Treatment of gastrointestinal disorders

Abstract

To provide peptides, compositions and methods for treating gastrointestinal disorders.SOLUTION: The invention features peptides, compositions, and related methods for treating gastrointestinal disorders and conditions, including but not limited to, irritable bowel syndrome (IBS), gastrointestinal motility disorders, functional gastrointestinal disorders, gastroesophageal reflux disease (GERD), duodenogastric reflux, Crohn's disease, ulcerative colitis, inflammatory bowel disease, functional heartburn, dyspepsia, visceral pain, gastroparesis, chronic intestinal pseudo-obstruction (or colonic pseudo-obstruction), disorders and conditions associated with constipation, and other conditions and disorders are described herein, using peptides and other agents that activate the guanylate cyclase C (GC-C) receptor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to peptides, compositions, and methods for treating gastrointestinal disorders. [Background technology]

[0002] Gastrointestinal (GI) disorders include irritable bowel syndrome (IBS), a common chronic intestinal disorder that affects 20 to 60 million people in the United States alone (Lehman Brothers' September 1999 Industry Update on Irritable Bowel Syndrome in Global Healthcare). IBS is the most common disorder diagnosed by gastroenterologists and accounts for 12% of visits to primary care physicians (Camilleri 2001, Gastroenterology 120:652-668). In the United States, the economic impact of IBS is estimated to be $25 billion annually due to direct medical costs and indirect costs from missed work (Talley 1995, Gastroenterology 109:1736-1741). Patients with IBS are three times more likely to miss work and report a reduced quality of life. Because few prescription options exist to treat IBS, there is a significant unmet medical need for patients suffering from IBS.

[0003] Patients with IBS suffer from abdominal pain and impaired bowel patterns. Based on the predominant bowel habit, three subgroups of IBS patients have been defined: constipation-predominant irritable bowel syndrome (c-IBS), diarrhea-predominant irritable bowel syndrome (d-IBS), or alternating 2 types of irritable bowel syndrome (a-IBS). Estimates of the number of people suffering from c-IBS range from 20 to 50% of IBS patients, with a more frequent estimate being 30%. In contrast to the other two subgroups, which have similar sex ratios, c-IBS is more prevalent in women (3:1 ratio) (Talley et al. 1995, Am J Epidemiol 142:76-83).

[0004] The definition and diagnostic criteria for IBS have been formalized in the "Rome Criteria" (Drossman et al. 1999, Gut 45:Suppl II:1-81) and are widely accepted in clinical practice. Recently, there has been increasing evidence regarding the role of inflammation in the pathogenesis of IBS. Reports have shown that a subset of IBS patients have colonic inflammation and small but significant increases in mast cells, inducible nitric oxide (NO) and synthase (iNOS), and altered expression of inflammatory cytokines (report from Talley 2000 at DDW Week with Medscape Coverage).

[0005] Digestive disorders can also include constipation, with as many as 34 million Americans suffering from symptoms related to chronic constipation (CC), and 8.5 million patients seeking medical treatment. Patients with CC often experience hard, lumpy stools, straining during bowel movements, a feeling of incomplete bowel movement, and fewer than three bowel movements per week. The discomfort and bloating of CC significantly impact patients' quality of life by reducing their ability to work and participate in typical daily activities.

[0006] Half of CC patients are dissatisfied with currently available CC treatments. Thus, there remains a need for new compounds and methods for treating CC.

[0007] Patent Document 1 (U.S. Patent No. 7,304,036) and Patent Document 2 (U.S. Patent No. 7,371,727) disclose peptides that act as agonists of the guanylate cyclase C (GC-C) receptor for the treatment of gastrointestinal disorders. One particular peptide disclosed is linaclotide, which consists of the following amino acid sequence: CysCysGluTyrCysCysAsnProAlaCysThrGlyCysTyr. These patents also disclose methods for preparing linaclotide and related peptides.

[0008] Linaclotide has the following amino acid structure: [ka]

[0009] Linaclotide, an orally administered drug currently undergoing clinical trials for the treatment of irritable bowel syndrome with constipation (IBS-c) and chronic constipation (CC), has profound effects on GI physiology, including (1) reducing visceral pain, (2) reducing bloating, and (3) increasing gastrointestinal transit, potentially leading to increased bowel movement frequency and improved stool consistency. Orally administered linaclotide acts locally by activating GC-C in the luminal area; no detectable levels of linaclotide are found systemically after oral administration at therapeutic dose levels. Thus, the results of clinical trials of linaclotide, as well as preclinical studies conducted with linaclotide and related peptides, suggest that GC-C peptide agonists may be useful therapeutically.

