, and ease, spinal cord lesions, Neurofibromatosis, autonomic ulcerative colitis. neuropathy, Chagas disease, Hirschsprung disease and cystic 0.125. In one embodiment, the invention provides methods fibrosis. Constipation may also be the result of surgery or due for treating or preventing gastrointestinal motility disorder, to the use of drugs such as analgesics (like opioids), antihy irritable bowel syndrome, a functional gastrointestinal disor pertensives, anticonvulsants, antidepressants, antispasmod der, gastroesophageal reflux disease, duodenogastric reflux, ics and antipsychotics. functional heartburn, dyspepsia, functional dyspepsia, non- I0127. In various embodiments, the constipation is associ ulcer dyspepsia, gastroparesis, chronic intestinal pseudo-ob- ated with use of a therapeutic agent; the constipation is asso struction, colonic pseudo-obstruction, obesity, congestive ciated with a neuropathic disorder, the constipation is post heart failure, or benign prostatic hyperplasia. Surgical constipation; the constipation is associated with a US 2015/0366935 A1 Dec. 24, 2015 gastrointestinal disorder, the constipation is idiopathic (func gastrointestinal system inflammation (e.g., Crohn's disease tional constipation or slow transit constipation); the consti and ulcerative colitis); necrotizing enterocolitis (NEC); pan pation is associated with neuropathic, metabolic or endocrine creatic inflammation (e.g., pancreatis), lung inflammation disorder (e.g., diabetes mellitus, hypothyroidism, hyperthy (e.g., bronchitis or asthma) or skin inflammation (e.g., pso roidism, hypocalcaemia, Multiple Sclerosis, Parkinson's dis riasis, eczema). ease, spinal cord lesions, neurofibromatosis, autonomic neu I0135) Lung Disorders include for example chronic ropathy, Chagas disease, Hirschsprung disease or cystic obstructive pulmonary disease (COPD), and fibrosis. fibrosis). Constipation may also be the result of Surgery or due 0.136 Cancer includes tissue and organ carcinogenesis to the use of drugs such as analgesics (e.g., opioids), antihy including metastases such as for example gastrointestinal pertensives, anticonvulsants, antidepressants, antispasmod cancer, (e.g., gastric cancer, esophageal cancer, pancreatic ics and antipsychotics. cancer colorectal cancer, intestinal cancer, anal cancer, liver 0128. In one embodiment, the invention provides methods cancer, gallbladder cancer, or colon cancer; lung cancer, thy for treating or preventing chronic idiopathic constipation and roid cancer, skin cancer (e.g., melanoma); oral cancer, uri increasing gastrointestinal motility in a Subject in need nary tract cancer (e.g. bladder cancer or kidney cancer); blood thereof by administering an effective amount of a GCC ago cancer (e.g. myeloma or leukemia) or prostate cancer. nist formulation to the subject. 0.137 Cardiac disorders include for example, congestive 0129. The term “treating as used herein refers to a reduc heart failure, trachea cardia hypertension, high cholesterol, or tion, apartial improvement, amelioration, or a mitigation of at high triglycerides. Cardiovascular disorders include for least one clinical symptom associated with the gastrointesti example aneurysm, angina, atherosclerosis, cerebrovascular nal disorders being treated. The term “preventing refers to an accident (stroke), cerebrovasculardisease, congestive heart inhibition or delay in the onset or progression of at least one failure, coronary artery disease, myocardial infarction (heart clinical symptom associated with the gastrointestinal disor attack), or peripheral vascular disease. ders to be prevented. The term “effective amount’ as used 0.138 Liver disorders include for example cirrhosis and herein refers to an amount that provides some improvement fibrosis. In addition, GC-C agonist may also be useful to or benefit to the subject. In certain embodiments, an effective facilitate liver regeneration in liver transplant patients. Eye amount is an amount that provides some alleviation, mitiga disorders include for example increased intra-ocular pres tion, and/or decrease in at least one clinical symptom of the Sure, glaucoma, dry eyes retinal degeneration, disorders of gastrointestinal disorder to be treated. In other embodiments, tear glands or eye inflammation. Skin disorders include for the effective amount is the amount that provides some inhi example Xerosis. Oral disorders include for example dry bition or delay in the onset or progression of at least one mouth (xerostomia), Sjögren's syndrome, gum diseases (e.g., clinical symptom associated with the gastrointestinal disor periodontal disease), or salivary gland duct blockage or mal der to be prevented. The therapeutic effects need not be com function. Prostate disorders include for example benign pro plete or curative, as long as Some benefit is provided to the static hyperplasia (BPH). Endocrine disorders include for subject. The term “subject’ preferably refers to a human example diabetes mellitus, hyperthyroidism, hypothyroid Subject but may also refer to a non-human primate or other ism, and cystic fibrosis. mammal preferably selected from among a mouse, a rat, a I0139 1.3.1 Therapeutically Effective Dosages dog, a cat, a cow, a horse, or a pig. 0140 Disorders are treated, prevented or alleviated by 0130. The invention also provides methods for treating administering to a subject, e.g., a mammal Such as a human in gastrointestinal cancer in a Subject in need thereof by admin need thereof, a therapeutically effective dose of a GCC ago istering an effective amount of a GCC agonist formulation to nist peptide. The present invention is based in part on the the Subject. Non-limiting examples of gastrointestinal can unexpected results of clinical trials in humans which demon cers that can be treated according to the methods of the strated that the formulations of the invention are therapeuti invention include gastric cancer, esophageal cancer, pancre cally effective at much lower doses than predicted based on atic cancer, colorectal cancer, intestinal cancer, anal cancer, animal studies. In accordance with one aspect of the inven liver cancer, gallbladder cancer, or colon cancer. tion, the therapeutically effective dose is between 0.01 milli 0131 The invention also provides methods for treating grams (mg) and 10 mg per unit dose. The term “unit dose' lipid metabolism disorders, biliary disorders, inflammatory refers to a single drug delivery entity, e.g., a tablet, capsule, disorders, lung disorders, cancer, cardiac disorders including Solution or inhalation formulation. In one embodiment, the cardiovascular disorders, eye disorders, oral disorders, blood effective dose is between 0.01 mg and 9 mg. In another disorders, liver disorders, skin disorders, prostate disorders, embodiment, the effective dose is between 0.01 mg and 5 mg. endocrine disorders, and obesity. In another embodiment, the effective dose is between 0.01 mg 0132 Lipid metabolism disorders include, but are not lim and 3 mg. In another embodiment, the effective dose is ited to, dyslipidemia, hyperlipidemia, hypercholesterolemia, between 0.10 mg and 5 mg. In another embodiment, the hypertriglyceridemia, sitosterolemia, familial hypercholes effective dose is between 0.10 mg and 3 mg. In one embodi terolemia, Xanthoma, combined hyperlipidemia, lecithin cho ment, the unit dose is 0.01 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 lesterol acyltransferase deficiency, tangier disease, abetalipo mg, 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 5 mg. or proteinemia, erectile dysfunction, fatty liver disease, and 10 mg. In one embodiment, the unit dose is 0.3 mg, 1.0 mg. hepatitis. 3.0 mg, 9.0 mg. or 9.5 mg. 0.133 Billary disorders include gallbladder disorders such 0.141. The GCC agonist peptides may be in a pharmaceu as for example, gallstones, gallbladder cancer cholangitis, or tical composition in unit dose form, together with one or more primary Sclerosing cholangitis; or bile duct disorders such as pharmaceutically acceptable excipients. The amount of pep for example, cholecystitis, bile duct cancer or fascioliasis. tide present should be sufficient to have a positive therapeutic 0134) Inflammatory disorders include tissue and organ effect when administered to a patient. What constitutes a inflammation Such as kidney inflammation (e.g., nephritis), “positive therapeutic effect” will depend upon the particular US 2015/0366935 A1 Dec. 24, 2015 34 condition being treated and will include any significant for the treatment or prevention of a gastrointestinal disease or improvement in a condition readily recognized by one of skill disorder. In some embodiments, the GCC agonist formulation in the art. comprises one or more additional therapeutic agents. In other 0142. The GCC agonists for use in the methods described embodiments, the GCC agonist is formulated separately from above are preferably administered orally. Dosage forms the one or more additional therapeutic agents. In accordance include Solutions, Suspensions, emulsions, tablets, and cap with this embodiment, the GCC agonist is administered either Sules. simultaneously, sequentially, or at a different time than the 0143. The total daily dose can be administered to the one or more additional therapeutic agents. In one embodi patient in a single dose, or in multiple Sub-doses. Typically, ment, the GCC agonist formulation is administered in com Sub-doses can be administered two to six times per day, pref bination with one or more additional therapeutic agents erably two to four times per day, and even more preferably selected from the group consisting of phosphodiesterase two to three times per day. Preferably, a single daily dose is inhibitors, cyclic nucleotides (such as cCMP and cAMP), a administered. laxative (such as SENNA or METAMUCIL), a stool softner, 0144. The GCC agonists may be administered as either the an anti-tumor necrosis factor alpha therapy for IBD (Such as sole active agent or in combination with one or more addi REMICADE, ENBREL, or HUMIRA), and anti-inflamma tional active agents. In all cases, additional active agents tory drugs (such as COX-2 inhibitors, sulfasalazine, 5-ASA should be administered at a dosage that is therapeutically derivatives and NSAIDS). In certain embodiments, the GCC effective using the existing art as a guide. The GCC agonists agonist formulation is administered in combination with an may be administered in a single composition or sequentially effective dose of an inhibitor of c(GMP-specific phosphodi with the one or more additional active agents. In one embodi esterase (cGMP-PDE) either concurrently or sequentially ment, the GCC agonist is administered in combination with with said GCC agonist. c6MP-PDE inhibitors include, for one or more inhibitors of c(GMP dependent phosphodi example, Suldinac Sulfone, Zaprinast, motapizone, Vardenifil. esterase such as Suldinac Sulfone, Zaprinast, motapizone, and sildenafil. In another embodiment, the GCC agonist for Vardenafil, or sildenifil. In another embodiment, the GCC mulation is administered in combination with inhibitors of agonist is administered in combination with one or more cyclic nucleotide transporters. Further examples of therapeu chemotherapeutic agents. In another embodiment, the GCC tic agents that may be administered in combination with the agonist is administered in combination with one or more or GCC agonist formulations of the invention are given in the anti-inflammatory drugs such as Steroids or non-steroidal following sections. anti-inflammatory drugs (NSAIDS), such as aspirin. 0145 Combination therapy can be achieved by adminis 1.3.2.1 Agents to Treat Gastrointestinal Cancers tering two or more agents, e.g., a GCC agonist peptide 0150. The GCC agonist formulations described herein can described herein and another compound, each of which is be used in combination with one or more antitumor agents formulated and administered separately, or by administering including but not limited to alkylating agents, epipodophyl two or more agents in a single formulation. Other combina lotoxins, nitrosoureas, anti-metabolites, vinca alkaloids, tions are also encompassed by combination therapy. For anthracycline antibiotics, nitrogen mustard agents, and the example, two agents can be formulated together and admin like. Particular antitumor agents include tamoxifen, taxol. istered in conjunction with a separate formulation containing etoposide, and 5-fluorouracil. In one embodiment, the GCC a third agent. While the two or more agents in the combination agonist formulations are used in combination with an antivi therapy can be administered simultaneously, they need not be. ral agent or a monoclonal antibody. For example, administration of a first agent (or combination 0151. Non-limiting examples of antitumor agents that can of agents) can precede administration of a second agent (or be used in combination with the GCC agonist formulations of combination of agents) by minutes, hours, days, or weeks. the invention for the treatment of colon cancer include anti Thus, the two or more agents can be administered within proliferative agents, agents for DNA modification or repair, minutes of each other or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 DNA synthesis inhibitors, DNA/RNA transcription regula hours of each other or within 1,2,3,4,5,6,7,8,9, 10, 12, 14 tors, RNA processing inhibitors, agents that affect protein days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks expression, synthesis and stability, agents that affect protein of each other. In some cases even longer intervals are pos localization or their ability to exert their physiological action, sible. While in many cases it is desirable that the two or more agents that interfere with protein-protein or protein-nucleic agents used in a combination therapy be present in within the acid interactions, agents that act by RNA interference, recep patient’s body at the same time, this need not be so. tor binding molecules of any chemical nature (including 0146 The GCC agonist peptides described herein may be Small molecules and antibodies), targeted toxins, enzyme combined with phosphodiesterase inhibitors, e.g., Sulindae activators, enzyme inhibitors, gene regulators, HSP-90 sulfone, Zaprinast, sildenafil. Vardenafil or tadalafil to further inhibitors, molecules interfering with microtubules or other enhance levels of c(3MP in the target tissues or organs. cytoskeletal components or cell adhesion and motility, agents 0147 Combination therapy can also include two or more for phototherapy, and therapy adjuncts. administrations of one or more of the agents used in the 0152 Representative anti-proliferative agents include combination. For example, if agent X and agent Y are used in N-acetyl-D-sphingosine (C. Sub.2 ceramide), apigenin, ber a combination, one could administer them sequentially in any berine chloride, dichloromethylenediphosphonic acid diso combination one or more times, e.g., in the order X-Y-X. dium salt, loe-emodine, emodin, HA 14-1, N-hexanoyl-D- X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc. sphingosine (C. sub.6 ceramide), 7b-hydroxycholesterol, 0148 1.3.2 Exemplary Agents for Combination Therapy 25-hydroxycholesterol, hyperforin, parthenolide, and rapa 014.9 The GCC agonist formulations of the invention may mycin. be administered alone or in combination with one or more 0153. Representative agents for DNA modification and additional therapeutic agents as part of a therapeutic regimen repair include aphidicolin, bleomycin Sulfate, carboplatin, US 2015/0366935 A1 Dec. 24, 2015 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 0154 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 0163. 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 GCC agonist formulation in captopurine, and 6-thioguanine. combination with one or more antitumor agent selected from 0155 Representative DNA/RNA transcription regulators the group consisting of paclitaxel, docetaxel, tamoxifen, include actinomycin D, daunorubicin hydrochloride, 5,6- Vinorelbine, gemcitabine, cisplatin, etoposide, topotecan, dichlorobenzimidazole 1-b-D-ribofuranoside, doxorubicin irinotecan, anastrozole, rituximab, trastuzumab, fludarabine, hydrochloride, homoharringtonine, and idarubicin hydro cyclophosphamide, gentuZumab, carboplatin, interferons, chloride. and doxorubicin. In a particular embodiment the antitumor 0156 Representative enzyme activators and inhibitors agent is paclitaxel. In a further embodiment, the method include forskolin, DL-aminoglutethimide, apicidin, Bow further comprises an antitumor agent selected from the group man-Birk Inhibitor, butein, (S)-(+)-camptothecin, curcumin, consisting of 5-FU, doxorubicin, vinorelbine, Cytoxan, and (-)-deguelin, (-)-depudecin, doxycycline hyclate, etoposide, cisplatin. formestane, fostriecin Sodium salt, hispidin, 2-limino-1-imi dazolidineacetic acid (Cyclocreatine), oxamflatin, 4-phenyl 1.3.2.2 Agents that Treat Crohn's Disease butyric acid, roscovitine, sodium valproate, trichostatin A, 0164. In one embodiment, a GCC agonist formulation of tyrphostin AG 34, tyrphostin AG 879, urinary trypsin inhibi the invention is administered as part of a combination therapy tor fragment, Valproic acid (2-propylpentanoic acid), and with one or more additional therapeutic agents for the treat XK469. ment of Crohn's disease. Non-limiting examples of the one or 0157 Representative gene regulators include 5-aza-2'- more additional therapeutic agents include sulfasalazine and deoxycytidine, 5-azacytidine, cholecalciferol (Vitamin D3), other mesalamine-containing drugs, generally known as ciglitiZone, cyproterone acetate, 15-deoxy-D. Sup.12, 14 5-ASA agents, such as Asacol, Dipentum, or Pentasa, or prostaglandin J. Sub.2, epitestosterone, flutamide, glycyr infliximab (REMICADE). In certain embodiments, the one or rhizic acid ammonium salt (glycyrrhizin), 4-hydroxytamox more additional agents is a corticosteroid or an immunosup ifen, mifepristone, procainamide hydrochloride, raloxifene pressive agent such as 6-mercaptopurine or azathioprine. In hydrochloride, all trans-retinal (vitamin Aaldehyde), retinoic another embodiment, the one or more additional agents is an acid (vitamin A acid), 9-cis-retinoic acid, 13-cis-retinoic antidiarrheal agent Such as diphenoxylate, loperamide, or acid, retinoic acid p-hydroxyanilide, retinol (Vitamin A), codeine. tamoxifen, tamoxifen citrate salt, tetradecylthioacetic acid, and troglitaZone. 1.3.2.3 Agents that Treat Ulcerative Colitis 0158 Representative HSP-90 inhibitors include 17-(ally lamino)-17-demethoxygeldanamycin and geldanamycin. 0.165. In one embodiment, a GCC agonist formulation of 0159 Representative microtubule inhibitors include the invention is administered as part of a combination therapy colchicines, dolastatin 15, nocodazole, taxanes and in par with one or more additional therapeutic agents for the treat ticular paclitaxel, podophyllotoxin, rhizoxin, vinblastine Sul ment of ulcerative colitis. The agents that are used to treat fate salt, Vincristine Sulfate salt, and vindesine Sulfate salt and ulcerative colitis overlap with those used to treat Chrohn's vinorelbine (Navelbine) ditartrate salt. Disease. Non-limiting examples of the one or more additional 0160 Representative agents for performing phototherapy therapeutic agents that can be used in combination with a include photoactive porphyrin rings, hypericin, 5-methoxyp GCC agonist formulation of the invention include aminosali Soralen, 8-methoxypsoralen, psoralen and urSodeoxycholic cylates (drugs that contain 5-aminosalicyclic acid (5-ASA)) acid. Such as Sulfasalazine, olSalazine, mesalamine, and bal 0161 Representative agents used as therapy adjuncts Salazide. Other therapeutic agents that can be used include include amifostine, 4-amino-1.8-naphthalimide, brefeldin A, corticosteroids, such as prednisone and hydrocortisone, cimetidine, phosphomycin disodium salt, leuprolide (leupro immunomodulators, such as azathioprine, 6-mercapto-purine relin) acetate salt, luteinizing hormone-releasing hormone (6-MP), cytokines, interleukins, and lymphokines, and anti (LH-RH) acetate salt, lectin, papaverine hydrochloride, TNF-alpha agents, including the thiazolidinediones or glita pifithrin-a, (-)-scopolamine hydrobromide, and thapsigargin. Zones such as rosiglitaZone and pioglitaZone. In one embodi 0162 The agents can also be anti-VEGF (vascular endot ment, the one or more additional therapeutic agents includes helial growth factor) agents, as Such are known in the art. both cyclosporine A and 6-MP or azathioprine for the treat Several antibodies and small molecules are currently in clini ment of active, severe ulcerative colitis. cal trials or have been approved that function by inhibiting 1.3.2.4 Agents that Treat Constipation/Irritable VEGF, such as Avastin (Bevacizumab), SU5416, SU11248 Bowel Syndrome and BAY 43-9006. The agents can also be directed against growth factor receptors such as those of the EGF/Erb-B fam 0166 In one embodiment, a GCC agonist formulation of ily such as EGF Receptor (Iressa or Gefitinib, and Tarceva or the invention is administered as part of a combination therapy US 2015/0366935 A1 Dec. 24, 2015 36 with one or more additional therapeutic agents for the treat 006007, WO03/007887, WO03/020217, WO03/026647, ment of constipation, Such as that associated with irritable WO03/026648, WO03/027069, WO03/027076, WO03/ bowel syndrome. Non-limiting examples of the one or more 027114, WO03/037332, WO03/04.01.07, WO03/086940, additional therapeutic agents include laxatives such as WO03/084943 and EP658546; CCK-A (cholecystokinin-A) SENNA, MIRALAX, LACTULOSE, PEG, or calcium poly agonists, such as AR-R 15849, GI 181771 (GSK), JMV-180, carbophil), stool softeners (such as mineral oil or COLACE), A-71378, A-71623 and SR146131 (Sanofi), and those bulking agents (such as METAMUCIL or bran), agents such described in U.S. Pat. No. 5,739,106; CNTF (Ciliary neu as ZELNORM (also called tegaserod), and anticholinergic rotrophic factors), such as GI-181771 (Glaxo-Smith-Kline), medications such as BENTYL and LEVSIN. SR146131 (Sanofi Synthelabo), butabindide, PD 170,292, and PD 149164 (Pfizer); CNTF derivatives, such as AXok 1.3.2.5 Agents for the Treatment of Postoperative ineR (Regeneron), and those disclosed in WO94/09134, Ileus WO98/22128, and WO99/43813; dipeptidyl peptidase IV 0167. In one embodiment, a GCC agonist formulation of (DP-IV) inhibitors, such as isoleucine thiazolidide, valine the invention is administered as part of a combination therapy pyrrolidide, NVP-DPP728, LAF237, P93/01, P3298, TSL with one or more additional therapeutic agents for the treat 225 (tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic ment of postoperative ileus. Non-limiting examples of the one acid; disclosed byYamada et al. Bioorg. & Med. Chem. Lett. or more additional therapeutic agents include ENTEREG 8 (1998) 1537-1540), TMC-2A/2B/2C, CD26 inhibtors, FE (alvimopan; formerly called adolor/ADL 8-2698), conivap 999011, P9310/K364, VIP 0177, SDZ 274-444, 2-cyanopy tan, and related agents describes in U.S. Pat. No. 6,645,959. rrolidides and 4-cyanopyrrolidides as disclosed by Ashworth et al. Bioorg. & Med. Chem. Lett. Vol. 6, No. 22, pp 1163 1.3.2.6 Anti-Obesity Agents 1166 and 2745-2748 (1996) and the compounds disclosed patent publications. WO99/38501, WO99/46272, WO99/ 0.168. In one embodiment, a GCC agonist formulation of 67279 (Probiodrug), WO99/67278 (Probiodrug), WO99/ the invention is administered as part of a combination therapy 61431 (Probiodrug), WO02/083128, WO02/062764, WO03/ with one or more additional therapeutic agents for the treat 000180, WO03/000181, WO03/000250, WO03/002530, ment of obesity. Non-limiting examples of the one or more WO03/002531, WO03/002553, WO03/002593, WO03/ additional therapeutic agents include 1 13 HSD-I (11-beta 004498, WO03/004496, WO03/017936, WO03/024942, hydroxy steroid dehydrogenase type 1) inhibitors, such as WO03/024965, WO03/033524, WO03/037327 and BVT 3498, BVT 2733, 3-(1-adamantyl)-4-ethyl-5-(eth EP1258476; growth hormone secretagogue receptor ago ylthio)-4H-1,2,4-triazole, 3-(1-adamantyl)-5-(3,4,5-tri nists/antagonists, such as NN703, hexarelin, MK-0677 methoxyphenyl)-4-methyl-4H-1,2,4-triazole, 3-adamanta (Merck), SM-130686, CP-424391 (Pfizer), LY 444,711 (Eli nyl-4,5,6,7,8,9,10,11,12.3a-decahydro-1,2,4-triazolo 4.3-a Lilly), L-692,429 and L-163.255, and such as those disclosed 11 annulene, and those compounds disclosed in WO01/ in U.S. Ser. No. 09/662,448, U.S. provisional application 90091, WOO 1/90090, WOO 1/90092 and WO02/072084; 60/203,335, U.S. Pat. No. 6,358,951, US20020491.96, 5HT antagonists such as those in WO03/037871, WO03/ US2002/022637, WO01/56592 and WO02/32888; H3 (his 037887, and the like; 5HTIa modulators such as carbidopa, tamine H3) antagonist/inverse agonists, such as thioperam benserazide and those disclosed in U.S. Pat. No. 6,207,699, ide, 3-(1H-imidazol-4-yl)propyl N-(4-pentenyl)carbamate), WO03/031439, and the like; 5HT2c (serotonin receptor 2c) clobenpropit, iodophenpropit, imoproxifan, GT2394 (Gliat agonists, such as BVT933, DPCA37215, IK264, PNU 22394, ech), and A331440, O-3-(1H-imidazol-4-yl)propanol car WAY 161503, R-1065, SB 243213 (GlaxoSmith Kline) and bamates (Kiec-Kononowicz, K. et al., Pharmazie, 55:349-55 YM 348 and those disclosed in U.S. Pat. No. 3,914,250, (2000)), piperidine-containing histamine H3-receptor WO00/77010, WO02/36596, WO02/48124, WO02/10169, antagonists (Lazewska, D. et al., Pharmazie, 56:927-32 WO01/66548, WO02/44152, WO02/51844, WO02/40456, (2001), benzophenone derivatives and related compounds and WO02/40457; 5HT6 receptor modulators, such as those (Sasse, A. et al., Arch. Pharm. (Weinheim) 334:45-52 in WO03/030901, WO03/035061, WO03/039547, and the (2001)), substituted N-phenylcarbamates (Reidemeister, S. et like; acyl-estrogens, such as oleoyl-estrone, disclosed in del al., Pharmazie, 55:83-6 (2000)), and proxifan derivatives Mar-Grasa, M. et al., Obesity Research, 9:202-9 (2001) and (Sasse, A. et al., J. Med. Chem. 43:3335-43 (2000)) and Japanese Patent Application No. JP 2000256190; anorectic histamine H3 receptor modulators such as those disclosed in bicyclic compounds such as 1426 (Aventis) and 1954 (Aven WO02/15905, WO03/024928 and WO03/024929; leptin tis), and the compounds disclosed in WO00/18749, WO01/ derivatives, such as those disclosed in U.S. Pat. No. 5,552, 32638, WO01/62746, WO01/62747, and WO03/015769; CB 524, U.S. Pat. No. 5,552,523, U.S. Pat. No. 5,552,522, U.S. 1 (cannabinoid-1 receptor) antagonist/inverse agonists such Pat. No. 5,521,283, WO96/23513, WO96/23514, WO96/ as rimonabant (Acomplia; Sanofi), SR-147778 (Sanofi), 23515, WO96/23516, WO96/23517, WO96/23518, WO96/ SR-141716 (Sanofi), BAY 65-2520 (Bayer), and SLV 319 23519, and WO96/23520; leptin, including recombinant (Solvay), and those disclosed in patent publications U.S. Pat. human leptin (PEG-OE, Hoffman La Roche) and recombi No. 4,973,587, U.S. Pat. No. 5,013,837, U.S. Pat. No. 5,081, nant methionyl human leptin (Amgen); lipase inhibitors, such 122, U.S. Pat. No. 5,112,820, U.S. Pat. No. 5,292,736, U.S. as tetrahydrolipstatin (orlistat/Xenical(R), Triton WR1 339, Pat. No. 5,532,237, U.S. Pat. No. 5,624,941, U.S. Pat. No. RHC80267, lipstatin, teasaponin, diethylumbelliferyl phos 6,028,084, U.S. Pat. No. 6,509,367, U.S. Pat. No. 6,509,367, phate, FL-386, WAY-121898, Bay-N-3176, valilactone, WO96/33159, WO97/29079, WO98/31227, WO98/33765, esteracin, ebelactone A, ebelactone B, and RHC 80267, and WO98/37061, WO98/41519, WO98/43635, WO98/43636, those disclosed in patent publications WO01/77094, U.S. Pat. WO99/02499, WO00/10967, WO00/10968, WO01/09120, No. 4,598,089, U.S. Pat. No. 4,452,813, U.S. Pat. No. 5,512, WO01/58869, WO01/64.632, WO01/64633, WO01/64634, 565, U.S. Pat. No. 5,391,571, U.S. Pat. No. 5,602,151, U.S. WO01/70700, WO01/96330, WO02/076949, WO03/ Pat. No. 4,405,644, U.S. Pat. No. 4,189,438, and U.S. Pat. No. US 2015/0366935 A1 Dec. 24, 2015 37

4,242.453; lipid metabolism modulators such as maslinic WO01/09120, WO02/20488, WO02/22592, WO02/48152, acid, erythrodiol, ursolic acid uvaol, betulinic acid, betulin, WO02/49648, WO02/051806, WO02/094789, WO03/ and the like and compounds disclosed in WO03/01 1267; 009845, WO03/014083, WO03/022849, WO03/028726 and Mc4r (melanocortin 4 receptor) agonists, such as Norman et al., J. Med. Chem. 43:4288-4312 (2000); opioid CHIR86036 (Chiron), ME-10142, ME-10145, and HS-131 antagonists, such as nalmefene (REVEXR), 3-methoxynal (Melacure), and those disclosed in PCT publication Nos. trexone, methylnaltrexone, naloxone, and naltrexone (e.g. WO99/64002, WO00/74679, WOO 1/991752, WOO PT901; Pain Therapeutics, Inc.) and those disclosed in 1/25192, WOO 1/52880, WOO 1/74844, WOO 1/70708, US20050004155 and WO00/215.09: orexin antagonists, such WO01/70337, WO01/91752, WO02/059095, WO02/ as SB-334867-A and those disclosed in patent publications 059107, WO02/059108, WO02/059117, WO02/06276, WO01/96302, WO01/68609, WO02/44.172, WO02/51232, WO02/12166, WO02/11715, WO02/12178, WO02/15909, WO02/51838, WO02/089800, WO02/090355, WO03/ WO02/38544, WO02/068387, WO02/068388, WO02/ 023561, WO03/032991, and WO03/037847; PDE inhibitors 067869, WO02/081430, WO03/06604, WO03/007949, (e.g. compounds which slow the degradation of cyclic AMP WO03/009847, WO03/009850, WO03/013509, and WO03/ (cAMP) and/or cyclic GMP (cGMP) by inhibition of the 031410; McSr (melanocortin 5 receptor) modulators, such as phosphodiesterases, which can lead to a relative increase in those disclosed in WO97/19952, WO00/15826, WOOO/ the intracellular concentration of cAMP and coMP; possible 15790, US20030092041; melanin-concentrating hormone 1 PDE inhibitors are primarily those substances which are to be receptor (MCHR) antagonists, such as T-226296 (Takeda), numbered among the class consisting of the PDE3 inhibitors, SB 568849, SNP-7941 (Synaptic), and those disclosed in the class consisting of the PDE4 inhibitors and/or the class patent publications WOO 1/21169, WO01/82925, WO01/ consisting of the PDE5 inhibitors, in particular those sub 87834, WO02/051809, WO02/06245, WO02/076929, stances which can be designated as mixed types of PDE3/4 WO02/076947, WO02/04433, WO02/.51809, WO02/ inhibitors or as mixed types of PDE3/4/5 inhibitors) such as 083134, WO02/094799, WO03/004027, WO03/13574, those disclosed in patent publications DE 1470341, WO03/15769, WO03/028641, WO03/035624, WO03/ DE2108438, DE2123328, DE2305339, DE2305575, 033476, WO03/033480, JP13226269, and JP1437059; DE2315801, DE2402908, DE2413935, DE2451417, mGluR5 modulators such as those disclosed in WOO3/ DE2459090, DE2646469, DE2727481, DE2825048, 029210, WO03/047581, WO03/048137, WO03/051315, DE2837161, DE2845220, DE2847621, DE2934747, WO03/051833, WO03/053922, WO03/059904, and the like; DE3021792, DE3038166, DE3044568, EP000718, serotoninergic agents, such as fenfluramine (such as Pondi EP0008.408, EP0010759, EP0059948, EP0075436, min(R) (Benzeneethanamine, N-ethyl-alpha-methyl-3-(trif EP0096517, EPO112987, EP01 16948, EPO150937, luoromethyl)-, hydrochloride), Robbins), dexfenfluramine EP0158380, EPO161632, EPO161918, EP0167121, (such as Redux(R) (Benzeneethanamine, N-ethyl-alpha-me EP0199127, EP0220044, EP0247725, EP0258191, thyl-3-(trifluoromethyl)-, hydrochloride), Interneuron) and EP0272910, EP0272914, EP0294647, EP0300726, sibutramine ((MeridiaR), Knoll/ReductiltM) including race EP0335386, EP0357788, EP038.9282, EP0406958, mic mixtures, as optically pure isomers (+) and (-), and EP0426180, EP0428302, EP0435811, EP0470805, pharmaceutically acceptable salts, solvents, hydrates, clath EP0482208, EP0490823, EP0506194, EP0511865, rates and prodrugs thereof including sibutramine hydrochlo EP0527117, EP0626939, EP0664289, EP0671389, ride monohydrate salts thereof, and those compounds dis EP0685474, EP0685475, EP0685479, JP92234389, closed in U.S. Pat. No. 4,746,680, U.S. Pat. No. 4,806,570, JP94329652, JP950.10875, U.S. Pat. No. 4,963,561, U.S. Pat. and U.S. Pat. No. 5,436,272, US20020006964, WOO No. 5,141,931, WO9117991, WO9200968, WO9212961, 1/27068, and WOO 1/62341; NE (norepinephrine) transport WO9307146, WO9315044, WO9315045, WO93.18024, inhibitors, such as GW 320659, despiramine, talsupram, and WO9319068, WO9319720, WO931.9747, WO9319749, nomifensine: NPY 1 antagonists, such as BIBP3226, WO9319751, WO9325517, WO9402465, WO9406423, J-115814, BIBO 3304, LY-357897, CP-671906, WO9412461, WO9420455, WO9422852, WO9425437, GI-264879A, and those disclosed in U.S. Pat. No. 6,001,836, WO9427947, WO9500,516, WO950.1980, WO9503794, WO96/14307, WO01/23387, WO99/51600, WO01/85690, WO9504045, WO9504046, WO9505386, WO9508534, WO01/85098, WO01/85173, and WO01/89528: NPY5 (neu WO9509623, WO9509624, WO9509627, WO9509836, ropeptideYY5) antagonists, such as 152,804, GW-569180A, WO9514667, WO9514680, WO9514681, WO9517392, GW-594884A, GW-587081X, GW-548118X, FR235208, WO9517399, WO9519362, WO9522520, WO9524381, FR226928, FR24.0662, FR252384, 1229U91, GI-264879A, WO9527692, WO9528926, WO953.5281, WO9535282, CGP71683A, LY-377897, LY-366377, PD-160170, WO9600218, WO9601825, WO9602541, WO96/1917, SR-120562A, SR-120819A, JCF-104, and H409/22 and DE3142982, DE 1116676, DE2162096, EP0293063, those compounds disclosed in patent publications U.S. Pat. EP0463756, EP0482208, EP0579496, EP0667345 U.S. Pat. No. 6,140,354, U.S. Pat. No. 6,191,160, U.S. Pat. No. 6,218, No. 6,331.543, US20050004222 (including those disclosed 408, U.S. Pat. No. 6,258,837, U.S. Pat. No. 6,313,298, U.S. in formulas I-XIII and paragraphs 37-39, 85-0545 and 557 Pat. No. 6,326,375, U.S. Pat. No. 6,329,395, U.S. Pat. No. 577), WO9307124, EP0163965, EP0393500, EP0510562, 6,335,345, U.S. Pat. No. 6,337,332, U.S. Pat. No. 6,329,395, EP0553174, WO950.1338 and WO9603399, as well as PDE5 U.S. Pat. No. 6,340,683, EP01010691, EP-01044970, WO97/ inhibitors (such as RX-RA-69, SCH-51866, KT-734, 19682, WO97/20820, WO97/20821, WO97/20822, WO97/ vesnarinone, Zaprinast, SKF-96231, ER-21355, BF/GP-385, 20823, WO98/27063, WO00/107409, WO00/185714, NM-702 and sildenafil (ViagraTM)), PDE4 inhibitors (such as WO00/185730, WO00/64880, WO00/68197, WO00/69849, etazolate, IC163197, RP73401, imazolidinone (RO-20 WO/0113917, WO01/09120, WO01/14376, WO01/85714, 1724), MEM 1414 (R1533/R1500; Pharmacia Roche), den WO01/85730, WO01/07409, WO01/02379, WO01/23388, bufylline, rolipram, oxagrelate, nitraduaZone, Y-590, WO01/23389, WOO 1/44201, WO01/62737, WO01/62738, DH-6471, SKF-94120, motapizone, lixazinone, indolidan, US 2015/0366935 A1 Dec. 24, 2015 olprinone, atizoram, KS-506-G, dipamfylline, BMY-43351, zine ((or (2S,3S)-3,4-Dimethyl-2phenylmorpholine L-(+)- atizoram, arofylline, filaminast, PDB-093, UCB-29646, tartrate (1:1)) such as Metra R. (Forest), PlegineR (Wyeth-Ay CDP-840, SKF-107806, piclamilast, RS-17597, RS-25344 erst), Prelu-2R (Boehringer Ingelheim), and StatobeXR 000, SB-207499, TIBENELAST, SB-210667, SB-211572, (Lemmon), phendamine tartrate (such as ThephorinR (2.3.4. SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, 9-Tetrahydro-2-methyl-9-phenyl-1H-indenol.2.1-cpyridine mopidamol, anagrelide, ibudilast, amrinone, pimobendan, L-(+)-tartrate (1:1), Hoffmann-LaRoche), methamphet cilostazol, quaZinone and N-(3,5-dichloropyrid-4-yl)-3-cy amine (such as Desoxyn R, Abbot ((S)- N, (alpha)-dimeth clopropylmethoxy-4-difluoromethoxybenzamide, PDE3 ylbenzeneethanamine hydrochloride)), and phendimetrazine inhibitors (such as IC1153, 100, bemorandane (RWJ 22867), tartrate (such as Bontril.R Slow-Release Capsules, Amarin MCI-154, UD-CG 212, Sulmazole, ampizone, cilostamide, (-3,4-Dimethyl-2-phenylmorpholine Tartrate); fatty acid carbazeran, piroXimone, imaZodan, CI-930, SiguaZodan, oxidation upregulator/inducers such as Famoxin R (Genset); adibendan, saterinone, SKF-95654, SDZ-MKS-492, 349-U- monamine oxidase inhibitors including but not limited to 85, emoradan, EMD-53998, EMD-57033, NSP-306, NSP befloxatone, moclobemide, brofaromine, phenoxathine, esu 307, revizinone, NM-702, WIN-62582 and WIN-63291, prone, befol, toloxatone, pirlindol, amiflamine, Sercloremine, enoximone and milrinone, PDE3/4 inhibitors (such as baZinaprine, lazabemide, millacemide, caroXaZone and other benafentrine, trequinsin, ORG-3.0029, Zardaverine, certain compounds as disclosed by WO01/12176; and other L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, and anti-obesity agents such as 5HT-2 agonists, ACC (acetyl-CoA tolafentrine) and other PDE inhibitors (such as vinpocetin, carboxylase) inhibitors such as those described in WO03/ papaverine, enprofylline, cilomilast, fenoXimone, pentoxifyl 072197, alpha-lipoic acid (alpha-LA), AOD9604, appetite line, roflumilast, tadalafil (Cialis(R), theophylline, and Vard suppressants such as those in WO03/40107, ATL-962 (Al enafil(Levitra(R); Neuropeptide Y2 (NPY2) agonists include izyme PLC), benzocaine, benzphetamine hydrochloride (Di but are not limited to: polypeptide YY and fragments and drex), bladderwrack (focus vesiculosus), BRS3 (bombesin variants thereof (e.g. YY3-36 (PYY3-36)(N. Engl. J. Med. receptor Subtype 3) agonists, bupropion, caffeine, CCK ago 349:941, 2003; IKPEAPGE DASPEELNRY YASL nists, chitosan, chromium, conjugated linoleic acid, corti RHYLNLVTRQRY (SEQID NO:XXX)) and PYYagonists cotropin-releasing hormone agonists, dehydroepiandroster such as those disclosed in WO02/47712, WO03/026591, one, DGAT1 (diacylglycerol acyltransferase 1) inhibitors, WO03/057235, and WO03/027637; serotonin reuptake DGAT2 (diacylglycerol acyltransferase 2) inhibitors, dicar inhibitors, such as, paroxetine, fluoxetine (ProzacTM), fluvox boxylate transporter inhibitors, ephedra, exendin-4 (an amine, sertraline, citalopram, and imipramine, and those dis inhibitor of glp-1) FAS (fatty acid synthase) inhibitors (such closed in U.S. Pat. No. 6,162,805, U.S. Pat. No. 6,365,633, as Cerulenin and C75), fat resorption inhibitors (such as those WO03/00663, WOO 1/27060, and WOO 1/162341; thyroid in WO03/053451, and the like), fatty acid transporter inhibi hormone Bagonists, such as KB-2611 (KaroBioBMS), and tors, natural water soluble fibers (such as psyllium, plantago, those disclosed in WOO2/15845, WO97/21993, WO99/ guar, oat, pectin), galanin antagonists, galega (Goat's Rue, 00353, GB98/284425, U.S. Provisional Application No. French Lilac), garcinia cambogia, germander (teucrium 60/183.223, and Japanese Patent Application No. JP chamaedrys), ghrelin antibodies and ghrelin antagonists 2000256190; UCP-I (uncoupling protein-1), 2, or 3 activa (such as those disclosed in WO01/87335, and WO02/08250), tors, such as phytanic acid, 4-(E)-2-(5,6,7,8-tetrahydro-5.5, polypeptide hormones and variants thereof which affect the 8,8-tetramethyl-2-napthalenyl)-1-propenylbenzoic acid islet cell secretion, such as the hormones of the secretin/ (TTNPB), retinoic acid, and those disclosed in WO99/00123; gastric inhibitory polypeptide (GIP)/vasoactive intestinal B3 (beta adrenergic receptor 3) agonists, such as AJ9677/ polypeptide (VIP)/pituitary adenylate cyclase activating TAK677 (Dainippon/Takeda), L750355 (Merck), CP331648 polypeptide (PACAP)/glucagon-like polypeptide II (GLP (Pfizer), CL-316,243, SB 418790, BRL-37344, L-796568, II)/glicentin/glucagon gene family and/or those of the BMS-196085, BRL-35135A, CGP12177A, BTA-243, GW adrenomedullinfamylin/calcitonin gene related polypeptide 427353, Trecadrine, Zeneca D7114, N-5984 (Nisshin (CGRP) gene family includingGLP-1 (glucagon-like Kyorin), LY-377604 (Lilly), SR 59119A, and those disclosed polypeptide 1) agonists (e.g. (1) exendin-4, (2) those GLP-I in U.S. Pat. No. 5,541,204, U.S. Pat. No. 5,770,615, U.S. Pat. molecules described in US20050130891 including GLP-1 (7- No. 5,491,134, U.S. Pat. No. 5,776,983, US488.064, U.S. Pat. 34), GLP-1 (7-35), GLP-1 (7-36) or GLP-1 (7-37) in its C-ter No. 5,705,515, U.S. Pat. No. 5,451,677, WO94/18161, minally carboxylated or amidated form or as modified GLP-I WO95/2915.9, WO97/.46556, WO98/04526 and WO98/ polypeptides and modifications thereof including those 32753, WO01/74782, WO02/32897, WO03/014113, WO03/ described in paragraphs 17-44 of US20050130891, and 016276, WO03/016307, WO03/024948, WO03/024953 and derivatives derived from GLP-1-(7-34)COOH and the corre WO03/037881; noradrenergic agents including, but not lim sponding acid amide are employed which have the following ited to, diethylpropion (Such as Tenuate R (1-propanone, general formula: R NH-HAEGTFTSDVSYLEGOAAKE 2-(diethylamino)-1-phenyl-, hydrochloride), Merrell), dex FIAWLVK-CONH wherein R—H oran organic compound troamphetamine (also known as dextroamphetamine Sulfate, having from 1 to 10 carbonatoms. Preferably, R is the residue dexamphetamine, dexedrine, Dexampex, Ferndex, Oxydess of a carboxylic acid. Particularly preferred are the following II, Robese, Spancap #1), mazindol (or 5-(p-chlorophenyl)- carboxylic acid residues: formyl, acetyl, propionyl, isopro 2,5-dihydro-3H-imidazo[2,1-aisoindol-5-ol) such as San pionyl, methyl, ethyl, propyl, isopropyl. n-butyl, sec-butyl, orex R, Novartis or Mazanor R, Wyeth Ayerst), phenylpro tert-butyl.) and glp-1 (glucagon-like polypeptide-1), gluco panolamine (or Benzenemethanol, alpha-(1-aminoethyl)-. corticoid antagonists, glucose transporter inhibitors, growth hydrochloride), phentermine ((or Phenol, 3-4,5-duhydro hormone secretagogues (such as those disclosed and specifi 1H-imidazol-2-yl)ethyl(4-methylpheny-1)amino, mono cally described in U.S. Pat. No. 5,536,716), interleukin-6 hydrochloride) such as Adipex-PR), Lemmon, FASTINR), (IL-6) and modulators thereof (as in WO03/057237, and the SmithKline Beecham and IonaminO, Medeva), phendimetra like), L-carnitine, Mc3r (melanocortin 3 receptor) agonists, US 2015/0366935 A1 Dec. 24, 2015 39