[0010] The contents of US Pat. Nos. 7,304,036 and 7,371,727 are incorporated herein by reference in their entireties. The present invention features peptides that can be modified at their α-amine group to ketone derivatives capable of activating guanylate cyclase-C (GC-C) receptors and / or binding to the receptors with different affinities. The present invention also features peptides that can be modified with a cysteine bond to an additional sulfur atom and further modified at their α-amine group. Although low levels of GC-C have been detected in other tissues, GC-C is an important regulator of mammalian intestinal function. GC-C responds to the endogenous hormones guanylin and uroguanylin, as well as enterobacterial peptides (ST peptides) from the heat-stable enterotoxin family. Binding of agonists to GC-C results in an increase in the second messenger cyclic GMP (c-GMP) and increased chloride and bicarbonate secretion, leading to increased intestinal fluid secretion. In some examples of the present invention, the peptides described herein can result in increased c-GMP levels, providing a therapeutic option for treating gastrointestinal disorders. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 7,304,036 [Patent Document 2] U.S. Patent No. 7,371,727 Summary of the Invention [Means for solving the problem]

[0012] The present invention features peptides, compositions, and related methods for treating gastrointestinal diseases and conditions, including but not limited to, irritable bowel syndrome (IBS), gastrointestinal motility disorders, constipation, functional gastrointestinal disorders, gastroesophageal reflux disease (GERD), duodenogastric reflux, Crohn's disease, ulcerative colitis, inflammatory bowel disease, functional heartburn, dyspepsia, visceral pain, gastroparesis, chronic intestinal pseudo-obstruction (or colonic pseudo-obstruction), and other conditions and disorders described herein, using peptides and compositions that activate guanylate cyclase C (GC-C) receptors.

[0013] One aspect of the present invention provides a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, wherein Cys of the peptide 1 The α-amine of amino acids is deaminated by oxidative or enzymatic reactions (Cys 1 -α-ketone peptides).

[0014] In one embodiment, the peptide is [ka] or a pharmaceutically acceptable salt thereof.

[0015] In another embodiment, the peptide is [ka] It comprises an amino acid structure or a pharmaceutically acceptable salt thereof.