MCH2R (melanin concentrating hormone 2R) agonist/an WO9603399. PDE5 inhibitors which may be mentioned by tagonists, melanin concentrating hormone antagonists, mel way of example are RX-RA-69, SCH-51866, KT-734, anocortinagonists (such as Melanotan II or those described in vesnarinone, Zaprinast, SKF-96231, ER-21355, BF/GP-385, WO99/64002 and WO00/74679), nomame herba, phosphate NM-702 and sildenafil (Viagra R). PDE4 inhibitors which transporter inhibitors, phytopharm compound 57 (CP 644, may be mentioned by way of example are RO-20-1724, MEM 673), pyruvate, SCD-I (stearoyl-CoA desaturase-1) inhibi 1414 (R1533/R1500; Pharmacia Roche), DENBUFYL tors, T71 (Tularik, Inc., Boulder Colo.), Topiramate (Topi LINE, ROLIPRAM, OXAGRELATE, NITRAQUAZONE, maX(R), indicated as an anti-convulsant which has been shown Y-590, DH-6471, SKF-94120, MOTAPIZONE, LIXAZI to increase weight loss), transcription factor modulators (such NONE, INDOLIDAN, OLPRINONE, ATIZORAM, as those disclosed in WO03/026576), B-hydroxy steroid KS-506-G, DIPAMFYLLINE, BMY-43351, ATIZORAM, dehydrogenase-1 inhibitors (3-HSD-I), f-hydroxy-3-meth AROFYLLINE, FILAMINAST, PDB-093, UCB-29646, ylbutyrate, p57 (Pfizer), Zonisamide (ZonegranTM, indicated CDP-840, SKF-107806, PICLAMILAST, RS-17597, as an anti-epileptic which has been shown to lead to weight RS-25344-000, SB-207499, TIBENELAST, SB-210667, loss), and the agents disclosed in US200301 19428 para SB-211572, SB-211600, SB-212066, SB-212179, graphs 20-26. GW-3600, CDP-840, MOPIDAMOL, ANAGRELIDE, IBUDILAST, AMRINONE, PIMOBENDAN, CILOSTA 1.3.2.7. Phosphodiesterase Inhibitors ZOL, QUAZINONE and N-(3,5-dichloropyrid-4-yl)-3-cy clopropylmethoxy-4-difluoromethoxybenzamide. PDE3 0169. In certain embodiments, the regimen of combina inhibitors which may be mentioned by way of example are tion therapy includes the administration of one or more phos SULMAZOLE, AMPIZONE, CILOSTAMIDE, CARBAZ phodiesterase (“PDE') inhibitors. PDE inhibitors slow the ERAN, PIROXIMONE, IMAZODAN, CI-930, SIGUA degradation of cyclic AMP (cAMP) and/or cyclic GMP ZODAN, ADIBENDAN, SATERINONE, SKF-95654, SDZ (cGMP) by inhibiting phosphodiesterases, which can lead to MKS-492, 349-U-85, EMORADAN, EMD-53998, EMD a relative increase in the intracellular concentration of cAMP 57033, NSP-306, NSP-307, REVIZINONE, NM-702, WIN and/or cGMP. Non-limiting examples of PDE inhibitors that 62582 and WIN-63291, ENOXIMONE and MILRINONE. can be used in combination with the GCC agonists of the PDE3/4 inhibitors which may be mentioned by way of invention include PDE3 inhibitors, PDE4 inhibitors and/or example are BENAFENTRINE, TREQUINSIN, ORG PDE5 inhibitors, in particular those substances which can be 3.0029, ZARDAVERINE, L-686398, SDZ-ISQ-844, ORG designated as mixed types of PDE3/4 inhibitors or as mixed 20241, EMD-54622, and TOLAFENTRINE. Other PDE types of PDE3/4/5 inhibitors. Non-limiting examples of such inhibitors include: cilomilast, pentoxifylline, roflumilast, tad PDE inhibitors are described in the following patent applica alafil (Cialis(R), theophylline, and Vardenafil(Levitra(R), Zap tions and patents: DE 1470341, DE2108438, DE2123328, rinast (PDE5 specific). GCC AGONIST DE2305339, DE2305575, DE2315801, DE2402908, DE2413935, DE2451417, DE2459090, DE2646469, 1.3.2.8 Analgesic Agents DE2727481, DE2825048, DE2837161, DE2845220, DE2847621, DE2934747, DE3021792, DE3038166, 0170 In certain embodiments, the regimen of combina DE3044568, EP000718, EP0008.408, EP0010759, tion therapy includes the administration of one or more anal EP0059948, EP0075436, EP00965.17, EPO112987, gesic agents, e.g., an analgesic compound or an analgesic EP0116948, EPO150937, EP0158380, EPO161632, polypeptide. In some embodiments, the GCC agonist formu EPO161918, EP0167121, EP019.9127, EP0220044, lation is administered simultaneously or sequentially with EP0247725, EP0258191 EP0272910, EP0272914, one or more analgesic agents. In other embodiments, the EP0294647, EP0300726, EP0335386, EP0357788, GCC agonist is covalently linked or attached to an analgesic EP0389282, EP0406958, EP0426180, EP0428302, agent to create a therapeutic conjugate. Non-limiting EP0435811, EP0470805, EP0482208, EP0490823, examples of analgesic agents that can be used include calcium EP0506194, EP0511865, EP0527117, EP0626939, channel blockers, 5HT receptor antagonists (for example EP0664289, EP0671389, EP0685474, EP0685475, 5HT3, 5HT4 and 5HT1 receptor antagonists), opioid receptor EP0685479, JP92234389, JP94329652, JP950.10875, U.S. agonists (loperamide, fedotozine, and fentanyl), NK1 recep Pat. Nos. 4,963,561, 5,141,931, WO91/7991, WO9200968, tor antagonists, CCK receptor agonists (e.g., loxiglumide), WO92/2961, WO9307146, WO9315044, WO9315045, NK1 receptor antagonists, NK3 receptor antagonists, norepi WO9318024, WO9319068, WO9319720, WO931.9747, nephrine-serotonin reuptake inhibitors (NSRI), Vanilloid and WO9319749, WO9319751, WO9325517, WO9402465, cannabanoid receptor agonists, and sialorphin. Further WO9406423, WO9412461, WO9420455, WO9422852, examples of analgesic agents in the various classes are known WO9425437, WO9427947, WO9500,516, WO950.1980, in the art. WO9503794, WO9504045, WO9504046, WO9505386, 0171 In one embodiment, the analgesic agent is an anal WO9508534, WO9509623, WO9509624, WO9509627, gesic polypeptide selected from the group consisting of Sia WO9509836, WO9514667, WO9514680, WO9514681, lorphin-related polypeptides, including those comprising the WO9517392, WO9517399, WO9519362, WO9522520, amino acid sequence QHNPR (SEQID NO: 239), including: WO9524381, WO9527692, WO9528926, WO9535281, VQHNPR (SEQ ID NO: 240); VRQHNPR (SEQ ID NO: WO9535282, WO9600218, WO9601825, WO9602541, 241); VRGQHNPR (SEQIDNO: 242):VRGPQHNPR (SEQ WO96/1917, DE3142982, DE1116676, DE2162096, ID NO: 243); VRGPRQHNPR (SEQ ID NO: 244); VRG EP0293063, EP0463756, EP0482208, EP0579496, PRRQHNPR (SEQ ID NO. 245); and RQHNPR (SEQ ID EP0667345 U.S. Pat. No. 6,331,543, US20050004222 (in NO: 246). Sialorphin-related polypeptides bind to neprilysin cluding those disclosed in formulas I- XIII and paragraphs and inhibit neprilysin-mediated breakdown of substance P 37-39, 85-0545 and 557-577) and WO9307124, EP0163965, and Met-enkephalin. Thus, compounds or polypeptides that EP0393500, EP0510562, EP0553174, WO950.1338 and are inhibitors of neprilysin are useful analgesic agents which US 2015/0366935 A1 Dec. 24, 2015 40 can be administered with the GCC agonists described herein 0.174. In addition to sialorphin-related polypeptides, anal or covalently linked to a GCC agonist to form a therapeutic gesic polypeptides include: AspPhe, endomorphin-1, endo conjugate. Sialorphin and related polypeptides are described morphin-2, nocistatin, dalargin, lupron, Ziconotide, and Sub in U.S. Pat. No. 6,589,750; U.S. 20030078200 A1; and WO stance P. O2/O51435 A2. 1.3.2.9 Insulin and Insulin Modulating Agents 0172. In another embodiment, a GCC agonist formulation of the invention is administered as part of a regimen of com 0.175. The GCC agonist peptides described herein can be bination therapy with an opioid receptor antagonist or ago used in combination therapy with insulin and related com nist. In one embodiment, the GCC agonist and the opioid pounds including primate, rodent, or rabbit insulin including receptor antagonist or agonist are linked via a covalent bond. biologically active variants thereof including allelic variants, Non-limiting examples of opioid receptorantagonists include more preferably human insulin available in recombinant naloxone, naltrexone, methyl naloZone, nalmefene, cypri form. Sources of human insulin include pharmaceutically dime, beta funaltrexamine, naloxonazine, naltrindole, nor acceptable and sterile formulations such as those available binaltorphimine, enkephalin pentapeptide (HOE825: Tyr-D- from Eli Lilly (Indianapolis, Ind. 46285) as HumulinTM (hu man insulin rDNA origin). See, the THE PHYSICIANS Lys-Gly-Phe-L-homoserine), trimebutine, vasoactive DESK REFERENCE, 55.sup.th Ed. (2001) Medical Eco intestinal polypeptide, gastrin, glucagons. Non-limiting nomics, Thomson Healthcare (disclosing other Suitable examples of opioid receptoragonists include fedotozine, asi human insulins). madoline, and ketocyclazocine, the compounds described in 0176 The GCC peptides described herein can also be used WO03/097051 and WO05/007626, morphine, diphenyloxy in combination therapy with agents that can boost insulin late, frakefamide (H-Tyr-D-Ala-Phe(F)-Phe-NH 2: WO effects or levels of a subject upon administration, e.g. glipiz 01/019849 A1), and loperamide. ide and/or rosiglitaZone. The polypeptides and agonistsde 0173 Further non-limiting examples of analgesic agents scribed herein can be used in combitherapy with SYMLINR) that can be used in a regimen of combination therapy along (pramlintide acetate) and Exenatide(R) (Synthetic exendin-4; a with the GCC agonist formulations of the invention include 39 aa polypeptide). the dipeptide Tyr-Arg (kyotorphin); the chromogranin-de rived polypeptide (CgA 47-66: See, e.g., Ghia et al. 2004 1.3.2.10 Anti-Hypertensive Agents Regulatory polypeptides 119:199); CCK receptor agonists (0177. The GCC agonist peptides described herein can be Such as caerulein; conotoxin polypeptides; peptide analogs of used in combination therapy with an anti-hypertensive agent thymulin (FRApplication 2830451); CCK (CCKa or CCKb) including but not limited to: (1) diuretics, such as thiazides, receptor antagonists, including loxiglumide and dexloxiglu including chlorthalidone, chlorthiazide, dichlorophenamide, mide (the R-isomer ofloxiglumide) (WO 88/05774): 5-HT4 hydroflumethiazide, indapamide, polythiazide, and hydro agonists such as tegaserod (Zelnorm R), mosapride, metoclo chlorothiazide; loop diuretics, such as bumetanide, pramide, Zacopride, cisapride, renzapride, benzimidazolone ethacrynic acid, furosemide, and torsemide; potassium spar derivatives such as BIMU 1 and BIMU 8, and lirexapride: ing agents, such as amiloride, and triamterene; carbonic calcium channel blockers such as Ziconotide and related com anhydrase inhibitors, osmotics(such as glycerin) and aldos pounds described in, for example, EP625162B1, U.S. Pat. terone antagonists, such as spironolactone, epirenone, and the No. 5,364,842, U.S. Pat. No. 5,587,454, U.S. Pat. No. 5,824, like; (2) beta-adrenergic blockers such as acebutolol. 645, U.S. Pat. No. 5,859,186, U.S. Pat. No. 5,994,305, U.S. atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, car Pat. No. 6,087,091, U.S. Pat. No. 6,136,786, WO 93/13128 teolol, carvedilol, celiprolol, esmolol, indenolol, metaprolol. A1, EP1336409 A1 EP 835126A1 EP 835126 B1, U.S. Pat. nadolol, nebivolol, penbutolol, pindolol, propanolol, Sotalol, No. 5,795,864, U.S. Pat. No. 5,891,849, U.S. Pat. No. 6,054, tertatolol, tilisolol, and timolol, and the like; (3) calcium 429, WO 97/01351 A1, NK-I, receptor antagonists such as channel blockers such as amlodipine, aranidipine, azelnid aprepitant (Merck & Co Inc), Vofopiitant, ezlopitant (Pfizer, ipine, barnidipine, benidipine, bepridil, cinaldipine, clevid Inc.), R-673 (Hoffmann-La Roche Ltd), SR-48968 (Sanofi ipine, diltiazem, efonidipine, felodipine, gallopamil, israd Synthelabo), CP-122,721 (Pfizer, Inc.), GW679769 (Glaxo ipine, lacidipine, lemildipine, lercanidipine, nicardipine, Smith Kline), TAK-637 (Takeda/Abbot), SR-14033, and nifedipine, nilvadipine, nimodepine, nisoldipine, nitren related compounds described in, for example, EP873753 A1, dipine, manidipine, pranidipine, and Verapamil, and the like; US 20010006972 A1, US 2003.0109417 A1, WO 01/52844 (4) angiotensin converting enzyme (ACE) inhibitors such as A1 (for a review see Giardina et al. 2003. Drugs 6:758); NK-2 benazepril; captopril; ceranapril; cilaZapril; delapril; enala receptor antagonists such as nepadutant (Menarini Ricerche pril; enalopril; fosinopril; imidapril; lisinopril; losinopril; SpA), saredutant (Sanofi-Synthelabo), GW5975.99 (Glaxo moexipril; quinapril; quinaprilat, ramipril; perindopril; per Smith Kline), SR-144190 (Sanofi-Synthelabo) and indropril; quanipril; spirapril; tenocapril; trandolapril, and UK-290795 (Pfizer Inc); NK3 receptor antagonists such as Zofenopril, and the like; (5) neutral endopeptidase inhibitors osanetant (SR-142801; Sanofi-Synthelabo), SSR-241586, Such as omapatrilat, cadoxatril and ecadotril, fosidotril, Sam talnetant and related compounds described in, for example, patrilat, AVE7688, ER4030, and the like: (6) endothelin WO 02/094187 A2, EP 876347 A1, WO 97/21680 A1, U.S. antagonists such as tezosentan, A3081.65, andYM62899, and Pat. No. 6,277,862, WO 98/1 1090, WO95/28418, WO the like; (7) vasodilators such as hydralazine, clonidine, 97/19927, and Boden et al. (JMedChem. 39:1664-75, 1996): minoxidil, and nicotinyl alcohol, and the like; (8) angiotensin norepinephrine-serotonin reuptake inhibitors (NSRI) such as II receptor antagonists such as aprosartan, candesartan, epro milnacipran and related compounds described in WO Sartan, irbesartan, losartan, olmesartan, pratosartan, tasosar 03/077897; and vanilloid receptor antagonists such as arvanil tan, telmisartan, Valsartan, and EXP-3137, FI6828K, and and related compouds described in WO 01/642 12 A1. RNH6270, and the like; (9) C/B adrenergic blockers such as US 2015/0366935 A1 Dec. 24, 2015

nipradillol, arotinolol and amosulalol, and the like: (10) alpha ethy)-amino-3-(1-naphthalenyloxy)-hydrochloride (CAS 1 blockers, such as teraZosin, urapidil, prazosin, tamsulosin, RN 13071-11-9), diacetolol hydrochloride (Acetamide, bunaZosin, trimaZosin, doxazosin, naftopidil, indoramin, N-3-acetyl-4-2-hydroxy-3-(1-methyl-ethyl)aminopro WHP 164, and XENOIO, and the like: (11) alpha 2 agonists poxyphenyl-, monohydrochloride CAS RN 69796-04-9), Such as lofexidine, tiamenidine, moxonidine, rillmenidine and dilevalol hydrochloride (Benzamide, 2-hydroxy-5-1-hy guanobenz, and the like: (12) aldosterone inhibitors, and the droxy-2-1-methyl-3-phenylpropyl)aminoethyl-, monohy like; and (13) angiopoietin-2-binding agents such as those drochloride, CAS RN 75659-08-4), exaprolol hydrochloride disclosed in WO03/030833. Specific anti-hypertensive (2-Propanol, 1-(2-cyclohexylphenoxy)-3-(1-methylethyl) agents that can be used in combination with polypeptides and amino-, hydrochloride CAS RN 59333-90-3), flestolol sul agonists described herein include, but are not limited to: fate (Benzoic acid, 2-fluro-3-2-aminocarbonyl)amino diuretics, such as thiazides (e.g., chlorthalidone, cyclothiaz dimethylethylamino-2-hydroxypropyl ester, (+)-Sulfate ide (CAS RN 2259-96-3), chlorothiazide (CAS RN 72956 (1:1) (salt), CAS RN 88844-73-9; metalol hydrochloride 09-3, which may be prepared as disclosed in U.S. Pat. No. (Methanesulfonamide, N-(4-1-hydroxy-2-(methylamino) 2.809,194), dichlorophenamide, hydroflumethiazide, inda propylphenyl-, monohydrochloride CAS RN 7701-65-7), pamide, polythiazide, bendroflumethazide, methyclothazide, metoprolol 2-Propanol. 1-4-(2-methoxyethyl)phenoxy-3- polythiazide, trichlormethazide, chlorthalidone, indapamide, 1-methylethyl)amino-: CAS RN 37350-58-6), metoprolol metolazone, quinethazone, althiazide (CAS RN 5588-16-9, tartrate (such as 2-Propanol. 1-4-(2-methoxyethyl)phe which may be prepared as disclosed in British Patent No. noxy-3-(1-methylethyl)amino-, e.g., Lopressor R, Novar 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)amino 1, (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 396-01-0)), 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) 1,4- nadolol (e.g. Nadolol, Mylan), practolol (CAS RN 6673-35 dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine 4, see also U.S. Pat. No. 3,408,387), propranolol hydrochlo dicarboxylate, e.g. NimotopR, Bayer), felodipine (such as ride (CAS RN 318-98-9), sotalol hydrochloride (e.g. Beta ethyl methyl 4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dim pace AFTM, Berlex), timolol (2-Propanol, 1-(1,1- ethyl-3,5-pyridinedicarboxylate-, e.g. Plendil R. Extended dimethylethyl)amino-3-4-4-(4-morpholinyl)-1,2,5- Release, AstraZeneca LP), nilvadipine (3.5-Pyridinedicar thiadiazol-3-yl)oxy-, hemihydrate, (S) . CAS RN 91524 boxylic acid, 2-cyano-1,4-dihydro-6-methyl-4-(3- 16-2), timolol maleate (S)-I-(1,1-dimethylethyl) amino nitrophenyl)-3-methyl 5-(1-methylethyl) ester, also see U.S. 3-4-(4-morpholinyl)-1,2,5-thiadiazol-3-yloxy-2- Pat. No. 3,799,934), nifedipine (such as 3.5-pyridinedicar propanol (Z)-2-butenedioate (1:1) salt, CAS RN 26921-17 boxylic acid, 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)- 5), bisoprolol (2-Propanol. 1-4-2-(1-methylethoxyl) dimethyl ester, e.g., Procardia XLR) Extended Release Tab ethoxy-methylphenoxyl-3-(1-meth-ylethyl)amino-, (t), lets, Pfizer), diltiazem hydrochloride (such as 1.5-Benzothi CAS RN 66722-44-9), bisoprolol fumarate (such as (+)-1-4- azepin-4 (5H)-one.3-(acetyloxy)-5-[2-(dimethylamino) 2-(1-Methylethoxyl)ethoxymethylphenoxy-3-(1-meth ethyl-2,-3-dihydro-2(4-methoxyphenyl)- ylethyl)amino-2-propanol (E)-2-butenedioate (2:1) (salt), monohydrochloride, (+)-cis., e.g., TiaZac(R), Forest), Vera e.g., ZebetaTM, Lederle Consumer), nebivalol (2H-1-Ben pamil hydrochloride (such as benzeneacetronitrile, (alpha)- Zopyran-2-methanol, C.C.'-iminobis(methylene)bis6 3-2-(3,4-dimethoxyphenyl)ethylmethylaminopropyl fluoro-3,4-dihydro-, CAS RN 992.00-09-6 see also U.S. Pat. 3,4-dimethoxy-(alpha)-(1-methylethyl) hydrochloride, e.g., No. 4,654.362), cicloprolol hydrochloride, such 2-Propanol, Isoptin R. SR, Knoll Labs), teludipine hydrochloride (3.5- 1-4-2-(cyclopropylmethoxy)ethoxyphenoxy-3-1-meth Pyridinedicarboxylic acid, 2-(dimethylamino)methyl4-2- ylethyl)amino-, hydrochloride, A.A.S. RN 63686-79-3), (1E)-3-(1,1-dimethylethoxy)-3-oxo-1-propenylphenyl-1, dexpropranolol hydrochloride (2-Propanol. 1-1-methyl 4-dihydro-6-methyl-, diethyl ester, monohydrochloride) US 2015/0366935 A1 Dec. 24, 2015 42

CAS RN 108700-03-4), belfosdil (Phosphonic acid, 2-(2- FEBS Lett. 165:201 (1984)), CI925 (Pharmacologist 26:243, phenoxy ethyl)-1,3-propane-diyl)bis-, tetrabutyl ester CAS 266 (1984)), WY-44221 (Wyeth, see J. Med. Chem. 26:394 RN 103486-79–9), fostedil (Phosphonic acid, 4-(2-ben (1983)), and those disclosed in US2003006922 (paragraph Zothiazolyl)phenyl)methyl-, diethyl ester CAS RN 75889 28), U.S. Pat. No. 4,337,201, U.S. Pat. No. 4,432,971 (phos 62-2), aranidipine, azelnidipine, barnidipine, benidipine, phonamidates); neutral endopeptidase inhibitors such as bepridil, cinaldipine, clevidipine, efonidipine, gallopamil, omapatrilat (Vanlev(R), CGS 30440, cadoxatrilandecadotril, lacidipine, lemildipine, lercanidipine, monatepil maleate fasidotril (also known as aladotril or alatriopril), Sampatrilat, (1-Piperazinebutanamide, N-(6,11-dihydrodibenzo(b.e)thi mixanpril, and gemopatrilat, AVE7688, ER4030, and those epin-11-yl)4-(4-fluorophenyl)-, (+)-, (Z)-2-butenedioate disclosed in U.S. Pat. No. 5,362,727, U.S. Pat. No. 5,366,973, (1:1) (+)-N-(6,11-Dihydrodibenzo(b.e)thiep-in-11-yl)-4-(p- U.S. Pat. No. 5,225,401, U.S. Pat. No. 4,722,810, U.S. Pat. fluorophenyl)-1-piperazinebutyramide maleate (1:1) CAS No. 5,223,516, U.S. Pat. No. 4,749,688, U.S. Pat. No. 5,552, RN 132046-06-1), nicardipine, nisoldipine, nitrendipine, 397, U.S. Pat. No. 5,504,080, U.S. Pat. No. 5,612,359, U.S. manidipine, pranidipine, and the like; T-channel calcium Pat. No. 5,525,723, EP0599444, EP0481522, EP0599444, antagonists such as mibefradil; angiotensin converting EP0595610, EP0534363, EP534396, EP534492, enzyme (ACE) inhibitors such as benazepril, benazepril EP0629627; endothelin antagonists such as tezosentan, hydrochloride (such as 3-1-(ethoxycarbonyl)-3-phenyl-(1 A308165, and YM62899, and the like; vasodilators such as S)-propylamino)-2,3,4,5-tetrahydro-2-oxo-1H-1-(3 S)-ben hydralazine (apresoline), clonidine (clonidine hydrochloride Zazepine-1-acetic acid monohydrochloride, e.g., Lotrel (R), (1H-Imidazol-2-amine, N-(2,6-dichlorophenyl)4,5-dihy Novartis), captopril (such as 1-(2S)-3-mercapto-2-methyl dro-, monohydrochloride CAS RN 4205-91-8), catapres, propionyl-L-proline, e.g., Captopril, Mylan, CAS RN minoxidil (loniten), nicotinyl alcohol (roniacol), diltiazem 62571-86-2 and others disclosed in U.S. Pat. No. 4,046,889), hydrochloride (such as 1.5-Benzothiazepin-4(5H)-one.3- ceranapril (and others disclosed in U.S. Pat. No. 4,452.790), (acetyloxy)-5-[2-(dimethylamino)ethyl-2,-3-dihydro-2(4- cetapril (alacepril, Dainippon disclosed in Eur. Therap. Res. methoxyphenyl)-, monohydrochloride, (+)-cis, e.g., 39:671 (1986): 40:543 (1986)), cilazapril (Hoffman TiazacR, Forest), isosorbide dinitrate (such as 1.4:3,6-dian LaRoche) disclosed in J. Cardiovasc. Pharmacol. 9:39 hydro-D-glucitol 2,5-dinitrate e.g., Isordil R. Titradose(R), (1987), indalapril (delapril hydrochloride (2H-1,2,4-Ben Wyeth-Ayerst), sosorbide mononitrate (such as 1.4:3,6-dian Zothiadiazine-7-sulfonamide, 3-bicyclo[2.2.1]hept-5-en-2- hydro-D-glucito-1,5-nitrate, an organic nitrate, e.g., Ismo (R), yl-6-chloro-3,4-dihydro-, 1,1-dioxide CAS RN 2259-96-3): Wyeth-Ayerst), nitroglycerin (such as 2.3 propanetriol trini disclosed in U.S. Pat. No. 4.385,051), enalapril (and others trate, e.g., Nitrostat(R) Parke-Davis), Verapamil hydrochloride disclosed in U.S. Pat. No. 4.374,829), enalopril, enaloprilat, (such as benzeneacetonitrile, (+)-(alpha)3-2-(3.4 fosinopril, (such as L-proline, 4-cyclohexyl-1-(2-methyl dimethoxypheny 1)ethylmethylaminopropyl-3,4- 1-(1-oxopropoxyl) propoxy (4-phenylbutyl) phosphinyl dimethoxy-(alpha)-(1-methylethyl) hydrochloride, e.g., acetyl-, sodium salt, e.g., Monopril, Bristol-Myers Squibb Covera HS(R) Extended-Release, Searle), chromonar (which and others disclosed in U.S. Pat. No. 4,168.267), fosinopril may be prepared as disclosed in U.S. Pat. No. 3,282.938), sodium (L-Proline, 4-cyclohexyl-1-((R)-(1S)-2-methyl-1- clonitate (Annalen 1870 155), droprenilamine (which may be (1-ox-opropoxy)propox), imidapril, indolapril (Schering, prepared as disclosed in DE2521113), lidoflazine (which disclosed in J. Cardiovasc. Pharmacol. 5:643, 655 (1983)), may be prepared as disclosed in U.S. Pat. No. 3,267,104); lisinopril (Merck), losinopril, moexipril, moexipril hydro prenylamine (which may be prepared as disclosed in U.S. Pat. chloride (3-Isoquinolinecarboxylic acid, 2-(2S)-2-(1S)- No. 3,152.173), propatyl nitrate (which may be prepared as 1-(ethoxycarbonyl)-3-phenylpropylamino-1-oxopropyl disclosed in French Patent No. 1,103,113), mioflazine hydro 1.-2,3,4-tetrahydro-6,7-dimethoxy-, monohydrochloride, chloride (1-Piperazineacetamide, 3-(aminocarbonyl)4-4.4- (3S)-CAS RN 82586-52-5), quinapril, quinaprilat, ramipril bis(4-fluorophenyl)butyl-N-(2,6-dichlorophenyl)-, dihydro (Hoechsst) disclosed in EP 79022 and Curr. Ther. Res. 40:74 chloride CAS RN 83898-67-3), mixidine (1986), perindopril erbumine (such as 2S,3aS,7aS-1-(S)- (Benzeneethanamine, 3,4-dimethoxy-N-(1-methyl-2-pyrro N—(S)-1-Carboxybutylalanylhexahydro-indolinecar lidinylidene)-Pyrrolidine, 2-(3,4-dimethoxyphenethyl) boxylic acid, 1-ethyl ester, compound with tert-butylamine imino-1-methyl-1-Methyl-2-(3,4-dimethoxyphenethyl) (1:1), e.g., Aceon R, Solvay), perindopril (Servier, disclosed iminolpyrrolidine CAS RN 27737-38-8), molsidomine (1.2, in Eur. J. din. Pharmacol. 31:519 (1987)), quanipril (disclosed 3-Oxadiazolium, 5-(ethoxycarbonyl)amino-3-(4- in U.S. Pat. No. 4.344.949), spirapril (Schering, disclosed in morpholinyl)-, inner salt CAS RN 25717-80-0), isosorbide Acta. Pharmacol. Toxicol. 59 (Supp. 5): 173 (1986)), tenoca mononitrate (D-Glucitol, 1.4:3,6-dianhydro-, 5-nitrate CAS pril, trandolapril, Zofenopril (and others disclosed in U.S. Pat. RN 16051-77-7), erythrityl tetranitrate (1,2,3,4-Butanetetrol, No. 4.316,906), rentiapril (fentiapril, disclosed in Clin. Exp. tetranitrate, (2R,3S)-rel-CAS RN 7297-25-8), clonitrate(1,2- Pharmacol. Physiol. 10:131 (1983)), pivopril, YS980, tepro Propanediol, 3-chloro-, dinitrate (7CI, 8CI, 9CI) CAS RN tide (Bradykinin potentiator BPP9a CAS RN 35115-60-7), 2612-33-1), dipyridamole Ethanol, 2.2.2".2"-(4,8-di-1-pip BRL 36,378 (Smith Kline Beecham, see EP80822 and eridinylpyrimido5,4-dipyrimidine-2,6-diyl)dinitrilotet EP60668), MC-838 (Chugai, see CA. 102:72588v and Jap.J. rakis-CAS RN 58-32-2), nicorandil (CAS RN 65141-46-0 Pharmacol. 40:373 (1986), CGS 14824 (Ciba-Geigy, 3-(1- 3-), pyridinecarboxamide (N-2-(nitrooxy)ethyl-Nisol ethoxycarbonyl-3-phenyl-(1S)-propylamino)-2,3,4,5-tet dipine3.5-Pyridinedicarboxylic acid, 1,4-dihydro-2,6-dim rahydro-2-ox-o-1-(3S)-benzazepine-1 acetic acid HCl, see ethyl-4-(2-nitrophenyl)-, methyl 2-methylpropyl ester CAS U.K. Patent No. 2103614), CGS 16,617 (Ciba-Geigy, 3(S)- RN 63675-72-9), nifedipine3,5-Pyridinedicarboxylic acid, (1S)-5-amino-1-carboxypentyl)amino-2,3,4,-5-tetrahy 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester dro-2-oxo-1H-1-benzazepine-1-ethanoic acid, see U.S. Pat. CAS RN 21829-25-4), perhexiline maleate (Piperidine, 2-(2, No. 4,473,575), Ru 44570 (Hoechst, see Arzneimittelfors 2-dicyclohexylethyl)-, (2Z)-2-butenedioate (1:1) CAS RN chung 34:1254 (1985)), R 31-2201 (Hoffman-LaRoche see 6724-53-4), oxprenolol hydrochloride (2-Propanol, 1-(1-

US 2015/0366935 A1 Dec. 24, 2015 44

WO93/03033, WO91/16313, WO92/00068, WO92/02510, amino-, hydrochloride CAS RN 63659-19-8), butoprozine WO92/09278, WO92/0179, WO92/10180, WO92/10186, hydrochloride (Methanone, 4-3(dibutylamino)propoxy WO92/10181, WO92/10097, WO92/10183, WO92/10182, phenyl (2-ethyl-3-indolizinyl)-, monohydrochloride CAS WO92/10187, WO92/10184, WO92/10188, WO92/10180, RN 62134-34-3), cinepazet maleatel-Piperazineacetic acid, WO92/10185, WO92/20651, WO93/03722, WO93/06828, 4-1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl-, ethyl WO93/03040, WO92/19211, WO92/22533, WO92/06081, ester, (2Z)-2-butenedioate (1:1) CAS RN 50679-07-7), tosifen (Benzenesulfonamide, 4-methyl-N-(1S)-1-methyl WO92/05784, WO93/00341, WO92/04343, WO92/04059, 2-phenylethylaminocarbonyl-CAS RN32295-184), Vera U.S. Pat. No. 5,104,877, U.S. Pat. No. 5,187,168, U.S. Pat. pamilhydrochloride (Benzeneacetonitrile, C-3-2-(3,4- No. 5,149,699, U.S. Pat. No. 5,185,340, U.S. Pat. No. 4,880, dimethoxyphenyl)ethylmethylaminopropyl-3,4- 804, U.S. Pat. No. 5,138,069, U.S. Pat. No. 4,916,129, U.S. dimethoxy-O-(1-methylethyl)-, monohydrochloride CAS Pat. No. 5,153,197, U.S. Pat. No. 5,173,494, U.S. Pat. No. RN 152-114), molsidomine (1,2,3-Oxadiazolium, 5,137,906, U.S. Pat. No. 5,155,126, U.S. Pat. No. 5,140,037, 5-(ethoxycarbonyl)amino-3-(4-morpholinyl)-, inner salt U.S. Pat. No. 5,137,902, U.S. Pat. No. 5,157,026, U.S. Pat. CAS RN25717-80-0), and ranolazine hydrochloride (1-Pip No. 5,053,329, U.S. Pat. No. 5,132,216, U.S. Pat. No. 5,057, erazineacetamide, N-(2,6-dimethylphenyl)4-2-hydroxy-3- 522, U.S. Pat. No. 5,066,586, U.S. Pat. No. 5,089,626, U.S. (2-meth-oxyphenoxy)propyl-, dihydrochloride CAS RN Pat. No. 5,049,565, U.S. Pat. No. 5,087,702, U.S. Pat. No. 95635-56-6); tosifen (Benzenesulfonamide, 4-methyl-N- 5,124,335, U.S. Pat. No. 5,102,880, U.S. Pat. No. 5,128,327, III(1S)-1-methyl-2-phenylethylaminocarbonyl-CAS RN U.S. Pat. No. 5,151,435, U.S. Pat. No. 5,202,322, U.S. Pat. 32295-184); adrenergic stimulants such as guanfacine hydro No. 5,187,159, U.S. Pat. No. 5,198,438, U.S. Pat. No. 5,182, chloride (such as N-amidino-2-(2,6-dichlorophenyl)aceta 288, U.S. Pat. No. 5,036,048, U.S. Pat. No. 5,140,036, U.S. mide hydrochloride, e.g., Tenex R. Tablets available from Pat. No. 5,087,634, U.S. Pat. No. 5,196,537, U.S. Pat. No. Robins); methyldopa-hydrochlorothiazide (such as levo-3- 5,153,347, U.S. Pat. No. 5,191,086, U.S. Pat. No. 5,190,942, (3,4-dihydroxyphenyl)-2-methylalanine) combined with U.S. Pat. No. 5,177,097, U.S. Pat. No. 5,212,177, U.S. Pat. Hydrochlorothiazide (such as 6-chloro-3,4-dihydro-2H-1.2. No. 5,208,234, U.S. Pat. No. 5,208,235, U.S. Pat. No. 5,212, 4-benzothiadiazine-7-sulfonamide 1,1-dioxide, e.g., the 195, U.S. Pat. No. 5,130,439, U.S. Pat. No. 5,045,540, U.S. combination as, e.g., Aldoril R. Tablets available from Merck), Pat. No. 5,041,152, and U.S. Pat. No. 5,210,204, and phar methyldopa-chlorothiazide (such as 6-chloro-2H-1,2,4-ben maceutically acceptable salts and esters thereof; C/B adren 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'(C))-. 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- 1.3.2.11 Agents for the Treatment of Respiratory (2-methoxyphenyl)-1-piperazinylethyl-, (2R,3R)-2,3-di Disorders hydroxybutanedioate (1:1) CAS RN 5591-43-5), Zolertine hydrochloride (Piperazine, 1-phenyl4-2-(1H-tetrazol-5-yl) 0.178 The GCC agonist peptides described herein can be ethyl-, monohydrochloride (8C1,9C1) CAS RN 7241-94-3) used in combination therapy with one or more of the follow and the like; a adrenergic receptor blockers, such as alfuZosin ing agents useful in the treatment of respiratory and other (CAS RN: 81403-68-1), terazosin, urapidil, prazosin (Mini disorders including but not limited to: (1) B-agonists includ press.(R), tamsulosin, bunaZosin, trimaZosin, doxazosin, naf ing but not limited to: albuterol (PROVENTIL(R), S ALBUT topidil, indoramin, WHP 164, XENOIO, fenspiride hydro AMOIR, VENTOLINR), bambuterol, bitoterol, clenbuterol, chloride (which may be prepared as disclosed in U.S. Pat. No. fenoterol, formoterol, isoetharine (BRONKOSOL(R), 3.399,192), proroxan (CAS RN33743-96-3), and labetalol BRONKOMETER(R), metaproterenol (ALUPENTR), hydrochloride and combinations thereof a 2 agonists such as METAPREL(R), pirbuterol (MAXAIRR), reproterol, rimit methyldopa, methyldopa HCL, lofexidine, tiamenidine, erol, salmeterol, terbutaline (BRETHAIRE(R), BRETHINER, moXonidine, rillmenidine, guanobenz, and the like; aldoster BRICANYL(R), adrenalin, isoproterenol (ISUPREL(R), epi one inhibitors, and the like; renin inhibitors including nephrine bitartrate (PRIMATENE(R), ephedrine, orcipren Aliskiren (SPP100; Novartis/Speedel); angiopoietin-2-bind line, fenoterol and isoetharine; (2) steroids, including but not ing agents such as those disclosed in WO03/030833; anti limited to beclomethasone, beclomethasone dipropionate, angina agents such as ranolazine (hydrochloride 1-Pipera betamethasone, budesonide, bunedoside, butiXocort, dexam zineacetamide, N-(2,6-dimethylphenyl)-4-2-hydroxy-3-(2- ethasone, flunisolide, fluocortin, fluticasone, hydrocortisone, methoxyphenoxyl)propyl-, dihydrochloride CAS RN methyl prednisone, mometaSone, predonisolone, predo 95635-56-6), betaxolol hydrochloride (2-Propanol. 1-4-2 nisone, tipredane, tiXocortal, triamcinolone, and triamcino (cyclopropylmethoxy)ethylphenoxy-3-(1-methylethyl) lone acetonide; (3)(32-agonist-corticosteroid combinations US 2015/0366935 A1 Dec. 24, 2015