[0016] The details of one or more embodiments of the invention are set forth in the accompanying description. In certain embodiments, for example, the following are provided: (Item 1) A peptide or a pharmaceutically acceptable salt thereof, wherein the peptide comprises an amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, and wherein Cys of the peptide 1 The peptide or a pharmaceutically acceptable salt thereof, wherein the α-amine of the amino acid is deaminated. (Item 2) The peptide is [ka] 2. The peptide according to item 1, or a pharmaceutically acceptable salt thereof, comprising the amino acid structure: (Item 3) The peptide is [ka] 2. The peptide according to item 1, or a pharmaceutically acceptable salt thereof, comprising the amino acid structure: (Item 4) A peptide or a pharmaceutically acceptable salt thereof, wherein the peptide consists of the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, and the Cys of the peptide 1 A peptide or a pharmaceutically acceptable salt thereof, wherein the α-amine of the amino acid is deaminated. (Item 5) The peptide is [ka] 5. The peptide according to item 4, or a pharmaceutically acceptable salt thereof, having an amino acid structure as follows: (Item 6) The peptide is [ka] 5. The peptide according to item 4, or a pharmaceutically acceptable salt thereof, having an amino acid structure as follows: (Item 7) 7. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 1 to 6, wherein the peptide activates guanylate cyclase C receptor. (Item 8) 4. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 1 to 3, wherein the peptide comprises 30 or fewer amino acids. (Item 9) 4. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 1 to 3, wherein the peptide comprises 20 or fewer amino acids. (Item 10) 4. The peptide or pharmaceutically acceptable salt thereof according to any one of items 1 to 3, wherein fewer than 5 amino acids precede the first Cys residue in the amino acid sequence. (Item 11) 11. The peptide or pharmaceutically acceptable salt thereof according to any one of items 1 to 10, wherein the peptide or pharmaceutically acceptable salt thereof is isolated. (Item 12) 12. The peptide or pharmaceutically acceptable salt thereof according to any one of items 1 to 11, wherein the peptide or pharmaceutically acceptable salt thereof is purified. (Item 13) 13. A pharmaceutical composition comprising the peptide or a pharmaceutically acceptable salt thereof according to any one of items 1 to 12. (Item 14) 13. A pharmaceutical composition comprising two or more peptides or pharmaceutically acceptable salts thereof according to any one of items 1 to 12. (Item 15) i. The peptide is [ka] a peptide or a pharmaceutically acceptable salt thereof, comprising the amino acid structure ii. The peptide is [ka] a peptide or a pharmaceutically acceptable salt thereof, comprising the amino acid structure and iii. The peptide is [ka] or a pharmaceutically acceptable salt thereof, wherein the peptide comprises an amino acid structure of (Item 16) The peptide is [ka] ; containing the amino acid structure A pharmaceutical composition comprising linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide or a pharmaceutically acceptable salt thereof accounts for less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight compared to the weight of linaclotide. (Item 17) The peptide is [ka] ; containing the amino acid structure A pharmaceutical composition comprising linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide or a pharmaceutically acceptable salt thereof accounts for less than 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, or 1% by weight compared to the weight of linaclotide. (Item 18) The peptide is [ka] , It consists of the amino acid structure A pharmaceutical composition comprising a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide accounts for at least 90% by weight compared to the weight of linaclotide or another guanylate cyclase C agonist. (Item 19) The peptide is [ka] ; containing the amino acid structure A pharmaceutical composition consisting essentially of a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide accounts for at least 90% by weight compared to the weight of linaclotide or another guanylate cyclase C agonist. (Item 20) The peptide is [ka] containing the amino acid structure A pharmaceutical composition comprising linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide or a pharmaceutically acceptable salt thereof accounts for less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight compared to the weight of linaclotide. (Item 21) The peptide is [ka] ; containing the amino acid structure A pharmaceutical composition comprising linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide or a pharmaceutically acceptable salt thereof accounts for less than 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, or 1% by weight compared to the weight of linaclotide. (Item 22) The peptide is [ka] , It consists of the amino acid structure A pharmaceutical composition comprising a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide accounts for at least 90% by weight compared to the weight of linaclotide or another guanylate cyclase C agonist. (Item 23) The peptide is [ka] ; containing the amino acid structure A pharmaceutical composition consisting essentially of a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide accounts for at least 90% by weight compared to the weight of linaclotide or another guanylate cyclase C agonist. (Item 24) Linaclotide and Cys 1 -α-ketone peptide, i. The peptide is [ka] Peptides containing the amino acid structure of 1 -IMD") or a pharmaceutically acceptable salt thereof; ii. The peptide is [ka] Hydrolyzed peptides ("Asp") containing the amino acid structure 7 ") or a pharmaceutically acceptable salt thereof; iii. The peptide is [ka] Acetylated peptides ("Cys") containing the amino acid structure 1 -N-acetyl") or a pharmaceutically acceptable salt thereof; iv. The peptide is Cys Cys Glu Tyr Cys Cys Asn Pro a linaclotide trisulfide peptide or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence Ala Cys Thr Gly Cys Tyr, wherein an additional sulfur atom may be attached to any one of the six cysteinyl sulfurs; v. The peptide [ka] Peptides ("Des-Tyr") containing the amino acid structure 14 ") or a pharmaceutically acceptable salt thereof, and one or more peptides selected from: (Item 25) The peptide has a Cys 1 25. The pharmaceutical composition according to item 24, which is an IMD peptide. (Item 26) The peptide is hydrolyzed Asp 7 25. The pharmaceutical composition according to item 24, which is a peptide. (Item 27) The peptide has an acetylated Cys 1 25. The pharmaceutical composition according to item 24, wherein the compound is an N-acetyl peptide. (Item 28) 25. The pharmaceutical composition of item 24, wherein the peptide is linaclotide trisulfide peptide. (Item 29) The peptide is Des-Tyr 14 Item 30. The pharmaceutical composition according to Item 24, which is a peptide. (i) Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K. + , Na + Or Al 3+ 30. The pharmaceutical composition according to any one of items 13 to 29, further comprising one or more agents selected from (i) a cation selected from: (Item 31) a pharmaceutically acceptable carrier, a peptide according to any one of items 1 to 12, and (i) Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K. + , Na + Or Al3+ or (ii) one or more agents selected from sterically hindered primary amines. (Item 32) The agent is Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K. + , Na + , or Al 3+ 32. The pharmaceutical composition according to item 30 or 31, wherein (Item 33) Said Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K. + , Na + , or Al 3+ 33. The pharmaceutical composition of item 32, wherein the hydroxybenzoate is provided as magnesium acetate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium acetate, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganese acetate, manganese chloride, manganese phosphate, manganese sulfate, potassium acetate, potassium chloride, potassium phosphate, potassium sulfate, sodium acetate, sodium