e.g., salmeterol-fluticasone (AD V AIRR), formoterol human recombinant DNase 1. (Pulmozyme(R); (20) nonste budesonid (SYMBICORTR); (4) leukotriene D4 receptor roidal anti-inflammatory agents (acemetacin, acetami antagonists/leukotriene antagonists/LTD4 antagonists (i.e., nophen, acetyl salicylic acid, alclofenac, alminoprofen, apa any compound that is capable of blocking, inhibiting, reduc Zone, aspirin, benoxaprofen, bezpiperylon, bucloxic acid, ing or otherwise interrupting the interaction between leukot carprofen, clidanac, diclofenac, diclofenac, diflunisal, diflu rienes and the Cys LTI receptor) including but not limited to: sinal, etodolac, fenbufen, fenbufen, fenclofenac, fenclozic Zafhiukast, montelukast, montelukast sodium (SINGU acid, fenoprofen, fentiazac, feprazone, flufenamic acid, LAIRR), pranlukast, iralukast, pobilukast, SKB-106,203 and flufenisal, flufenisal, fluprofen, flurbiprofen, flurbiprofen, furofenac, ibufenac, ibuprofen, indomethacin, indomethacin, compounds described as having LTD4 antagonizing activity indoprofen, isoxepac, isoxicam, ketoprofen, ketoprofen, described in U.S. Pat. No. 5,565,473; (5) 5-lipoxygenase ketorolac, meclofenamic acid, meclofenamic acid, mefe inhibitors and/or leukotriene biosynthesis inhibitors (e.g., namic acid, mefenamic acid, miroprofen, mofebutaZone, zileuton and BAY1005 (CA registry 128253-31-6); (6) his nabumetone oxaprozin, naproxen, naproxen, niflumic acid, tamine H1 receptor antagonists/antihistamines (i.e., any com oxaprozin, Oxpinac, oxyphenbutaZone, phenacetin, phenylb pound that is capable of blocking, inhibiting, reducing or utaZone, phenylbutaZone, piroxicam, piroxicam, pirprofen, otherwise interrupting the interaction between histamine and pranoprofen, Sudoxicam, tenoxican, Sulfasalazine, Sulindac, its receptor) including but not limited to: astemizole, acrivas Sulindac. Suprofen, tiaprofenic acid, tiopinac, tioxaprofen, tine, antazoline, azatadine, azelastine, astamizole, bromophe tolfenamic acid, tolmetin, tolmetin, Zidometacin, Zomepirac, niramine, bromopheniramine maleate, carbinoxamine, care and Zomepirac); and (21) aerosolized antioxidant therapeu bastine, cetirizine, chlorpheniramine, chloropheniramine tics such as S-Nitrosoglutathione. maleate, cimetidine clemastine, cyclizine, cyproheptadine, descarboethoxyloratadine, dexchlorpheniramine, dimethin dene, diphenhydramine, diphenylpyraline, doxylamine Suc 1.3.2.12 Anti-Diabetic Agents cinate, doxylamine, ebastine, efletirizine, epinastine, famoti 0179 The GCC agonist peptides described herein can be dine, fexofenadine, hydroxy Zine, hydroxy Zine, ketotifen, used in therapeutic combination with one or more anti-dia levocabastine, levocetirizine, levocetirizine, loratadine, betic agents, including but not limited to: PPARY agonists meclizine, mepyramine, meduitazine, methdilazine, such as glitazones (e.g., WAY-120.744, AD 5075, balaglita mianserin, mizolastine, noberastine, norasternizole, noraZ Zone, ciglitazone, darglitazone (CP-86325, Pfizer), englita temizole, phenindamine, pheniramine, picumast, promethaZ Zone (CP-68722, Pfizer), isaglitazone (MIT/J&J), MCC-555 ine, pynlamine, pyrilamine, ranitidine, temelastine, terfena (Mitsibishi disclosed in U.S. Pat. No. 5,594,016), pioglita dine, trimeprazine, tripelenamine, and triprolidine; (7) an Zone (such as such as ActosTM pioglitaZone; Takeda), rosigli anticholinergic including but not limited to: atropine, benz taZone (AvandiaTM; Smith Kline Beecham), rosiglitazone tropine, biperiden, flutropium, hyoscyamine (e.g. Levsin(R); maleate, troglitazone (Rezulin(R), disclosed in U.S. Pat. No. LevbidR; Levsin/SL(R), AnaspaZR), Levsinex Timecaps.(R), 4.572,912), rivoglitazone (CS-OI 1, Sankyo), GL-262570 NuLev(R), ilutropium, ipratropium, ipratropium bromide, (Glaxo Welcome), BRL49653 (disclosed in WO98/05331), methScopolamine, oxybutinin, rispenzepine, Scopolamine, CLX-0921, 5-BTZD, GW-0207, LG-100641, JJT-501 and tiotropium; (8) an anti-tussive including but not limited (JPNT/P&U), L-895.645 (Merck), R-1 19702 (Sankyo/ to: dextromethorphan, codeine, and hydromorphone; (9) a Pfizer), NN-2344 (Dr. Reddy/NN), YM-440 (Yamanouchi), decongestant including but not limited to: pseudoephedrine LY-300512, LY-519818, R483 (Roche), T131 (Tularik), and and phenylpropanolamine; (10) an expectorant including but the like and compounds disclosed in U.S. Pat. No. 4,687,777. not limited to: guafenesin, guaicolsulfate, terpin, ammonium U.S. Pat. No. 5,002,953, U.S. Pat. No. 5,741,803, U.S. Pat. chloride, glycerol guaicolate, and iodinated glycerol; (11) a No. 5,965,584, U.S. Pat. No. 6,150,383, U.S. Pat. No. 6,150, bronchodilator including but not limited to: theophylline and 384 U.S. Pat. No. 6,166,042, U.S. Pat. No. 6,166,043, U.S. aminophylline; (12) an anti-inflammatory including but not Pat. No. 6,172,090, U.S. Pat. No. 6,211,205, U.S. Pat. No. limited to: fluribiprofen, diclophenac, indomethacin, keto 6,271,243, U.S. Pat. No. 6,288,095, U.S. Pat. No. 6,303,640, profen, S-ketroprophen, tenoxicam; (13) a PDE (phosphodi U.S. Pat. No. 6,329,404, U.S. Pat. No. 5,994,554, WO97/ esterase) inhibitor including but not limited to those disclosed 10813, WO97/27857, WO97/28115, WO97/28137, WO97/ herein; (14) a recombinant humanized monoclonal antibody 27847, WO00/76488, WO03/000685, WO03/027112, e.g. Xolair (also called omalizumab), rhuMab, and tali WO03/035602, WO03/048130, WO03/055867, and pharma Zumab); (15) a humanized lung Surfactant including recom ceutically acceptable salts thereof; biguanides Such as met binant forms of surfactant proteins SP-B, SP-C or SP-D formin hydrochloride (N,N-dimethylimidodicarbonimidic e.g. SURFAXINR), formerly known as disc-104 (Discovery diamide hydrochloride, such as GlucophageTM, Bristol-My Laboratories), (16) agents that inhibit epithelial sodium ers Squibb); metforminhydrochloride with glyburide, such as channels (ENaC) such as amiloride and related compounds; GlucovanceTM, Bristol-Myers Squibb); buformin (Imidodi (17) antimicrobial agents used to treat pulmonary infections carbonimidic diamide, N-butyl-): etoformine (1-Butyl-2-eth Such as acyclovir, amikacin, amoxicillin, doxycycline, trime ylbiguanide, Schering A. G.); other metformin salt forms thoprin Sulfamethoxazole, amphotericin B, azithromycin, (including where the salt is chosen from the group of acetate, clarithromycin, roXithromycin, clarithromycin, cephalospor benzoate, citrate, ftimarate, embonate, chlorophenoxyac ins(ceffoxitin, cefimetazole etc), ciprofloxacin, ethambutol, etate, glycolate, palmoate, aspartate, methaneSulphonate, gentimycin, ganciclovir, imipenem, iSoniazid, itraconazole, maleate, parachlorophenoxyisobutyrate, formate, lactate, penicillin, ribavirin, rifampin, rifabutin, amantadine, riman Succinate, Sulphate, tartrate, cyclohexanecarboxylate, hex tidine, Streptomycin, tobramycin, and Vancomycin; (18) anoate, octanoate, decanoate, hexadecanoate, octodecanoate, agents that activate chloride secretion through Ca++ depen benzenesulphonate, trimethoxybenzoate, paratoluene dent chloride channels (such as purinergic receptor (P2Y(2) Sulphonate, adamantanecarboxylate, glycoxylate, glutamate, agonists); (19) agents that decrease sputum viscosity, such as pyrrolidonecarboxylate, naphthalenesulphonate, 1-glucose US 2015/0366935 A1 Dec. 24, 2015 46 phosphate, nitrate, Sulphite, dithionate and phosphate), and ile formulations such as those available from Eli Lilly (India phenformin; protein tyrosine phosphatase-IB (PTP-IB) napolis, Ind. 46285) as HumulinTM (human insulin rDNA inhibitors, such as A-401,674, KR 61639, OC-060062, origin), also see the THE PHYSICIAN'S DESK REFER OC-83839, OC-297962, MC52445, MC52453, ISIS 113715, ENCE, 55.sup.th Ed. (2001) Medical Economics, Thomson and those disclosed in WO99/585521, WO99/58518, WO99/ Healthcare (disclosing other Suitable human insulins); non 58522, WO99/61435, WO03/032916, WO03/032982, thiazolidinediones such as JT-501 and farglitazar (GW-2570/ WO03/041729, WO03/055883, WO02/26707, WO02/ GI-262579), and pharmaceutically acceptable salts and esters 26743, JP2002114768, and pharmaceutically acceptable thereof; PPARO/Y dual agonists such as AR-HO39242 (AZ salts and esters thereof. Sulfonylureas such as acetohexamide traZeneca), GW-409544 (Glaxo-Wellcome), BVT-142, CLX (e.g. Dymelor, Eli Lilly), carbutamide, chlorpropamide (e.g. 0940, GW-1536, GW-1929, GW-2433, KRP-297 (Kyorin Diabinese(R), Pfizer), gliamilide (Pfizer), gliclazide (e.g. Merck; 5-(2,4-Dioxo thiazolidinyl)methylmethoxy-N4-4- Diamcron, Servier Canada Inc), glimepiride (e.g. disclosed in (trifluoromethyl)phenylmethylbenzamide), L-796449, U.S. Pat. No. 4.379,785, such as Amaryl, Aventis), glipentide, LR-90, MK-0767 (Merck/Kyorin/Banyu), SB 219994, mura glipizide (e.g. Glucotrol or Glucotrol XL Extended Release, glitazar (BMS), tesaglitzar (AstraZeneca), reglitazar (JTT Pfizer), gliquidone, glisolamide, glyburide/glibenclamide 501) and those disclosed in WO99/16758, WO99/19313, (e.g. Micronase or Glynase Prestab, Pharmacia & Upjohn and WO99/20614, WO99/38850, WO00/23415, WO00/23417, Diabeta, Aventis), tolaZamide (e.g. Tolinase), and tolbuta WO00/23445, WO00/50414, WO01/00579, WO01/79150, mide (e.g. Orinase), and pharmaceutically acceptable salts WO02/062799, WO03/004.458, WO03/016265, WO03/ and esters thereof: meglitinides Such as repaglinide (e.g. 018010, WO03/033481, WO03/033450, WO03/033453, PranidinC), Novo Nordisk), KAD1229 (PF/Kissei), and WO03/043985, WO 031053976, U.S. application Ser. No. nateglinide (e.g. Starlix R, Novartis), and pharmaceutically 09/664,598, filed Sep. 18, 2000, Murakamietal. Diabetes 47. acceptable salts and esters thereof a glucoside hydrolase 1841-1847 (1998), and pharmaceutically acceptable salts and 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; GSK3 B/GSK3 inhibitors such as 4-2-(2- (2R.3R.4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperi 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 14, and the compounds disclosed in U.S. Pat. No. 4,062,950, phosphorylase (HGLPa) inhibitors such as CP-368,296, U.S. Pat. No. 4,174,439, U.S. Pat. No. 4,254.256, U.S. Pat. CP-316,819, BAYR3401, and compounds disclosed in WOO No. 4,701,559, U.S. Pat. No. 4,639,436, U.S. Pat. No. 5,192, 1/94300, WO02/20530, WO03/037864, and pharmaceuti 772, U.S. Pat. No. 4,634,765, U.S. Pat. No. 5,157,116, U.S. cally acceptable salts or esters thereof; ATP consumption Pat. No. 5,504,078, U.S. Pat. No. 5,091,418, U.S. Pat. No. promotors such as those disclosed in WO03/007990; TRB3 5,217,877, US51091 and WOO 1/47528 (polyamines); inhibitors; Vanilloid receptor ligands such as those disclosed C.-amylase inhibitors such as tendamistat, trestatin, and in WO03/049702; hypoglycemic agents such as those dis A1-3688, and the compounds disclosed in U.S. Pat. No. closed in WO03/015781 and WO03/040114; glycogen syn 4,451,455, U.S. Pat. No. 4,623,714, and U.S. Pat. No. 4,273, thase kinase 3 inhibitors such as those disclosed in WOO3/ 765: SGLT2 inhibtors including those disclosed in U.S. Pat. 035663 agents such as those disclosed in WO99/51225, No. 6,414,126 and U.S. Pat. No. 6,515,117; an aP2 inhibitor US2003.0134890, WOO1/24786, and WO03/059870; insulin such as disclosed in U.S. Pat. No. 6,548,529; insulin secrea responsive DNA binding protein-1 (IRDBP-I) as disclosed in tagogues such as linogliride, A-4166, forskilin, dibutyrl WO03/057827, and the like; adenosine A2 antagonists such cAMP, isobutylmethylxanthine (IBMX), and pharmaceuti as those disclosed in WO03/035639, WO03/035640, and the cally acceptable salts and esters thereof fatty acid oxidation like: PPARö agonists such as GW 501516, GW 590735, and inhibitors, such as clomoxir, and etomoxir, and pharmaceu compounds disclosed in JP10237049 and WO02/14291; tically acceptable salts and esters thereof; A2 antagonists, dipeptidyl peptidase IV (DP-IV) inhibitors, such as isoleu Such as midaglizole, isaglidole, deriglidole, idazoxan, ear cine thiazolidide, NVP-DPP728A (1-2-(5-cyanopyridin oxan, and fluparoxan, and pharmaceutically acceptable salts 2-yl)aminoethylaminoacetyl-2-cyano-(S)-pyrrolidine, and esters thereof insulin and related compounds (e.g. insu disclosed by Hughes et al. Biochemistry, 38(36), 11597 lin mimetics) such as biota, LP-100, novarapid, insulin 11603, 1999), P32/98, NVP-LAF-237, P3298, TSL225 (tryp detemir, insulin lispro, insulin glargine, insulin Zinc suspen tophyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, sion (lente and ultralente), Lys-Pro insulin, GLP-I (1-36) disclosed by Yamada et al. Bioorg. & Med. Chem. Lett. 8 amide, GLP-I (73-7) (insulintropin, disclosed in U.S. Pat. No. (1998) 1537-1540), valine pyrrolidide, TMC-2A/2B/2C, 5,614,492), LY-315902 (Lilly), GLP-I (7-36)-NH2), AL-401 CD-26 inhibitors, FE999011, P9310/K364, VIP 0177, DPP4, (Autoimmune), certain compositions as disclosed in U.S. Pat. SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides No. 4,579,730, U.S. Pat. No. 4,849,405, U.S. Pat. No. 4,963, as disclosed by Ashworth et al. Bioorg. & Med. Chem. Lett. 526, U.S. Pat. No. 5,642,868, U.S. Pat. No. 5,763,396, U.S. Vol. 6, No. 22, pp 1163-1166 and 2745-2748 (1996), and the Pat. No. 5,824,638, U.S. Pat. No. 5,843.866, U.S. Pat. No. compounds disclosed in U.S. Pat. No. 6,395,767, U.S. Pat. 6,153,632, U.S. Pat. No. 6,191,105, and WO 85/05029, and No. 6,573.287, U.S. Pat. No. 6,395,767 (compounds dis primate, rodent, or rabbit insulin including biologically active closed include BMS-4771 18, BMS-471211 and BMS 538, variants thereof including allelic variants, more preferably 305), WO99/38501, WO99/46272, WO99/67279, WO99/ human insulin available in recombinant form (sources of 67278, WO99/61431 WO03/004498, WO03/004496, human insulin include pharmaceutically acceptable and ster EP1258476, WO02/083128, WO02/062764, WO03/000250, US 2015/0366935 A1 Dec. 24, 2015 47

WO03/002530, WO03/002531, WO03/002553, WO03/ -continued 002593, WO03/000180, and WO03/000181; GLP-I agonists Such as exendin-3 and exendin-4 (including the 39 aa Arms Interventions polypeptide synthetic exendin-4 called Exenatide(R), and compounds disclosed in US2003087821 and NZ504256, and SP-304 0.3 mg: Experimental Subjects receiving SP-304 0.3 mg for 14 pharmaceutically acceptable salts and esters thereof; peptides consecutive days including amlintide and Symlin R (pramlintide acetate); and 0182. Subjects diagnosed with CIC were screened for the glycokinase activators such as those disclosed in anticipated 4 cohorts to yield 80 randomized subjects for US2002103199 (fused heteroaromatic compounds) and enrollment. There were four dose cohorts (1.0 mg, 3.0 mg, 9.0 WO02/48106 (isoindolin-1-one-substituted propionamide mg and 0.3 mg) with 20 Subjects per dose cohort random compounds). ization ratio 3:1 (15 receive SP-304:5 receive placebo). Sub jects who continued to meet all the entry criteria and complete EXAMPLES the pre-treatment bowel movement (BM) diary received, in a double-blind, randomized fashion, SP-304 or matching pla Example 1 cebo. The entry criteria included (1) meeting modified ROME III criteria for chronic constipation (CC); (2) no significant finding in colonoscopy within past 5 years; (3) good health as Clinical Study for Safety and Efficacy in Humans for determined by physical examination, medical history, vital the Treatment of Chronic Idiopathic Constipation signs, ECG, clinical chemistry, hematology, urinalysis, drug screen and serology assessments; and (4) during 14-day pre 0180 A randomized, double-blind, placebo-controlled, treatment period, subjects reporting <6 SBM and <3 CSBM 14-day repeat oral, dose ranging study was conducted in in each pre-treatment week. All Subjects receiving at least one patients with chronic idiopathic constipation (CIC). The pri dose of SP-304 or matching placebo were considered evalu mary objective of this study was to evaluate the safety of able for the safety endpoints (78 total). If a subject did not SP-304 (1.0 mg, 3.0 mg, 9.0 mg and 0.3 mg) for 14 days in have a major protocol deviation, had at least 5 days of study patients with CIC. One secondary objective was to assess the treatment each week and corresponding entries for bowel pharmacokinetic profile of SP-304 in plasma. Other second habits, he/she was considered evaluable for efficacy param ary objectives included evaluations of pharmacodynamic eters (54-55 total). effects (efficacy) on parameters such as the time to first bowel 0183 The demographics of the subjects in the study are movement after daily dosing with SP-304, bowel habits over summarized in the table below.

Placebo 0.3 mg 1.0 mg 3.0 mg 9.0 mg Age 47.7 (14.6) 51.1 (12.0) 50.5 (10.6) 48.5 (16.1) 47.3 (12.7) Gender Female 18 (90.0%) 12 (85.7%) 14 (100%) 13 (86.7) 12 (80%) Male 2 (10.0%) 2 (14.3%) O 2 (13.3%) 3 (20%) Race White 17 (85.0%) 13 (92.9%) 12 (85.7%) 14 (93.3%) 12 (80.0%) African American 1 (5.0%) O 1 (7.1%) O 2 (13.3%) Asian 1 (5.0%) 1 (7.1%) 1 (7.1%) O 1 (6.7%) American Indian 1 (5.0%) O O O O Other O O O 1 (6.7%) O Walues for age are the mean (standard deviation); values for gender and race are the number (percentage of experimental arm), time for example, spontaneous bowel movements (SBMs), Results complete spontaneous bowel movements (CSBMs), and stool consistency using Bristol Stool Form Scale (BSFS)—and 0184 Pharmacokinetics and Safety: other patient reported outcomes Such as abdominal discom fort. 0185. There was no detectable systemic absorption of ple 0181. The study included five arms with assigned inter canatide (assay sensitivity >10 ng/mL). No serious adverse ventions as indicated in the table below. events (SAE) were reported in subjects receiving plecanatide and no deaths reported in this study. 10% (2/20) subjects who received placebo and 17.2% (10/58) subjects who received Arms Interventions SP-304 reported adverse events considered as related to the SP-304 1.0 mg: Experimental Subjects receiving SP-304 1.0 mg for 14 treatment. The majority of adverse events were mild/moder consecutive days SP-3043.0 mg: Experimental Subjects receiving SP-3043.0 mg for 14 ate and transient in nature. 10% (2/20) subjects who received consecutive days placebo and 5.2% (3/58) subjects who received SP-304 SP-3049.0 mg: Experimental Subjects receiving SP-3049.0 mg for 14 reported GI-related adverse events considered as related to consecutive days Placebo: Placebo Comparator Subjects receiving Placebo for 14 treatment. There was no diarrhea reported for any subject consecutive days receiving SP-304. The table below is a GI-related adverse event (AE) summary. US 2015/0366935 A1 Dec. 24, 2015 48

Example 4 Placebo 0.3 mg 1.0 mg 3.0 mg 9.0 mg n = 20 n = 14 n = 14 n = 15 n = 15 Excipient Compatibility Abdominal 1 (5.0%) O O O O (0190. Binary mixtures of SP-304 were prepared and Cramping stored in glass vials. For Solid excipients the binary mixtures Abdominal 1 (5.0%) O O O O were comprised of 9.1% or 50% excipient. Glass vials were Pain stored at 40 C/75RHopen or closed. The percent purity (mea Bloating O O O O 1 (6.7%) sured by HPLC) of the GCC agonist peptide (SP-304) after Diarrhea 1 (5.0%) O O O O storage for the time indicated in each column (i.e., 1, 2, or 3 Flatulence 2 (10.0%) O O O O months for the closed vial and 0.5, 1, 2, or 3 months for the Nausea O 1 (7.1%) O O Upset O O O 1 (6.7%) O open vials) is indicated by numerical values. Stomach