chloride, sodium phosphate, sodium sulfate, aluminum acetate, aluminum chloride, aluminum phosphate, or aluminum sulfate. (Item 34) 32. The pharmaceutical composition according to item 30 or 31, wherein the drug is a sterically hindered primary amine. (Item 35) 35. The pharmaceutical composition of claim 34, wherein the sterically hindered primary amine is an amino acid. (Item 36) 36. The pharmaceutical composition according to item 35, wherein the amino acid is a naturally occurring amino acid, a non-naturally occurring amino acid, or an amino acid derivative. (Item 37) 37. The pharmaceutical composition of item 36, wherein the naturally occurring amino acid is histidine, phenylalanine, alanine, glutamic acid, aspartic acid, glutamine, leucine, methionine, asparagine, tyrosine, threonine, isoleucine, tryptophan, or valine, or the unnatural amino acid is 1-aminocyclohexanecarboxylic acid, lanthanine, or theanine. (Item 38) The sterically hindered primary amine has the formula [ka] wherein R1, R2, and R3 are independently selected from H, C(O)OH, C1-C6 alkyl, C1-C6 alkyl ether, C1-C6 alkyl thioether, C1-C6 alkyl carboxylic acid, C1-C6 alkyl carboxylamide, and alkylaryl, any group may be mono- or polysubstituted with halogen or amino, provided that not more than one of R1, R2, and R3 is H. (Item 39) 39. The pharmaceutical composition according to item 38, wherein the sterically hindered primary amine is cyclohexylamine or 2-methylbutylamine. (Item 40) 35. The pharmaceutical composition of claim 34, wherein the sterically hindered primary amine is a polymeric amine. (Item 41) 41. The pharmaceutical composition of claim 40, wherein the polymeric amine is chitosan. (Item 42) The pharmaceutical composition contains Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K. + , Na + , or Al 3+ 42. The pharmaceutical composition according to any one of items 34 to 41, further comprising: (Item 43) Said Mg 2+ , Ca 2+ , Zn2+ , Mn 2+ , K. + , Na + , or Al 3+ 43. The pharmaceutical composition of item 42, wherein the hydroxybenzoate is provided as magnesium acetate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium acetate, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganese acetate, manganese chloride, manganese phosphate, manganese sulfate, potassium acetate, potassium chloride, potassium phosphate, potassium sulfate, sodium acetate, sodium chloride, sodium phosphate, sodium sulfate, aluminum acetate, aluminum chloride, aluminum phosphate, or aluminum sulfate. (Item 44) 44. The pharmaceutical composition according to any one of items 13 to 43, further comprising an antioxidant. (Item 45) 45. The pharmaceutical composition according to item 44, wherein the antioxidant is BHA, vitamin E, or propyl gallate. (Item 46) 40. The pharmaceutical composition according to any one of items 13 to 39, further comprising a pharmaceutically acceptable binder or excipient. (Item 47) 47. The pharmaceutical composition according to item 46, wherein the pharmaceutically acceptable binder or excipient is selected from polyvinyl alcohol, polyvinylpyrrolidone (povidone), starch, maltodextrin, or cellulose ether. (Item 48) 48. The pharmaceutical composition according to item 47, wherein the pharmaceutically acceptable binder or excipient is polyvinyl alcohol. (Item 49) 48. The pharmaceutical composition according to item 47, wherein the pharmaceutically acceptable binder or excipient is a cellulose ether. (Item 50) 50. The pharmaceutical composition of claim 49, wherein the cellulose ether is selected from methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose. (Item 51) 51. The pharmaceutical composition according to any one of items 13 to 50, further comprising a pharmaceutically acceptable filler. (Item 52) 52. The pharmaceutical composition according to item 51, wherein the pharmaceutically acceptable filler is cellulose, isomalt, mannitol, or calcium hydrogen phosphate. (Item 53) 53. The pharmaceutical composition according to item 52, wherein the cellulose is selected from fine cellulose and microcrystalline cellulose. (Item 54) 54. The pharmaceutical composition according to any one of items 13 to 53, further comprising an additional therapeutic agent. (Item 55) 55. The pharmaceutical composition of item 54, wherein the additional therapeutic agent is selected from one or more of an analgesic, an antidepressant, a prokinetic or prokinetic agent, an antiemetic, an antibiotic, a proton pump inhibitor, an acid blocker, a PDE5 inhibitor, an acid pump antagonist, a GABA-B agonist, a bile acid sequestrant, or a mucosal protectant. (Item 56) 24. A dosage unit comprising the pharmaceutical composition according to any one of items 13 to 23. (Item 57) 57. The dosage unit according to item 56, wherein the dosage unit is a capsule or a tablet. (Item 58) 58. The dosage unit according to item 57, wherein the dosage unit is a capsule. (Item 59) 58. The dosage unit according to item 57, wherein each of the dosage units contains 5 μg to 1 mg of linaclotide. (Item 60) 56. A dosage unit comprising the pharmaceutical composition according to any one of items 24 to 55. (Item 61) 61. The dosage unit according to item 60, wherein the dosage unit is a capsule or a tablet. (Item 62) 62. The dosage unit according to item 61, wherein the dosage unit is a capsule. (Item 63) 62. The dosage unit according to item 61, wherein each of the dosage units contains 5 μg to 1 mg of linaclotide. (Item 64) 64. The dosage unit according to item 63, wherein each of the dosage units contains 290 μg of linaclotide. (Item 65) 64. The dosage unit according to item 63, wherein each of the dosage units contains 145 μg of linaclotide. (Item 66) 56. A method for treating a digestive disorder, comprising administering the pharmaceutical composition according to any one of items 13 to 55. (Item 67) Item 67. The method of item 66, wherein the digestive disorder is selected from the group consisting of irritable bowel syndrome (IBS), constipation, functional gastrointestinal disorder, gastroesophageal reflux disease, functional heartburn, dyspepsia, visceral pain, gastroparesis, chronic intestinal pseudo-obstruction, colonic pseudo-obstruction, Crohn's disease, ulcerative colitis, and inflammatory bowel disease. (Item 68) Item 68. The method of item 67, wherein the digestive disorder is constipation. (Item 69) Item 69. The method of item 68, wherein the constipation is chronic constipation, idiopathic constipation, resulting from postoperative ileus, or caused by opiate use. (Item 70) Item 68. The method of item 67, wherein the digestive disorder is irritable bowel syndrome (IBS). (Item 71) Item 71. The method of item 70, wherein the irritable bowel syndrome is constipation-predominant irritable bowel syndrome (c-IBS), diarrhea-predominant irritable bowel syndrome (d-IBS), or alternating two types of irritable bowel syndrome (a-IBS). (Item 72) Item 68. The method of item 67, wherein the digestive disorder is dyspepsia. (Item 73) Item 68. The method of item 67, wherein the digestive disorder is gastroparesis. (Item 74) 74. The method of claim 73, wherein the gastroparesis is idiopathic, diabetic, or postoperative gastroparesis. (Item 75) Item 68. The method of item 67, wherein the digestive disease is chronic intestinal pseudo-obstruction. (Item 76) Item 68. The method of item 67, wherein the digestive disease is Crohn's disease. (Item 77) Item 68. The method of item 67, wherein the digestive disease is ulcerative colitis. (Item 78) Item 68. The method of item 67, wherein the digestive disease is inflammatory bowel disease. (Item 79) Item 68. The method of item 67, wherein the digestive disorder is visceral pain. (Item 80) 56. A method for increasing intestinal motility in a patient, comprising administering to the patient an effective amount of the pharmaceutical composition according to any one of items 13 to 55. (Item 81) 13. A method for increasing the activity of guanylate cyclase C (GC-C) receptor in a biological sample or an organism, the method comprising contacting the biological sample or an organism with the peptide according to any one of items 1 to 12. [Brief explanation of the drawings]