Walues are the number (percentage of experimental arm), PUR- EX- Closed Open 0186. Efficacy: POSE CIPIENT 1M 2M 3M O.SM 1M 2M 3M 0187 SP-304 (plecanatide) treatment decreased the time None None 914 88.2 84.1 93.7 91.2 88.2 84.8 to first bowel movement, increased stool frequency (SBM and Diluent Sorbitol 92.4 90.1. 87.2 92.2 90.8 87.1 80.9 CSBM), improved stool consistency, and reduced straining Mannitol 91.9 88.4 85.1 92.6 9 O.S 87.9 83.8 and abdominal discomfort. See FIGS. 1-6. ProSolw 92.2 89.6 86.3 93 9 O.S 87.8 83.7 Starch 91.4 88.7 85.4 92.S 90.5 87.9 83.7 Example 2 Binder Emdex 91.3 88.7 85.2 91.8 90.7 87.9 81.9 Plasdone 92.8 90.6 85.6 93.1 90.4 87.3 83 Composition of Wet Granulation Batch 10005 Disin- Explotab 91.9 89.4 87.1 92.2 90.3 84.7 78.3 egrant Poly- 92 89 85.6 93.S 90.3 87.4 83.1 0188 plasdone Glidant Cabosil 92.1 88.3 85.6 92.6 9 O.S 87.3 84 Lubricant Mg stearte 91.5 87.7 84.6 92.6 90.6 87.6 83.8 PRUV 92 88.3 85.7 92.2 90.S 87.5 83.8 Concentration compritol 90.8 87.1 84.4 92 9 O.S. 86.7 84.1 Item No. Ingredient Use % Wiw Excipient PEG 3350 90.9 87 83.3 91.5 89.4 84.4 77.5 1 SP3O4 O.23 Anti- Ascorbic 91.3 86.9 83 92.8 90 85.7 83.8 2 Mannogem EZ, Diluent 79.77 oxidant acid USP/EP (Mannitol) BHA 91.9 88.9 85.9 93.S 90.8 87.4 85.8 3 PROSOLVSMCC 90 Binder 1S.O BHT 90.8 87.2 84.6 92.4 90.3 86.6 83.6 EDTA 90.9 87.S 84.1 92.3 90.4 86.7 84.6 LM (silicified Capsule HPMC 92.2 89 85.2 92.3 90.2 86.4 83.5 microcrystalline capsule cellulose) Gelatin 91S 88.3 84.3 84.3 90.5 86.7 83.6 4 Purified Water vehicle na capsule (chilled to 5° C.), USP 5 Purified Water na Liquid Medium 90.4 (chilled to 5° C.), USP for chain 6 Explotab (Sodium Disintregant 4.0 liquid trig filled Starch Glycolate) capsule 7 Pruv (sodium stearyl Lubricant 1.O PG 89.3 fumarate) dicaprylo Total 100 caprate Vit E 90 Soybean 89.6 oil Cremaphor 79.7 Example 3 PG 3.4 PG 400 0.7 Composition of Wet Granulation Batch 10007 (0189 Example 5 Geometric Dry Mix for 0.3 mg Capsule Concentration Item No. Ingredient Use % Wiw 0191 Place 12 g mannitol in mortar. Add 4g SP-304 and gently mix until a visually uniform powder is obtained. Trans 1 SP3O4 O.3 fer to Turbula mixer. Rinse mortar with mannitol and transfer 3 PROSOLVSMCC 90 Binder 95.7 HD (silicified to Turbula mixer and mix at high speed for 10 minutes. Add microcrystalline about 150 g of mannitol to 4 quart V-shell mixer. Transfer the cellulose) contents of the Turbula mixer to the V-shell and add 150 g of 4 Purified Water vehicle na (chilled to 5° C.), USP mannitol mix. Discharge V-shell contents and screen through 5 Purified Water na 40 mesh and return to mixer. Add 586 g of mannitol to mixer (chilled to 5° C.), USP and mix for 20 minutes. 6 Explotab (Sodium Disintregant 4.0 Starch Glycolate) Example 6

Total 1OO Wet Granulation Process (0192 Batch 017-10005 comprised of mannitol and low moisture (2.4%) PROSOLV LM90 (0.33 g/mL) was sprayed US 2015/0366935 A1 Dec. 24, 2015 49 with SP-304 solution and fluid bed dried resulted in granula Example 9 tion water content of 0.35%. The final blend contained 1% water, flowed well, and filled capsules well. The 2nd proto Composition of Batch 1528-2855-RD (Capsules) type 017-1006 comprised of the same components was and Spray Coating and Drying Process adjusted to obtain a target capsule fill weight of 100 mg based on the results of the 1st batch. Water was sprayed onto powder 0195 blend with SP-304. The inlet temperature was 50 C and the granulation was dried for 1.5 hours and stopped when the Amount per Concentration product temperature reached 36 C. The 3rd (batch017-10006) Item No. Ingredient unit (mg) % Wiw and 4th (batch 017-10007) capsule prototypes will use PRO 1 SP-304 O.3246 O.3246 SOLV HD90, which is a higher density material with superior Microcrystalline cellulose 99.10 99.10 flow properties and higher moisture content of 5.5% than the (Celphere SCP-100) PROSOLV LM90. The moisture content of the PROSOLV 3 Calcium chloride dihydrate O.2622 O.2622 4 Leucine USP O. 1171 O. 1171 HD90 is readily removed by fluid bed drying. The density of 5 Hypromellose (Methocel E5 O.2OOO O.2OOO PROSOLV HD90 is about 0.55 g/mL. The PRUV lubricant PremLV) will be removed for these batches. 6 Purified Water, USP 7.2 mL. na Total 1OO 1OO Example 7 *The amount of water is calculated based on use of 119.0 mL purified water for the whole batch containing 5.356 g SP-304. Wet Granulation Stability 0196. The spray drying process of making the batch 2855 RD is described below. 0193 SP-304 was extracted from the capsules by sonica tion at either at room temperature (RT) or cold temperature Preparation of Coating Dispersion: and the amount of peptide was determined by HPLC. Initial percentages are based on the amount Stated on the label. 0197) Purified water was added to a glass container and stirred such that a liquid vortex was produced without intro ducing air. Then calcium chloride dihydrate was slowly added Batch % peptide (initial) % peptide (1 mos at RT) into the water. The mixture was stirred until the salt was dissolved or well dispersed. Next, leucine was slowly added and the resulting mixture was stirred until the amino acid was O17-1OOO6 101.1 (sonicated RT) 97.6 (sonicated cold) dissolved or well dispersed. Afterward, methocel was slowly O17-10008 97.5 (sonicated RT) 108.2 (sonciated cold) added and the mixture was stirred until methocel was com pletely dissolved. The solution could be warmed up to dis Solve methocel, if necessary. The resulting excipient Solution was allowed to cool to room temperature and pass through 80 Example 8 mesh screen. Then, 127.9 g of screened excipient solution was added to a glass container and placed in an ice bath for 0.5 to 1 hour until the solution reached 0° C. Next, SP-304 was 1M Capsule Stability in HDPE Bottles added into the cold excipient solution. The mixture was stir vigorously to allow the peptide to dissolve in the cold solu 0194 Capsules contained 0.3 mg SP-304 with the remain tion. The resulting peptide Solution was kept cold in the ice der of the fill weight (up to 5 mg) made up by mannitol bath as a spraying/coating solution. (Perlitol 300 DC). Each capsule contained 1.5% by weight SP-304 and 98.5% mannitol. The capsule shell was composed Drug Layering of HPMC. Amounts are relative to the amount specified on the label (i.e., 0.30 mg peptide). The indicated number of cap (0198 AGlatt GPCG-2 fluid bed processor (with top spray Sules was placed in a high density polyethylene bottle with an tower) with a Wurster insert was set up for drug layering onto induction seal and molecular sieve desiccant for 1 month at Celphere SCP-100 beads. After loading the Wurster column either 2-8C (first two columns) or 25 C and 60% relative with Celphere SCP-100 beads, bed temperature was raised to humidity (last two columns). The initial amount of peptide 35° C. and maintained for 30 minutes with minimum fluidi present was 101% of the label claim. The last row gives the zation of the beads. The bed temperature was reduced until an exhaust temperature of 35°C. was achieved. The pump tubing amount of peptide remaining after 1 month storage at the of the peristaltic pump used was primed by circulating the indicated temperature as determined by HPLC. spraying solution mentioned above. After the spraying appa ratus was adjusted to obtain a satisfactory spray pattern, the coating solution was sprayed onto Celphere SCP-100 beads 2-8 C. 2-8 C. 25 C.,6ORH 25 C.6ORH until all coating Solution was sprayed. Operating parameters were recorded. The bed temperature and fluidization were 1-capsule per 6-capsules per 1-capsule per 6-capsules per maintained until the beads were sufficiently dry. The fluidi bottle bottle bottle bottle zation was then reduced while the bed temperature was main 100% 92% 92% 98% tained at 35°C. for 10 minutes. 2 g of beads were sampled for moisture analysis when the bed temperature was kept at 35° C. When the moisture of the sampled beads reached <5% US 2015/0366935 A1 Dec. 24, 2015 50 moisture, the coated beads were discharged and loaded into a at 35° C. for 10 minutes. 2 g of beads were sampled for dry container. LOD (loss on drying) 2.399%. moisture analysis when the bed temperature was kept at 35° C. When the moisture of the sampled beads reached <5% Example 10 moisture, the coated beads were discharged and loaded into a dry container. LOD (loss on drying)<5%. Composition of Batch 1528-2851-RD (Tablets) and 0203 The net weight of the coated blend was determined Spray Coating and Drying Process for calculation of the amount of magnesium Stearate needed to lubricate the blend. Then the magnesium stearate was 0199. added to the coated blend and the mixture was blended for 1 minute. Amount per Concentration Item No. Ingredient unit (mg) % Wiw Compression 1 SP-304 O.3246 O.36O7 0204 A Fette tablet press was set up. Then the blend 2 Microcrystalline cellulose 88.88 98.75 mixture was loaded into the powder hopper and tooling was (Avicel PH 102) installed. The weight of each tablet was set to be 90 mg 5% 3 Calcium chloride dihydrate O.2622 O.2913 4 Leucine USP O. 1171 O.1301 and hardness to be 4-6 Kp. The weight, hardness and thick 5 Hypromellose (Methocel E5 O.2OOO O.2222 ness of tablets were measured and recorded every 5 to 10 PremLV) minutes. Friability measurement was also performed to 6 Magnesium Stearate O.225 O.2SOO ensure satisfactory product. 7 Purified Water, USP 7.2 mL. na

Total 90.0 1OO Example 11 *The amount of water is calculated based on use of 119.0 mL purified water for the whole Composition of Batch 1528-2850-RD (Capsules) batch containing 5.356 g SP-304. and Process 0200. The spray coating and drying process of making the 0205 batch 2851-RD is described below.

Preparation of Coating Dispersion: Concentration Item No. Ingredient % ww 0201 Purified water was added to a glass container and stirred such that a liquid vortex was produced without intro 1 SP-304 O.3246 2 Microcrystalline cellulose 99.43 ducing air. Then calcium chloride dihydrate was slowly added (Avicel PH 102) into the water. The mixture was stirred until the salt was 3 Magnesium Stearate O.2SOO dissolved or well dispersed. Next, leucine was slowly added 4 HPMC capsule shells na and the resulting mixture was stirred until the amino acid was dissolved or well dispersed. Afterward, methocel was slowly Total 100 added and the mixture was stirred until methocel was com pletely dissolved. The solution could be warmed up to dis (0206. The dry blend process of making the batch 2850-RD Solve methocel, if necessary. The resulting excipient Solution is described below. was allowed to cool to room temperature and pass through 80 mesh screen. Then, 127.9 g of screened excipient solution Blending: was added to a glass container and placed in an ice bath for 0.5 to 1 hour until the solution reached 0° C. Next, SP-304 was 0207 Avicel PH 102 was screened through a 60 mesh added into the cold excipient solution. The mixture was stir screen. V-blenders (1 Qt, 4 Qt, and 16 Qt) were then dusted by vigorously to allow the peptide to dissolve in the cold solu the screened Avicel PH 102. SP-304 was screened through a tion. The resulting peptide solution was kept cold in the ice 200 mesh screen and loaded into the 1-Qt V-blender. Then, bath as a spraying/coating solution. about 80 g Avicel PH 102 was added into the 1-Qt blender and the mixture was blended for 10 minutes at 25 rpm. The mix Drug Layering ture was then transferred to the 4-Qt V-blender which was pre-dusted by the screened Avicel PH 102. The 1-Qt blender 0202 AGlatt GPCG-2 fluid bed processor (with top spray was rinsed with Avicel and the rinse material was transferred tower) with a Wurster insert was set up for drug layering onto to the 4-Qt blender. The rinsing was repeated until all SP-304 Avicel PH 102 beads. After loading the Wurster column with was transferred to the 4-Qt blender. About 200 g Avicel was Avicel PH 102 beads, temperature was raised to 35° C. and added to the 4-Qt V-blender and the mixture was blended for maintained for 30 minutes with minimum fluidization of the 10 minutes. The resulting blend was then screened through a beads. The bed temperature was reduced until an exhaust 60 mesh screen and then transferred into the pre-dusted 16-Qt temperature of 35°C. was achieved. The pump tubing of the blender (dusted with 1500 g Avicel). The 4-Qt blender was peristaltic pump used was primed by circulating the spraying rinsed with Avicel and the rinse material was transferred to Solution mentioned above. After the spraying apparatus was the 16-Qt blender. The remaining Avicel was added to the adjusted to obtain a satisfactory spray pattern, the coating 16-Qt blender and the mixture was blended for 10 minutes. solution was sprayed onto Avicel PH 102 beads until all The resulting blend was passed through Comil and then coating solution was sprayed. Operating parameters were returned to the 16-Qt blender and was further blended for 5 recorded. The bed temperature and fluidization were main minutes. Properamount of magnesium Stearate was weighed, tained until the beads were sufficiently dry. The fluidization screened through a 60 mesh screen, and added into the 16-Qt was then reduced while the bed temperature was maintained blender. The resulting mixture was blended for 2 minutes. US 2015/0366935 A1 Dec. 24, 2015

Encapsulation Example 13 0208 A MG2 Planeta capsule filler was set up. Average Composition of Dry Blend Tablet Formulation weight of the empty capsule shells was determined and target 1528-3161-RD, 1 mg for Vacuum Drying capsule fill weight was calculated (+5%). The blend from the above process was added into the hopper of the capsule filler 0213 and encapsulation was started. Run weight parameters were manually adjusted. Resulting capsules were then Sorted Concentration according to the target fill weight. Item No. Ingredient % Wiw

1 SP-304 1.176 Example 12 2 Microcrystalline cellulose 98.57 (Avicel PH 102) Composition of Batch 1528-2850B-RD (Tablets) and 3 Magnesium Stearate O.2SOO Process Total 100 0209 Example 14 Concentration Item No. Ingredient % ww Composition of Dry Blend Tablet Formulation 1 SP-304 O.3246 1528-3162-RD, 1 mg with Low-Moisture Cellulose 2 Microcrystalline cellulose 99.43 (Avicel PH 102) 0214 3 Magnesium stearate O.2SOO

Total 100 Concentration Item No. Ingredient % Wiw 0210. The dry blend process of making the batch 2850B 1 SP-304 1.176 RD is described below. Microcrystalline cellulose 97.09 (Avicel PH 112) 3 Magnesium Stearate O.2SOO Blending: Total 100 0211 Avicel PH 102 was screened through a 60 mesh screen. V-blenders (1 Qt, 4 Qt, and 16 Qt) were then dusted by the screened Avicel PH 102. SP-304 was screened through a Example 15 200 mesh screen and loaded into the 1-Qt V-blender. Then, about 80 g Avicel PH 102 was added into the 1-Qt blender and Composition of Spray Coated Trehalose Granules the mixture was blended for 10 minutes at 25 rpm. The mix Tablet Formulation 1528-3170-RD, 1 mg ture was then transferred to the 4-Qt V-blender which was pre-dusted by the screened Avicel PH 102. The 1-Qt blender 0215 was rinsed with Avicel and the rinse material was transferred to the 4-Qt blender. The rinsing was repeated until all SP-304 Concentration was transferred to the 4-Qt blender. About 200 g Avicel was Item No. Ingredient % Wiw added to 4-Qt V-blender and the mixture was blended for 10 minutes. The resulting blend was then screened through a 60 1 SP-304 1.176 2 Trehalose granules 70.48 mesh screen and then transferred into the pre-dusted 16-Qt 3 Methocel ES Premium LV OSO blender (dusted with 1500 g Avicel). The 4-Qt blender was 4 Histidine (in coating O. 9225 rinsed with Avicel and the rinse material was transferred to Solution) the 16-Qt blender. The remaining Avicel was added to the 5 Calcium ascorbate O.100 6 Purified water NA 16-Qt blender and the mixture was blended for 10 minutes. 7 Trehalose powder (in 1.0176 The resulting blend was passed through Comil and then coating solution) returned to the 16-Qt blender and was further blended for 5 8 Microcrystalline cellulose 2S.OO minutes. Properamount of magnesium Stearate was weighed, (Avicel PH 200) screened through a 60 mesh screen, and added into the 16-Qt 9 Histidine 0.5535 blender. The resulting mixture was blended for 2 minutes. 10 Magnesium Stearate O.2SOO Total 100 Compression 0212. A Fette tablet press was set up. Then the blend 0216. The process for making spray coated trehalose mixture was loaded into the powder hopper and tooling was Granules tablet formulation 1528-3170-RD is described installed. The weight of each tablet was set to be 90 mg 5% below. and hardness to be 4-6 Kp. The weight, hardness, and thick Preparation of the Coating Dispersion ness of tablets were measured and recorded every 5 to 10 minutes. Friability measurement was also performed to 0217. Add purified water to labeled container and begin ensure satisfactory product. stirring. Stir such that a liquid vortex is produced without US 2015/0366935 A1 Dec. 24, 2015 52 introducing air into liquid. Slowly add Methocel to solution. Blending Stir until methocel is completely dissolved. Warm the solu 0219 Screen required Avicel and pass through 60 mesh tion if necessary to dissolve Methocel (s.50° C.). Solution screen. Setup 4 qt V-blender per SOP EQP-OCM-00056. must be cooled before adding other materials. Add Trehalose Weigh amount of Histidine needed and blend with small to solution. Stir until materials are dissolved. Add Calcium amount of Avicel weighed. Charge into 4 qt. V-blender. Trans Ascorbate to solution. Stir until materials are dissolved. fer Plecanatide Dried Granules into the V-Blender. Rinse 2-3 Adjust pH to 7.0 with 1N NaOH solution if pH >7.0. Record times the Lyoguard tray from Step 24 with adequate amount adjusted pH. Place the Coating Solution in an ice bath and of Weighed Avicel. Transferrinses into 4 qt. V-blender. Trans allow it stay in the batch for 0.5 to 1 hour until it reaches the fer all remaining Pre-weighed/screened Avicel into the ice temperature. Check with a thermometer to ensure at ice V-Blender. Mix for 15 minutes. Weigh and screen Magne temperature. Weigh portions of required amount of API on a sium Stearate through a 60 mesh screen. Charge Magnesium weighing boat and add each portion carefully to the cold Stearate to the 4 qt V-Blender. Ensure the cover is securely Excipient Solution. Stir vigorously to allow peptide wetting closed with no potential powder leakage during blending. and dissolving in the cold solution. Total amount of peptide Blend for 2 minutes. must equal 14.107 g. Continue stirring solution Such that a liquid Vortex is produced without introducing air into liquid. Compression Stir until PLECANATIDE is completely dissolved. Keep peptide solution cold all the time in the ice bath. Add Histi 0220 Set-up Korsch press per SOP EQP-OCM-00087. dine to solution. Stir not more than 10 min to dissolve the Install 0.250" Standard Concave Round Plain tolling. Obtain material. Obtain final pH of the Coating Solution. Obtain net blend Assay results and calculate Target Tablet Weight. weight of the Coating Solution. Coating Solution must be Acceptable weight range of tablets is +5.0%. Load the Final used within 30 minto avoid coloration. Blend into the powder hopper. Refill as necessary. Adjust fill weight to obtain tablets in the range of 95.0-105.0 mg and hardness in the range of 4-6 kP. Verify friability is NMT 1.0%. Drug Layering Check 5 tablet weights periodically every 5-10 minto ensure 0218 Setup Glatt GPCG2 with Wurster insert according tablet weight is within the range and record on form QRA to SOP EQP-OCM-064 for drug layering onto Trehalose DOC-00011-F6. After tablet weights are recorded, obtain and Granules with coating dispersion. Use Glatt GPCG2 In-pro record 3 tablet hardness and thickness during the periodic cess form, “EQP-OCM-064-F1 to record in-process infor weight check. Continue to compress acceptable tablets until mation. Turn unit on and preheat column. Fluid Bed Proces the blend is used up. Once press is running properly to achieve Sor: Glatt GPCG-2. Filter: 200 micron Screen. Product specifications above, perform final Friability test and record Container: 4" wurster, stainless steel. Insert height from bot results (Spec: NMT 1.0%). tom: 1". Spray direction: Top Spray. Fluid Nozzle Size/Type: 1 mm. Pump: Peristaltic, Master Flex LS. Tubing: Nalge #14 Example 16 Silicon. Bed Temperature: s40° C. Inlet air temperature: Adjust to meet bed temperature target. Outlet air temperature: Composition of Spray Coated Trehalose Granules Monitor & record. Spray rate: initial rate 4-6 g/min, adjust as Tablet Formulation 1528-3171-RD, 1 mg required. Atomizing air pressure: 20 psi. Airflow: 60 cmhand 0221) adjust for fluidization. Prepare double polyethylene bags large enough to hold drug layered Granules. Load column with Trehalose. Increase bed temperature to 35° C. and main Concentration tain for 30 minutes with minimum fluidization of the Gran Item No. Ingredient % Wiw ules. Reduce bed temperature until an exhaust temperature of 1 SP-304 1.167 35°C. is achieved. Prime pump tubing by circulating spray 2 Trehalose granules 70.31 ing Solution; must not use more than 40 g for tubing priming. 3 Methocel ES Premium LV OSO 4 Arginine 1.657 Adjust the spraying apparatus to obtain satisfactory spray 5 Calcium ascorbate O.100 pattern. Coating Solution Weight after priming should >317 6 Water for injection NA g. Recordinitial weight below before spraying onto trehalose. 7 Trehalose powder (in 1.0176 Start spraying the coating solution onto Trehalose Granules. coating solution) 8 Microcrystalline cellulose 2S.OO Record operating parameters on fluid bed processing form. (Avicel PH 200) Stop spraying when 297.2 g of coating solution has been 9 Magnesium Stearate O.2SOO sprayed. Maintain bed temperature and continue fluidization until Granules are sufficiently dry. Reduce fluidization and Total 100 maintain bed temperature at 35° C. for 10 minutes. Do not cool down the Granules. Sample 2 g for moisture analysis 0222. The process for making spray coated trehalose until moisture is below 1%. Discharge coated Granules into Granules tablet formulation 1528-3171-RD is described pre-prepared and labeled container (with tare weight) lined with double polyethylene bag. Calculate net weight of drug below. layered Granules. Setup Lyophilizer per SOP EQP-OCM 00002. Load drug layered granules into a Lyoguardtray (Save Preparation of Coating Solution bags). Use recipe 3 to dry blend overnight. Discharge dried 0223) Add purified water (Item 6) to labeled container and blend into saved polyethylene bags. Obtain final moisture of begin stirring. Stir Such that a liquid Vortex is produced with the dried granules. Record final Moisture (<1%). Calculate out introducing air into liquid. Slowly add Methocel to solu net weight of dried Granules. tion. Stir until methocel is completely dissolved. Warm the US 2015/0366935 A1 Dec. 24, 2015

solution if necessary to dissolve Methocel (s.50° C.). Record ene bags. Obtain final moisture of the dried granules. Calcu appearance of solution. Solution must be cooled before add late net weight of dried Granules. ing other materials. Add Trehalose to solution. Stir until mate rials are dissolved. Record appearance of solution. Add Argi Blending nine to solution. Stir until materials are dissolved. Record appearance of Solution. Add Calcium Ascorbate to solution. 0226 Screen required Avicel and pass through 60 mesh Stir until materials are dissolved. Record appearance of solu screen. Setup 4 qt V-blender. Transfer Plecanatide Dried tion. Adjust solution pH to pH 8.5-8.6 with concentrated HCl Granules into the V-Blender. Save bag for discharging final followed by adjust pH to 8.3-8.4 with 10N HC1. Record final blend. Rinse 2-3 times the Lyoguard tray and bag with adjusted pH. Place the Coating Solution in an ice bath and adequate amount of Weighed Avicel. Transferrinses into 4 qt. allow it stay in the batch for 0.5 to 1 hour until it reaches the V-blender. Transfer all remaining Pre-weighed/screened ice temperature. Check with a thermometer to ensure at ice Avicel into the V-Blender. Mix for 20 minutes. Weigh and temperature. Weigh portions of required amount of API on a screen Magnesium Stearate through a 60 mesh screen. weighing boat and add each portion carefully to the cold Charge Magnesium Stearate to the 4 qt V-Blender. Ensure the Excipient Solution. Stir vigorously to allow peptide wetting cover is securely closed with no potential powder leakage and dissolving in the cold solution. Total amount of peptide during blending. Blend for 2 minutes. Sample 3x350 mg of must equal 14.006 g. Continue stirring solution Such that a blend at three locations. Obtain exact weight of each sample liquid Vortex is produced without introducing air into liquid. that has been transferred into the sampling bottle. Stir until PLECANATIDE is completely dissolved. Keep Compression peptide solution cold all the time in the ice bath. Weigh 5.0 g of WFI to rinse API container. Carefully rinse the side of 0227 Set-up Korsch press per SOP EQP-OCM-00087. coating solution container and completely transfer the rinse Install 0.250" Standard Concave Round Plain tolling. Obtain back to the coating solution container. Obtain final pH of the blend Assay results and calculate Target Tablet Weight. Coating Solution. Obtain net weight of the Coating Solution Acceptable weight range of tablets is +5.0%. Load the Final (-360.3 g). Coating Solution must be used within as soon as Blend into the powder hopper. Refill as necessary. Adjust fill possible. weight to obtain tablets in the range of 95.0-105.0 mg and hardness in the range of 4-6 kP. Verify friability is NMT 1.0%. Drug Layering Check 5 tablet weights periodically every 5-10 minto ensure tablet weight is within the range. After tablet weights are 0224 Setup Glatt GPCG2 with Wurster insert according recorded, obtain and record 3 tablet hardness and thickness to SOP EQP-OCM-064 for drug layering onto Trehalose during the periodic weight check. Continue to compress Granules with coating dispersion. Use Glatt GPCG2 In-pro acceptable tablets until the blend is used up. Once press is cess form, “EQP-OCM-064-F1 to record in-process infor running properly to achieve specifications above, perform mation. Turn unit on and preheat column. final Friability test and record results (Spec: NMT 1.0%). 0225. Fluid Bed Processor: Glatt GPCG-2. Filter: 200 Example 17 micron screen. Product Container: 4" wurster, stainless steel. Insert height from bottom: 1". Spray direction: Top Spray. Fluid Nozzle Size/Type: 1 mm. Pump: Peristaltic, Master Composition of Spray Coated Trehalose Granules Flex LS. Tubing: Nalge #14 Silicon. Bed Temperature: s40° Tablet Formulation 1528-3172, 1 mg C. Inlet air temperature: Adjust to meet bed temperature 0228 target. Outlet air temperature: Monitor & record. Spray rate: initial rate 4-6 g/min, adjust as required. Atomizing air pres sure: 20 psi. Airflow: 60 cmhand adjust for fluidization. Load Concentration column with Trehalose G.Increase bed temperature to 35°C. Item No. Ingredient % Wiw and maintain for 30 minutes with minimum fluidization of the 1 SP-304 1.167 Granules. Reduce bed temperature until an exhaust tempera 2 Trehalose granules 70.81 ture of 35° C. is achieved. Prime pump tubing with coating 3 Methocel ES Premium LV OSO 4 TRIS 1.1524 Solution. Must not use more than 40 g for tubing priming. 5 Calcium ascorbate O.100 Adjust the spraying apparatus to obtain satisfactory spray 6 Water for injection NA pattern. Record initial weight below before spraying onto 7 Trehalose powder (in 1.0176 trehalose. Start spraying the coating Solution onto Trehalose coating solution) Granules. Record operating parameters on fluid bed process 8 Microcrystalline cellulose 2S.OO (Avicel PH 200) ing form. Stop spraying when 300.3 g of coating solution has 9 Magnesium Stearate O.2SOO been sprayed. Maintain bed temperature and continue fluidi zation until Granules are sufficiently dry. Reduce fluidization Total 100 and maintain bed temperature at 35°C. for 10 minutes. Do not cool down the Granules. Sample 2 g for moisture analysis 0229. The process for making spray coated trehalose gran until moisture is below 1%. Discharge coated Granules into ules tablet formulation 1528-3172-RD is described below. pre-prepared and labeled container (with tare weight) lined with double polyethylene bag. Calculate net weight of drug Preparation of Coating Solution layered Granules. If moisture is >1%, vacuum dry blend as follows: Setup Lyophilizer per SOPEQP-OCM-00002. Load 0230. Add purified water to labeled container and begin drug layered granules into a Lyoguard tray. Use recipe 3 to dry stirring. Stir such that a liquid vortex is produced without blend overnight. Discharge dried blend into saved polyethyl introducing air into liquid. Slowly add Methocel to solution. US 2015/0366935 A1 Dec. 24, 2015 54

Stir until methocel is completely dissolved. Warm the solu Example 19 tion if necessary to dissolve Methocel (s.50° C.). Record appearance of Solution. Composition of 3 mg Dry Blend Tablet Formulation 0231 Solution must be cooled before adding other mate 1528-2926-RD rials. Add Trehalose to solution. Stir until materials are dis solved. Record appearance of solution. Add TRIS to solution. 0234 Stir until materials are dissolved. Record appearance of solu tion. Add Calcium Ascorbate to solution. Stir until materials Concentration are dissolved. Record appearance of solution. Obtain solution Item No. Ingredient % Wiw pH: Adjust pH to pH 7.8-7.9 with concentrated HCl followed 1 SP-304 3.318 by adjust pH to 7.7-7.6 with 10N HC1. Record final adjusted 2 Microcrystalline cellulose 96.43 pH. Place the Coating Solution in an ice bath and allow it stay (Avicel PH 102) in the batch for 0.5 to 1 hour until it reaches the ice tempera 3 Magnesium Stearate O.2SOO ture. Check with a thermometer to ensure at ice temperature. Weigh portions of required amount of APIona weighing boat Total 100 and add each portion carefully to the cold Excipient Solution. Stir vigorously to allow peptide wetting and dissolving in the 0235. Other batches were prepared by the processes simi cold solution. Total amount of peptide must equal 14.006 g. lar to those described in Examples 9-12. Their compositions Continue stirring Solution Such that a liquid Vortex is pro are listed below. duced without introducing air into liquid. Stir until PLE 0236 Batch 500-55: 0.33% plecanatide, 95.17% micro CANATIDE is completely dissolved. Keep peptide solution cyrstalline cellulose, 4.0% sodium starch glycolate, and 0.5% cold all the time in the ice bath. Weigh 5.0 g of WFI to rinse magnesium Stearate. API container. Carefully rinse the side of coating solution 0237 Batches 1528-2907-RD and 2010F100A: 3.3.18% container and completely transfer the rinse back to the coat plecanatide, 96.43% Avicel, and 0.25% Mg stearate. ing solution container. Obtain final pH of the Coating Solu 0238 Batches 1528-2906-RD and 2010F099A: 1...106% tion. Obtain net weight of the Coating Solution (-354.2 g). plecanatide, 98.65% Avicel, and 0.25% Mg stearate. Coating Solution must be used as soon as possible. 0239 Batches 1528-2890-RD and 2010F101A: 0.324.6% 0232. The blending and compression processes for batch plecanatide, 99.43% Avicel, and 0.25% Mg stearate. 1528-3172-RD are similar to that described above for batch 0240 Formula compositions for batches 11H 141, 1528-3171-RD. 11H152, and 11H140 in this table below (not previously disclosed) are the same as the formula compositions for GMP Example 18 stability batches 2010F101A, 2010F099A, and 2010F100A, respectively. Composition of 1 mg Dry Blend Tablet Formulation 1528-2925-RD Example 20 0233 Plecanatide Tablet and Capsule Stability 0241 Capsules and tablets of different batches were tested Concentration for their stability and the results were provided. Unless oth Item No. Ingredient % ww erwise specified, 1M, 2M, 3M, or 4M in the tables below 1 SP-304 1106 denotes that the measurements were carried out at the end of Microcrystalline cellulose 98.64 1, 2, 3, or 4 month(s) of the storage period. (Avicel PH 102) 0242 Potency Summary: 3 Magnesium stearate O.2SOO 0243 This test was performed by taking a composite Total 100 sample of about 5 units to determine the average potency of the sample. The table below shows the stability of capsules or tablets in terms of potency (% of label claim). US 2015/0366935 A1 Dec. 24, 2015 55

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ponunuoo

£0I 66 00I 86 86

?OTI US 2015/0366935 A1 Dec. 24, 2015 57

0244 As demonstrated by the table above, there was little or no appreciable loss in potency after storage under acceler ated conditions (40 C/75RH or 30 C/65RH), which suggests that these capsules or tablets could be stable at room tempera ture for 18 months or for longer times if refrigerated or stored at 25 C. 0245 Water Content Summary: 0246 The table below shows that the water content was stable over the testing period in the packages evaluated for various capsule?tablet compositions. This further demon strated that products were stable. US 2015/0366935 A1 Dec. 24, 2015 58

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0247 Impurity Summary: 0248. The table below shows the product stability in terms of HPLC or UPLC oftotal impurities as a function of time and storage condition. The data in the table suggest that the increase in total impurities in tested batches except batch 500-55 be no greater than 7% at room temperature after 18 months. It also suggest that the increase in total impurities in all tested 1528-2855-RD batch in different packages be no greater than 7% at 30°C. for 18 months. It was also observed that the 1528-2855-RD batch had less impurity increase than the 1528-2850-RD batch or was more stable than the 1528 2850-RD batch. US 2015/0366935 A1 Dec. 24, 2015 61

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0249 Content Uniformity: 0250. This test was performed by placing 10 individual 0.3 m blend capsule 1528-2850-RD capsule/tablet units in 10 individual bottles and potency of Original Re-preparation each unit was measured to show whether individual capsules Sample % LC % LC or tablets have uniform potency (% label claim or % LC). 1 82.73 85.87 2 84.57 89.45 3 80.29 91.39 4 84.88 88.45 0.3 mg Dry blend tablet 1528-285OB-RD 5 85.2 86.96 6 82.9 84.84 % LC 7 84.75 86.21 1528-285OB- 8 86.58 91.37 Sample RD (dry tabs) 9 8434 88.79 10 88.82 84.75 1 78.62 Mean 84.51 87.81 2 91.43 stol. dev 2.288.445 2.467121 3 86.52 % RSD 2.7 2.8 4 90.9 5 84.83 6 95.29 7 75.69 Conte1528- 1528 8 76.87 28SS-RD 28SOB-RD 9 84.92 Sample % LC Sample % LC 10 86.9 Mean 85.2 : s: : still 6.51 3 89.06 3 86.52 Po RSD 7.64 4 84.94 4 90.9 5 89.93 5 84.83 6 88.7 6 95.29 7 88.71 7 75.69 8 86.85 8 76.87 0.3 mg Coated particle tablet 1528-2851-RD 9 86.92 9 84.92 10 91.33 10 86.9 Weight % Label Mean 88.9 Mean 85.2 Sample (mg) Claim stol. dev 2.45 stol. dev 6.51 % RSD 2.76 % RSD 7.64 1 88.86 69.55 2 89 94.41 3 88.89 94.34 4 88.6 72.18 5 88.37 14252 500-55 6 88.76 14944 7 89.42 78.8 % label 8 88.56 131.08 Sample claim 9 89.08 102.55 10 88.78 99.13 1 96.90% Mean 103.4 2 99.40% St. Dew 28.53 3 103.20% % RSD 27.59 4 96.90% 5 100.00% 6 99.60% 7 96.90% 8 102.80% 3 mg Dry blend 1 mg Dry blend 9 96.8% 0.3 mg Dry blend capsule 1528- capsule 1528-2906- 10 93.9% capsule 1528-2890 2907-RD RD Mean 98.60% SD 2.91 Sample % LC Sample % LC Sample % LC RSD 3.00% AV 7.1 (PASS) 1 87.2 1 94.5 1 98.1 2 94.6 2 101.2 2 101.8 3 92.6 3 97.9 3 93.1 0251. The data in the tables above show that all of the 4 94.2 4 94.5 4 97.5 batches yield very good content uniformity acceptable for 25 g commercial product. 7 91.6 7 96.1 7 94.5 0252) Dissolution 50-Rpm Summary: 8 99 8 99 8 100.1 0253) The tables below are summaries of the dissolution of 9 91.8 9 93.8 9 98.1 drug from capsules or tablets in an unconventional Small 10 92.1 10 93.4 10 97.9 Mean 92.8 Mean 96.2 Mean 97.6 Volume apparatus needed to measure the Small amount of RSD 3.20% RSD 2.60% RSD 2.50% drug in the units using slow stirring to look for changes in AV(10)*** 12.8 AV(10) 8.4 AV(10) 6.8 dissolution over time. The test was performed by placing one M unit into a very small volume of water at 37 C with a paddle than***AV 15 =to acceptance pass USP<905> value used content for UPS uniformity, <905> content uniformity, Idealy AV should be less stirring at 50-rpm (which is slow) and data were collected at 15, 3045, and 60 minutes to show the drug release rate over US 2015/0366935 A1 Dec. 24, 2015 64 time. These tested products are “immediate release' oral solid dosage forms and a conventional requirement is to have about Dissolution (% label claim at 45 minutes) 75% released in about 45 minutes. The tables summarize the results at 45 minutes and indicate that dissolution was stable Lot Initial 40 C./75RH 30 C, 65RH over time. (description) bulk 1 M 2M 3 M

1528-2851 - Wessel 1 S8% 67 68 89 Dissolution (% label claim at 45 minutes) RD (coated Wessel 2 779, 84 78 124 particle tablet Wessel 3 57% 62 68 70 Lot 40 C. 3O C., HDPE bottle) Wessel 4 96% 110 84 105 (de- Initial 75 RH 6S RH 25 C. 5 C. Wessel 5 95% 65 107 61 Wessel 6 64% 103 76 51 Scription) bulk OM 1M 2M 3M 3M 4M Average 74% 82 8O 83 RSD 24% 26 18 33

asid y (SSC l . ". ". s 1528-2851 - Wessel 1 S8% 89 S4 118 (dry blend Vessel 3 88 79 85 79 91. 87 R t y s 0. f g . V-Cap Vessel 4 84 86 87 78 83 85 particle table (SSC . capsule Vessel 5 89 72 89 80 79 90 OxyGuard Wessel 4 96 o 68 67 73 HDPE Vessel 6 88 81 8S 82 88 83 bottle) Wessel 5 95. 76 162 96 bottle) Average 87 78 87 80 85 85 Wessel 6 64o 97 82 95 RSD 2 64 2.7 21 S.O 2.9 Average t 8O 91 89 1528- Wessel 1 85 69 89 79 88 82 RSD 24% 14 42 21 28SO-RD Vessel 2 87 75 89 87 81 85 (dry blend Vessel 3 88 77 87 86 84 86 Vcap Wessel 4 84 8O 87 83 83 8O capsule Vessel 5 89 71 88 89 84 84 Dissolution (% label claim at 45 minutes Oxyguard Vessel 6 88 76 88 79 86 89 bottle) Average 87 75 88 84 84 84 Lot Initial 40 C.75RH 3O C.f6SRH RSD 2 5.3 1.2 5.2 3.1 3.6 1528- Wessel 1 85 75 59 86 73 83 (description) bulk 1 M 2M 3 M 28SO-RD Wessel 2 87 89 77 79 81 81 1528-285OB- Vessel 1 90% 88 96 92 (dry Wessel 3 88 88 83 87 74 84 RD (dry blend Wessel 2 69% 79 82 92 blend Wessel 4 84 89 67 93 85 83 tablet HDPE Wessel 3 83% 76 1OO 85 V-cap Vessel 5 89 93 75 82 82 84 bottle) Vessel 4 % 96 86 94 capsule Vessel 6 88 90 82 90 67 87 y blister Average 87 87 74 86 77 84 Average 86% 85 92 88 strip) RSD 2 7 12.5 6.3 8.6 2.4 RSD 11% 8.2 8 S.6 1528-285OB- Vessel 1 90% 74 8O 91 RD (dry blend Wessel 2 69% 97 87 95 tablet Wessel 3 83% 91 86 90 OxyGuard Vessel 4 94% 94 91 90 Dissolution (% label claim at 45 minutes bottle) Wessel 5 88% 83 91 89 Wessel 6 92% 91 76 84 Lot Initial 40 C. 7SRH 30 C.f6S RH 25 C. ge s: s 6 s 9. O (description) bulk 1M 2M 3M 3M 0.

1528-2855-RD Wessel 1 104 85 100 79 83 (coated bead Vessel2 89 90 97 83 88 V-Cap capsule Vessel 3 91 84 71 91 50 HDPE bottle) Vessel 4 88 64 73 94 88 Dissolution (% label claim at 45 minutes Wessel 5 94 75 72 75 92 Wessel 6 93 8O 39 96 94 Lot Initial 40 C. 7SRH 3O C.f6S RH 25 C. Average 93 8O 75 86 83 RSD 6 12 29 9.7 20 (description) bulk OM 1M 2M 3M 3M 1528-2855RD Wessel 1 104 88 8O 87 78 (coated bead Vessel 2 89 79 91 86 94 500-55 (dry Vessel 1 95 90 92 91 89 V-cap capsule Vessel 3 91 84 63 92 74 blend V-Cap Vessel 2 98 85 98 97 98 OxyOuard Vessel 4 88 92 98 90 98 Plus capsule Vessel 3 69 85 96 94 76 bottle) Wessel 5 94 89 81 81 93 HDPE bottle) Vessel 4 94 89 9S 100 97 Wessel 6 93 44 99 81 78 Wessel 5 99 89 97 98 86 Average 93 79 85 86 86 Wessel 6 104 100 99 94 92 RSD 6 23 16 5.3 12.1 Average 93 89 96 96 90 1528-2855-RD Wessel 1 104 85 98 100 81 RSD 13.1 6.2 2.4 3.6 9.1 (coated bead Vessel 2 89 84 94 63 8O 500-55 (dry Vessel 1 95 84 103 99 94 V-cap capsule Vessel 3 91 97 96 82 87 blend V-Cap Vessel 2 98 97 101 95 103 blister strip) Vessel 4 88 94 96 55 74 Plus capsule Vessel 3 69 97 99 98 97 Wessel 5 94 64 75 95 66 OxyOuard Vessel 4 94 92 97 92 96 Wessel 6 93 96 102 89 82 bottle) Wessel 5 99 91 100 95 101 Average 93 87 93 81 78 Wessel 6 104 96 95 93 91 RSD 6 14 10 22.4 9.2 Average 93 93 99 95 97 RSD 13.1 5.3 2.7 2.7 4.3 US 2015/0366935 A1 Dec. 24, 2015 65

-continued -continued

Dissolution (% label claim at 45 minutes Dry blend l ng lot 1528-2906-RD 150-mL

Lot Initial 40 C. 7SRH 3O C.f6S RH 25 C. 15 min 30 min 45 min 60 min

(description) bulk OM 1M 2M 3M 3M Wessel 6 57 90 95 96 Average 50 89 96 98 500-55 (dry Vessel 1 95 98 99 89 98 RSD 18.8 7 2.8 2.4 blend V-Cap Vessel 2 98 101 88 94 87 Plus capsule Vessel 3 69 107 90 89 96 foil blister) Vessel 4 94 96 90 86 87 Wessel 5 99 99 68 89 94 Wessel 6 104 99 90 82 89 - Dry blend Onslot 5282800-RDSO-in Average 93 100 87 88 92 RSD 13.1 3.8 11.8 4.3 5.5 15 min 30 min 45 min 60 min Vessel 1 57 94 100 105 Wessel 2 60 96 100 105 Wessel 3 86 93 94 95 Dry blend 3 ng lot 1528-2907-RD 500-mL Vessel 4 76 90 91 101 Wessel 5 69 90 97 106 15 min 30 min 45 min 60 min Wessel 6 68 95 97 97 Average 69 93 97 102 Vessel 1 91 96 97 96 RSD 15.6 2.8 3.4 4.5 Wessel 2 96 95 97 96

Capsule Dissolution at 45 minutes

Lot 5 C. 25 C. 3O C. 40 C. (strength) COA 1M 2M 3M 1M 2M 3M 1M 2M 3M 1M 2M 3M 2011F101 98% 99%. 95% 95% 95% 92%. 95% 94% 93% 97% 93% 90% 92% A (0.3 mg) 2011F099 96% 95% 95% 95% 91% 93% 94% 93% 90% 95% 95% 92% 93% A (1 mg) 2011F1OO 99%. 1019-0. 97% 97%. 100% 95% 95% 98% 95% 95% 96% 93% 95% A (3 mg) 11H141 1019/o 102%. 1019 1019/o 105% 96%. 106%. 102% 97%. 103% 99% 96% 98% (0.3 mg) 11H152 96% 96% 99% 97% 96% 99% 97% 96% 96% 98% 96% 96% 98% (1 mg) 11H140 102%. 102%. 102%. 1019/o 105%. 100% 97%. 102% 99%. 102%. 1019/o 99% 96% (3 mg)

-continued (0254. Dissolution 75-Rpm: (0255. The tables below show a few examples where the Dry blend 3 mg lot 1528-2907-RD 500-mL stirring rate was increased slightly to 75-rpm to give more 15 min 30 min 45 min 60 min consistent results and indicates stable dissolution after accel- 0 erated storage of 1 or 2 months at 40C 75% relative humidity. Wessel 3 96 97 97 97 Vessel 4 95 102 1OO 1OO Wessel 5 97 96 96 97 Wessel 6 92 99 98 98 D; S.E. ed Average 94 97 98 97 -rpm Su-m RSD 2.7 2.5 1.1 1.4 15 min 30 min 45 min 60 min

Vessel 1 75 8O 8O 81 Wessel 2 61 75 8O 82 Dry blend l ng lot 1528-2906-RD 150-mL Wessel 3 65 81 83 84 15 min 30 min 45 min 60 min Vessel 4 78 86 84 85 Wessel 5 66 79 83 84 Vessel 1 65 92 96 99 Wessel 6 62 79 84 86 Wessel 2 49 91 95 96 Average 68 8O 82 84 Wessel 3 46 88 96 97 Vessel 4 44 96 101 102 RSD 10.3 4.5 2.3 2.2 Wessel 5 39 78 93 99 US 2015/0366935 A1 Dec. 24, 2015

0256 2855-RD Dissolution: Dry blend 1 mg lot 1528-2906A-RD (0257. The tables below are all the dissolution profiles of 2 M40 C./75RH 75-rpm 50-mL batch 1528-2850-RD and indicate stable drug release over t1me.