[0017] [Figure 1] 1 shows the specific binding of exemplary peptides of the invention to cell surface GC-C receptors on T84 cells in a competitive radioligand binding assay. [Figure 2] 1 shows the dose response of exemplary peptides of the invention in a c-GMP assay in T84 cells. [Figure 3] An example of the analysis of an exemplary peptide by RP-HPLC is shown, where "Cys1-α-ketone" refers to a linaclotide ketone derivative modified at the N-terminal α-amino group.

[0018] The drawings are provided as examples and are not intended to limit the scope of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] Guanylate cyclase C (GC-C) is a transmembrane receptor located on the apical surface of gastric and intestinal epithelial cells. The receptor has an extracellular ligand-binding domain, a single transmembrane region, and a C-terminal guanylyl cyclase domain. When a ligand binds to the extracellular domain of GC-C, the intracellular catalytic domain catalyzes the production of cGMP from GTP. In vivo, this increase in intracellular cGMP initiates a cascade of events that results in increased chloride and bicarbonate secretion into the intestinal lumen, elevated luminal pH, decreased luminal sodium absorption, increased fluid secretion, and facilitated intestinal transit. cGMP is secreted bidirectionally from the epithelium into the mucosa and lumen. The peptides and compositions of the present invention bind to the intestinal GC-C receptor, a regulator of intestinal fluid and electrolyte balance.

[0020] In some situations, it may be desirable to treat patients with mutant or modified peptides that bind to and activate intestinal GC-C receptors, but that have lower or higher activity than the non-mutated form of the peptide.Decreased activity can result from decreased affinity to the receptor, or decreased ability to activate the receptor once bound, or decreased stability of the peptide.Increased activity can result from increased affinity to the receptor, or increased ability to activate the receptor once bound, or increased stability of the peptide.

[0021] Description of Exemplary Peptides In various embodiments, the peptide has the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, and the Cys of the peptide 1 The α-amine of an amino acid is deaminated by oxidation or enzyme catalysis. This peptide can be prepared, for example, by deamination of Cys to form a Schiff base product. 1Oxidative deamination of linaclotide involving nucleophilic attack of the α-amine of the amino acid, followed by prototropic tautomerization of the Schiff base, and finally hydrolysis in acid, leads to the formation of α-Cys in equilibrium. 1 The mixture of these two peptides can be produced by obtaining the ketone and its hydrate. 1 -α-ketone" or "Cys 1 These peptides may be tautomeric and various tautomeric mixtures in different ratios may be useful and within the scope of the present invention.

[0022] In some embodiments, the peptide comprises the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, wherein Cys of the peptide 1 The α-amine of the amino acid is deaminated.

[0023] In one embodiment, the peptide is [ka] or a pharmaceutically acceptable salt thereof.

[0024] In another embodiment, the peptide is [ka] or a pharmaceutically acceptable salt thereof.

[0025] Those skilled in the art will appreciate that [ka] A peptide containing the amino acid structure [ka] It will be appreciated that the hydroxybenzoate may be in equilibrium with its geminal diol monohydrate form, which contains the amino acid structure As used herein, the term "Cys1-α-ketone peptide" or the structure [ka] is intended to include both the Cys1-α-ketone structure and the geminal diol monohydrate form.

[0026] In another embodiment, the peptide comprises the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, with an additional sulfur atom optionally attached to any one of the six cysteinyl sulfurs. In one embodiment, the peptide comprises a linaclotide trisulfide product (referred to herein as a "linaclotide trisulfide product" or "linaclotide trisulfide peptide") formed by the addition of a single sulfur atom to one of the three disulfide cysteinyl bonds of linaclotide.

[0027] In one embodiment, the peptide is [ka] , [ka] , and [ka] The amino acid structure is selected from:

[0028] In a further embodiment, the peptide consists of the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, wherein the Cys of the peptide 1 The α-amine of the amino acid is deaminated.

[0029] In one embodiment, the peptide is [ka] or a pharmaceutically acceptable salt thereof.

[0030] In one embodiment, the peptide is [ka] or a pharmaceutically acceptable salt thereof.

[0031] In another embodiment, the peptide consists of the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, with an additional sulfur atom optionally attached to any one of the six cysteinyl sulfurs.

[0032] In one embodiment, the peptide is [ka] , [ka] , and [ka] The amino acid structure is selected from the group consisting of:

[0033] In some embodiments, the peptide or a pharmaceutically acceptable salt thereof activates guanylate cyclase C receptors.

[0034] In other embodiments, the peptide or pharmaceutically acceptable salt thereof has fewer than 30 amino acids.

[0035] In a further embodiment, the peptide or pharmaceutically acceptable salt thereof has fewer than 20 amino acids.

[0036] In other embodiments, the peptide or pharmaceutically acceptable salt thereof comprises a peptide in which fewer than five amino acids precede the first Cys residue in the amino acid sequence.

[0037] In some embodiments, the peptide or a pharmaceutically acceptable salt thereof is isolated.

[0038] In other embodiments, the peptide or a pharmaceutically acceptable salt thereof is purified.

[0039] In some embodiments, pharmaceutically acceptable salts of the peptides are provided. In some cases, the pharmaceutically acceptable salt is a chloride salt.

[0040] Mutant or modified peptides In various embodiments, the peptide contains two Cys that form one disulfide bond, the peptide contains four Cys that form two disulfide bonds, or the peptide contains six Cys that form three disulfide bonds.

[0041] In various embodiments, the peptide contains two Cys that form one trisulfide bond, the peptide contains four Cys that form two trisulfide bonds, or the peptide contains six Cys that form three trisulfide bonds.

[0042] In some peptides, one or both members of one or both pairs of Cys residues that normally form disulfide bonds can be substituted with homocysteine, penicillamine, 3-mercaptoproline (Kolodziej et al. 1996 Int J Pept Protein Res 48:274); β,β dimethylcysteine (Hunt et al. 1993 Int J Pept Protein Res 42:249), or diaminopropionic acid (Smith et al. 1978 J Med Chem 21:117) to form alternative internal bridges at the positions of the normal disulfide bonds. In other embodiments, disulfide bonds can be replaced by hydrocarbon bridges (Schafmeister et al. 2000 J Am Chem Soc 122:5891; Patgiri et al. 2008 Acc Chem Res 41:1289, Henchey et al.2008 Curr Opin Chem Biol 12:692).

[0043] Peptide generation In one embodiment, the peptides or precursor peptides of the invention can be recombinantly produced in any known protein expression system, including, but not limited to, bacteria (e.g., Escherichia coli or Bacillus subtilis), insect cell systems (e.g., Drosophila Sf9 cell systems), yeast cell systems (e.g., Saccharomyces cerevisiae, Saccharomyces cerevisiae), or filamentous fungal expression systems, or animal cell expression systems (e.g., mammalian cell expression systems). In some embodiments, recombinantly produced peptides may be chemically modified after expression to form the peptides of the invention.

[0044] When the peptide or variant peptide is produced recombinantly, e.g., in E. coli, the nucleic acid molecule encoding the peptide can also encode a leader sequence that allows secretion of the mature peptide from the cell. Thus, the peptide-encoding sequence can include, for example, the pre- and pro-sequences of a naturally occurring bacterial ST peptide. The secreted mature peptide can be purified from the culture medium.

[0045] The sequences encoding the peptides described herein can be inserted into vectors that can deliver and maintain the nucleic acid molecule in bacterial cells. The DNA molecule can be inserted into a self-replicating vector (suitable vectors include, for example, pGEM3Z and pcDNA3 and their derivatives). The vector nucleic acid can be bacterial DNA or bacteriophage DNA, such as bacteriophage λ or M13 and its derivatives. Construction of a vector containing a nucleic acid described herein can be followed by transformation of a host cell, such as a bacterium. Suitable bacterial hosts include, but are not limited to, Escherichia coli, Bacillus subtilis, Pseudomonas, and Salmonella. In addition to the encoding nucleic acid molecule, the genetic construct also contains elements enabling expression, such as promoter and regulatory sequences. Expression vectors may contain transcriptional regulatory sequences that control transcription initiation, such as promoter sequences, enhancer sequences, operator sequences, and repressor sequences. Various transcriptional regulatory sequences are well known to those skilled in the art. Expression vectors can also contain translational regulatory sequences (e.g., untranslated 5' sequences, untranslated 3' sequences, or internal ribosome entry sites). The vector may be capable of autonomous replication or may integrate into the host DNA to ensure stability during peptide production.

[0046] Protein coding sequences containing the peptides described herein can also be fused to nucleic acids encoding peptide affinity tags, such as glutathione S-transferase (GST), maltose E-binding protein, protein A, FLAG tags, hexa-histidine, myc tags, or influenza HA tags, to facilitate purification. Affinity tag or reporter fusions link the reading frame of a peptide of interest to the reading frame of a gene encoding the affinity tag, creating a translational fusion. Expression of the fusion gene results in the translation of a single peptide containing both the peptide of interest and the affinity tag. In some cases where an affinity tag is used, a DNA sequence encoding a protease recognition site is fused between the reading frame of the affinity tag and the reading frame of the peptide of interest.

[0047] The peptides can also be produced in biological systems using suitable genetic constructs and methods for producing immature and mature forms of the peptides and variants described herein in protein expression systems other than bacteria that are well known to those skilled in the art.

[0048] In other embodiments, the above-mentioned peptides containing amino acids not normally incorporated by the translational machinery may be produced recombinantly by tRNA modification methods. Methods for modifying tRNAs are known in the art, including, but not limited to, modifying the anticodon, amino acid attachment site, and / or acceptor stem to allow for the incorporation of unnatural and / or arbitrary amino acids (Biochem. Biophys. Res. Comm. (2008) 372:480-485; Chem. Biol. (2009) 16:323-36; Nat. Methods (2007) 4:239-44; Nat. Rev. Mol. Cell Biol. (2006) 7:775-82; Methods (2005) 36:227-238; Methods (2005) 36:270-278; Annu. Rev. Biochem. (2004) 73:147-176; Nuc. Acids Res.(2004)32:6200-6211;Proc.Natl.Acad.Sci.USA(2003)100:6353-6357;Royal Soc.Chem.(2004)33:422-430).

[0049] In some embodiments, peptides may be produced chemically. Peptides can be synthesized by a number of different methods, including solution-phase synthesis and solid-phase synthesis using conventional BOC or FMOC protection. For example, peptides can be synthesized on 2-chlorotrityl or Wang resin using sequential amino acid coupling. The following protecting groups can be used: fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl (α-amino group, N-terminus); trityl or tert-butyl (thiol group of Cys); tert-butyl (γ-carboxyl of glutamic acid and hydroxyl group of threonine, if present); and trityl (β-amide function of asparagine side chain and phenolic group of tyrosine, if present). Coupling can be achieved using DIC and HOBt in the presence of a tertiary amine, and the peptide can be deprotected and cleaved from the solid support using cocktail K (81% by weight trifluoroacetic acid, 5% by weight phenol, 5% by weight thioanisole, 2.5% by weight 1,2-ethanedithiol, 3% by weight water, 2% by weight dimethyl sulfide, and 1.5% by weight ammonium iodide). After removal of trifluoroacetic acid and other volatiles, the peptide can be precipitated using an organic solvent. Disulfide bonds between Cys residues can be formed using dimethyl sulfoxide (Tam et al. (1991) J. Am. Chem. Soc. 113:6657-62) or using an air oxidation strategy. The resulting peptide can be purified by reverse-phase chromatography and lyophilized. In some embodiments, chemically synthesized peptides can be chemically modified after synthesis to form the peptides of the present invention.

[0050] These peptides can be prepared, isolated, or used in the form of either the base or a pharmaceutically acceptable salt thereof, including, but not limited to, acetate, chloride, sulfate, and phosphate salts of the peptides.

[0051] Compositions of peptides and GC-C receptor agonists In another aspect, pharmaceutical compositions are provided in which the peptides, alone or in combination, can be combined with any pharmaceutically acceptable carrier or vehicle.

[0052] In some embodiments, a pharmaceutical composition comprises a peptide described herein or a pharmaceutically acceptable salt thereof. A pharmaceutical composition may comprise two or more peptides described herein or a pharmaceutically acceptable salt thereof.

[0053] In some embodiments, the pharmaceutical composition comprises: i. The peptide is [ka] a peptide or a pharmaceutically acceptable salt thereof, comprising the amino acid structure ii. The peptide is [ka] a peptide or a pharmaceutically acceptable salt thereof, comprising the amino acid structure or iii. The peptide is [ka] or a pharmaceutically acceptable salt thereof,

[0054] In other embodiments, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] and the peptide or a pharmaceutically acceptable salt thereof accounts for less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight compared to the weight of linaclotide.

[0055] In a further embodiment, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] and the pharmaceutical composition comprises less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% by weight of the peptide or a pharmaceutically acceptable salt thereof relative to the weight of linaclotide.

[0056] In a further embodiment, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] and the pharmaceutical composition contains about 0.01 to 9% by weight (e.g., about 0.01 to 8% by weight, about 0.01 to 7% by weight, about 0.01 to 6% by weight, about 0.01 to 5% by weight, about 0.01 to 4% by weight, about 0.01 to 3% by weight, about 0.01 to 2% by weight, about 0.01 to 2% by weight, about 0.01 to 1% by weight) of the peptide or a pharmaceutically acceptable salt thereof, relative to the weight of linaclotide.

[0057] In other embodiments, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] and the peptide or a pharmaceutically acceptable salt thereof accounts for less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight compared to the weight of linaclotide.

[0058] In a further embodiment, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] and the peptide or a pharmaceutically acceptable salt thereof accounts for less than 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1% by weight, or 0.5% by weight compared to the weight of linaclotide.

[0059] In a further embodiment, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] ; and the pharmaceutical composition contains about 0.01 to 9% by weight (e.g., about 0.01 to 8% by weight, about 0.01 to 7% by weight, about 0.01 to 6% by weight, about 0.01 to 5% by weight, about 0.01 to 4% by weight, about 0.01 to 3% by weight, about 0.01 to 2% by weight, about 0.01 to 2% by weight, about 0.01 to 1% by weight) of the peptide or a pharmaceutically acceptable salt thereof, relative to the weight of linaclotide.

[0060] In other embodiments, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] and the peptide or a pharmaceutically acceptable salt thereof accounts for less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight compared to the weight of linaclotide.

[0061] In a further embodiment, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] and the peptide or a pharmaceutically acceptable salt thereof accounts for less than 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1% by weight, or 0.5% by weight compared to the weight of linaclotide.

[0062] In a further embodiment, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] and the pharmaceutical composition contains about 0.01 to 9% by weight (e.g., about 0.01 to 8% by weight, about 0.01 to 7% by weight, about 0.01 to 6% by weight, about 0.01 to 5% by weight, about 0.01 to 4% by weight, about 0.01 to 3% by weight, about 0.01 to 2% by weight, about 0.01 to 2% by weight, about 0.01 to 1% by weight) of the peptide or a pharmaceutically acceptable salt thereof, relative to the weight of linaclotide.

[0063] In other embodiments, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] and the peptide or a pharmaceutically acceptable salt thereof accounts for less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight compared to the weight of linaclotide.

[0064] In a further embodiment, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] and the peptide or a pharmaceutically acceptable salt thereof accounts for 9 wt%, 8 wt%, 7 wt%, 