15 min 30 min 45 min 60 min Initial Percent Dissolved Vessel 1 69 84 88 88 Vessel 15 30 45 60 Wessel 2 62 82 84 85 Wessel 3 65 82 85 85 1 84% 99% 1.04% 1.04% Vessel 4 58 70 8O 79 2 28% 80% 89% 92% 3 68% 83% 91% 95% Wessel 5 59 77 82 81 4 S6% 79% 88% 98% Wessel 6 68 8O 83 84 5 29% 83% 94% 98% Average 64 79 84 84 6 74% 85% 93% 96% Mean 57% 85% 93% 97% RSD 7.2 6.4 3.3 3.8 RSD 41.20% 8.50% 6.00% 4.20%

2M3O C.f6S RH 3M3O C.f6S RH 3M25 C.60 RH 1M40 C.75 RHOxyGuard Packaging OxyOuard OxyOuard OxyGuard

15 30 45 60 15 30 45 60 15 30 45 60 15 30 45 60 Vessel min min min min min min min min min min min min min min min min

1 35 74 88 93 47 67 8O 90 76 83 87 88 44 62 78 85 2 46 74 79 85 57 8O 91 95 65 79 86 91 70 89 94 97 3 39 78 84 88 43 55 63 71 64 84 92 97 48 62 74 79 4 59 82 92 94 753 92 98 101 71 85 90 94 65 92 98 103 5 22 82 89 92 38 64 81 92 60 75 81 87 72 86 93 96 6 4 2O 44 61 S4 94 99 101 55 74 81 87 53 74 78 84 Average 34 68 79 86 52 75 85 92 65 8O 86 91 59 78 86 91 RSD 57 35 23 14 25 21 16 12 11.7 5.7 5.3 4.6 20.1. 17.4 12.1 10.4

15 30 45 60 15 30 45 60 15 30 45 60 15 30 45 60 Vessel min min min min min min min min min min min min min min min min

1 61 78 85 89 78 97 100 103 S8 72 79 85 S4 70 83 92 2 63 83 90 92 77 93 97 98 S1 72 83 90 66 81 88 92 3 66 79 84 91 41 59 71 78 53 84 91 94 10 29 50 66 4 25 44 64 77 50 65 73 78 66 89 94 95 69 81 88 92 5 47 67 75 8O 37 59 72 83 48 66 75 81 68 83 92 97 6 57 71 8O 85 6 21 39 52 85 94 96 99 82 91 94 97 Average 53 70 8O 86 48 66 75 82 60 8O 86 91 58 73 83 89 RSD 28 2O 12 7 56 42 29 22 22.6 14 9.7 7.3 43 30.6 19.6 13.3

1M40 C.75 RH Blister Packaging 2M30 C.65 RH Blister 3M 3O C.f6S RH Blister 3M25 C.60 RHBlister

15 30 45 60 15 30 45 60 15 30 45 60 15 30 45 60 Vessel min min min min min min min min min min min min min min min min

1 36 69 85 90 61 91 98 100 82 95 100 102 53 71 81 90 2 41 69 84 88 57 82 94 100 31 48 63 74. 27 57 8O 87 3 67 96 97 98 63 87 96 100 69 77 82 85 70 78 87 92 4 S4 83 94 104 36 8O 96 100 29 41 55 69 52 66 74 87 5 10 46 64 79 45 61 75 83 84 94 95 97 25 48 66 8O 6 70 91 96 1OO 87 100 102 104 74 84 89 82 50 74 82 84 Average 47 76 87 93 58 83 93 98 62 73 81 85 46 66 78 87 RSD 48 25 14 10 30 16 10 8 4O.S. 32.1 224 14.9 37.O. 17.0 9.2 5.3

0258 Bathes 2850-RD, 2850B-RD, 2851-RD, and 500-55 were also tested in the similar fashion and all showed stable drug release over time.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 261

<21 Os SEQ ID NO 1 &211s LENGTH: 16 US 2015/0366935 A1 Dec. 24, 2015 67

- Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized

<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 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically 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 US 2015/0366935 A1 Dec. 24, 2015 68

- Continued

<210s, SEQ ID NO 7 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <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 <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: 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 US 2015/0366935 A1 Dec. 24, 2015 69

- Continued

<210s, SEQ ID NO 11 &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 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 O 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: US 2015/0366935 A1 Dec. 24, 2015 70

- Continued <223> OTHER INFORMATION: Chemically Synthesized

<4 OOs, SEQUENCE: 14 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys 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 US 2015/0366935 A1 Dec. 24, 2015 71

- Continued 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: (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 <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 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 US 2015/0366935 A1 Dec. 24, 2015 72

- Continued <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

<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 US 2015/0366935 A1 Dec. 24, 2015 73

- Continued polyethylene glycol

<4 OOs, SEQUENCE: 22 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 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 <222 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 US 2015/0366935 A1 Dec. 24, 2015 74

- Continued

<210s, SEQ ID NO 25 &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 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: 25 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 26 &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: (16) ... (16) <223> OTHER INFORMATION: wherein LEU is a D-amino acid

<4 OOs, SEQUENCE: 26 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 27 &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 x is 3-(2-naphthyl)alanine 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE US 2015/0366935 A1 Dec. 24, 2015 75

- Continued

<222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein x is a D-amino acid

<4 OOs, SEQUENCE: 27 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: MOD RES <222s. LOCATION: (8) (8) <223> OTHER INFORMATION: wherein the x is a 2-aminoisobutyric acid, Aib 22 Os. FEATURE <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein the x is a 2-aminoisobutyric acid, Aib 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: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: wherein x is an ornithine, Orn 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 US 2015/0366935 A1 Dec. 24, 2015 76

- Continued

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: 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: US 2015/0366935 A1 Dec. 24, 2015 77

- Continued NAME/KEY: MISC FEATURE LOCATION: (1) . . (1) OTHER INFORMATION: wherein ASN at position 1 is attached to polyethylene glycol 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 LEU is a D-amino acid

SEQUENCE: 33 Asn Asp Glu. Cys Glu Tyr Cys Val Asn Val Ala Cys Thr Gly Cys Lieu. 1. 5 15

SEQ ID NO 34 LENGTH: 16 TYPE PRT ORGANISM: Artificial Sequence FEATURE: 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: (16) . . (16) OTHER INFORMAT ON: wherein 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: 34 Asn Asp Glu. Cys Glu Cyr Cys Val Asn Val Ala Cys Thr Gly Cys Lieu. 1. 5 15

SEO ID NO 35 LENGTH: 16 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMAT ON: Chemically Synthesized FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) . . (1) OTHER INFORMAT ON: wherein 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: (16) . . (16) OTHER INFORMAT ON: wherein LEU is a D-amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (16) . . (16) OTHER INFORMAT ON: wherein LEU at position 16 is attached to polyethylene g ycol

SEQUENCE: 35 Asn Asp Glu. Cys Glu Ser Cys Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 15 US 2015/0366935 A1 Dec. 24, 2015 78

- Continued

<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 <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: 36 Asn Asp Glu. Cys Glu Ser Cys Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 37 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221. 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 OO > SEQUENCE: 37 Asn Asp Glu. Cys Glu Ser Cys Val Asn Val Ala Cys Thr Gly Cys Lieu 1. 5 1O 15

<210s, SEQ ID NO 38 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized

<4 OOs, SEQUENCE: 38 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Ser 1. 5 1O 15

<210s, SEQ ID NO 39 &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 US 2015/0366935 A1 Dec. 24, 2015 79

- Continued

22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein SER at position 16 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 39 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Ser 1. 5 1O 15

<210s, SEQ ID NO 4 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 at position 1 is attached to polyethylene glycol

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

<210s, SEQ ID NO 41 &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 SER at position 16 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 41 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Ser 1. 5 1O 15

<210s, SEQ ID NO 42 &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 SER is a D-amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein SER at position 16 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 42 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Ser 1. 5 1O 15

<210s, SEQ ID NO 43 US 2015/0366935 A1 Dec. 24, 2015 80

- Continued

&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 SER is a D-amino acid

<4 OOs, SEQUENCE: 43 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Ser 1. 5 1O 15

<210s, SEQ ID NO 44 &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 SER at position 16 is attached to polyethylene glycol 22 Os. FEATURE: <221. NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein SER is a D-amino acid

<4 OOs, SEQUENCE: 44 Asn Asp Glu. Cys Glu Lieu. CyS Val Asn Val Ala Cys Thr Gly Cys Ser 1. 5 1O 15

<210s, SEQ ID NO 45 &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: (5) . . (6) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue, and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (11 <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue, and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (13) . . (14) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue, and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue, and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid

<4 OOs, SEQUENCE: 45 US 2015/0366935 A1 Dec. 24, 2015 81

- Continued Asn Asp Glu. Cys Xaa Xaa Cys Xaa Xala Xala Xaa Cys Xaa Xala Cys Xaa 1. 5 1O 15

<210s, SEQ ID NO 46 &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 x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue that is zero or one residue in length and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue that is zero or one residue in length and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (6) <223> OTHER INFORMATION: wherein X is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (11 <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (13) . . (14) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue that is zero or one residue in length and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid

<4 OOs, SEQUENCE: 46 Xaa Xala Xala Cys Xaa Xaa Cys Xaa Xala Xala Xaa Cys Xaa Xala Cys Xaa 1. 5 1O 15

<210s, SEQ ID NO 47 &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 x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE US 2015/0366935 A1 Dec. 24, 2015 82

- Continued

<222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue that is zero or one residue in length and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue that is zero or one residue in length and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein x is a cysteine, penicillamine homocysteine, or 3-mercaptoproline 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein x is a cysteine, penicillamine homocysteine, or 3-mercaptoproline 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: wherein x is a cysteine, penicillamine homocysteine, or 3-mercaptoproline & 22 O FEATURE; <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: wherein x is a cysteine, penicillamine homocysteine, or 3-mercaptoproline 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue that is zero or one residue in length and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid

<4 OOs, SEQUENCE: 47 Xaa Xala Xala Xala Glu Xaa Xaa Val Asn. Wall Ala Xaa Thr Gly Xaa Xala 1. 5 1O 15

<210s, SEQ ID NO 48 &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 x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue that is zero or one residue in length and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue that is zero or one residue in length US 2015/0366935 A1 Dec. 24, 2015 83

- Continued and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein x is a cysteine, penicillamine homocysteine, or 3-mercaptoproline 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (6) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein x is a cysteine, penicillamine homocysteine, or 3-mercaptoproline 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (11 <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: wherein x is a cysteine, penicillamine homocysteine, or 3-mercaptoproline 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (13) . . (14) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: wherein x is a cysteine, penicillamine homocysteine, or 3-mercaptoproline 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue that is zero or one residue in length and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid

<4 OOs, SEQUENCE: 48

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 1. 5 1O 15

<210s, SEQ ID NO 49 &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: (5) . . (6) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (10) ... (11) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino US 2015/0366935 A1 Dec. 24, 2015 84

- Continued acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (13) . . (14) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid

<4 OOs, SEQUENCE: 49 Asn Asp Asp Cys Xaa Xaa Cys Xaa Asn Xala Xaa Cys Xaa Xala Cys Xaa 1. 5 1O 15

<210s, SEQ ID NO 50 &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 <222 LOCATION; (5) . . (6) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (10) ... (11) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (13) . . (14) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid

<4 OOs, SEQUENCE: 50 Asn Glu Glu. Cys Xaa Xaa Cys Xaa Asn Xala Xaa Cys Xaa Xala Cys Xaa 1. 5 1O 15

<210s, SEQ ID NO 51 &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 2015/0366935 A1 Dec. 24, 2015 85

- Continued

<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 GLU is a D-amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (6) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (10) ... (11) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (13) . . (14) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein X is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid

<4 OOs, SEQUENCE: 51 Asn Glu Asp Cys Xaa Xaa Cys Xaa Asn Xala Xaa Cys Xaa Xala Cys Xaa 1. 5 1O 15

<210s, SEQ ID NO 52 &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: (5) . . (6) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (10) ... (11) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino acid 22 Os. FEATURE: US 2015/0366935 A1 Dec. 24, 2015 86

- Continued <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (13) . . (14) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino ac id 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, or a methylated or an unmethylated amino ac id

<4 OOs, SEQUENCE: 52 Asn Asp Glu. Cys Xaa Xaa Cys Xaa Asn Xala Xaa Cys Xaa Xala Cys Xaa 1. 5 1O 15

<210s, SEQ ID NO 53 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221 NAMEAKEY:A MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein ASN is a D-amino acid 22 Os. FEATURE: <221 NAMEAKEY:A MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein ASP is a D-amino acid 22 Os. FEATURE: <221. 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: (5) . . (6) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, Co a D-amino acid, or a methylated or an unmethylated amino ac id 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, Co a D-amino acid, or a methylated or an unmethylated amino ac id 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (10) ... (11) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, Co a D-amino acid, or a methylated or an unmethylated amino ac id 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (13) . . (14) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, Co a D-amino acid, or a methylated or an unmethylated amino ac id 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, Co a D-amino acid, or a methylated or an unmethylated amino ac id

<4 OOs, SEQUENCE: 53 Asn Asp Glu. Cys Xaa Xaa Cys Xaa Tyr Xaa Xaa Cys Xaa Xala Cys Xaa 1. 5 1O 15

<210s, SEQ ID NO 54 &211s LENGTH: 16 212. TYPE: PRT US 2015/0366935 A1 Dec. 24, 2015 87

- Continued 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: (2) ... (2) OTHER INFORMATION: wherein GLU is a D-amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (3) ... (3) OTHER INFORMATION: wherein GLU is a D-amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (5) . . (6) OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, a methylated or an unmethylated amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (8) ... (8) OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, a methylated or an unmethylated amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (10) . . (11) OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, a methylated or an unmethylated amino acid FEATURE; NAME/KEY: MISC FEATURE LOCATION: (13) . . (14) OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, a methylated or an unmethylated amino acid FEATURE: NAME/KEY: MISC FEATURE LOCATION: (16) . . (16) OTHER INFORMATION: wherein x is any natural, or unnatural amino acid, or amino acid analogue and may be an L-amino acid, or a D-amino acid, a methylated or an unmethylated amino acid

SEQUENCE: 54 Asn Glu Glu. Cys Xaa Xaa Cys Xaa Tyr Xaa Xaa Cys Xaa Xala Cys Xaa 1. 5 1O 15

SEO ID NO 55 LENGTH: 14 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Chemically Synthesized

SEQUENCE: 55 Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1.

SEO ID NO 56 LENGTH: 13 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Chemically Synthesized

SEQUENCE: 56 Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys 1. US 2015/0366935 A1 Dec. 24, 2015 88

- Continued

<210s, SEQ ID NO 57 &211s LENGTH: 14 212. 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) <223s OTHER INFORMATION: wherein CYS at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (14) . . (14) <223s OTHER INFORMATION: wherein TYR at position 14 is attached to polyethylene glycol

<4 OO > SEQUENCE: 57 Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O

SEO ID NO 58 LENGTH: 16 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Chemically Synthesized

<4 OOs, SEQUENCE: 58 Asn Phe Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

SEO ID NO 59 LENGTH: 16 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Chemically Synthesized <4 OO > SEQUENCE: 59 Asn Phe Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

SEQ ID NO 60 LENGTH: 14 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Chemically Synthesized FEATURE: NAME/KEY: MISC FEATURE LOCATION: (14) . . (14) OTHER INFORMATION: wherein TYR is a D-amino acid

<4 OOs, SEQUENCE: 60 Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O

SEQ ID NO 61 LENGTH: 14 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Chemically Synthesized FEATURE: NAME/KEY: MISC FEATURE US 2015/0366935 A1 Dec. 24, 2015 89

- Continued

<222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein CYS at position 1 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 61 Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O

<210s, SEQ ID NO 62 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized

<4 OOs, SEQUENCE: 62 Asn Phe Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 63 &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 TYR is a D-amino acid

<4 OOs, SEQUENCE: 63 Asn Phe Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 64 &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: 64 Asn Phe Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 65 &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 TYR is a D-amino acid

<4 OOs, SEQUENCE: 65 Asn Phe Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15 US 2015/0366935 A1 Dec. 24, 2015 90

- Continued

<210s, SEQ ID NO 66 &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 TYR is a D-amino acid

<4 OOs, SEQUENCE: 66 Asn Phe Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 67 &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 OO > SEQUENCE: 67 Asn Phe Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 68 &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 TYR is a D-amino acid

<4 OOs, SEQUENCE: 68 Asn Phe Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 69 &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 TYR is a D-amino acid

<4 OOs, SEQUENCE: 69 Asn Phe Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 70 &211s LENGTH: 16 US 2015/0366935 A1 Dec. 24, 2015 91

- Continued

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 OO > SEQUENCE: 7 O Asn Phe Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 71 &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 TYR is a D-amino acid

<4 OOs, SEQUENCE: 71 Asn Phe Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 72 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: wherein TYR at position 14 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 72 Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O

<210s, SEQ ID NO 73 &211s LENGTH: 13 212. 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 CYS at position 1 is attached to polyethylene glycol 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: wherein CYS at position 13 is attached to polyethylene glycol

<4 OO > SEQUENCE: 73 Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys 1. 5 1O US 2015/0366935 A1 Dec. 24, 2015 92

- Continued

<210s, SEQ ID NO 74 &211s LENGTH: 13 212. 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 CYS at position 1 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 74 Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys 1. 5 1O

<210s, SEQ ID NO 75 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Chemically Synthesized 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: wherein CYS at position 13 is attached to polyethylene glycol

<4 OO > SEQUENCE: 75 Cys Cys Glu Tyr Cys CyS ASn Pro Ala Cys Thr Gly Cys 1. 5 1O

<210s, SEQ ID NO 76 &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 TYR at position 16 is attached to polyethylene glycol

<4 OO > SEQUENCE: 76 Asn Phe Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 77 &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

<4 OO > SEQUENCE: 77 Asn Phe Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15 US 2015/0366935 A1 Dec. 24, 2015 93

- Continued

<210s, SEQ ID NO 78 &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 TYR at position 16 is attached to polyethylene glycol

<4 OO > SEQUENCE: 78 Asn Phe Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 79 &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 <222 LOCATION: (16) . . (16) <223> OTHER INFORMATION: wherein TYR at position 16 is attached to polyethylene glycol

<4 OO > SEQUENCE: 79 Asn Phe Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 8O &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

<4 OOs, SEQUENCE: 80 Asn Phe Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1. 5 1O 15

<210s, SEQ ID NO 81 &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 TYR at position 16 is attached to polyethylene glycol

<4 OOs, SEQUENCE: 81 Asn Phe Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr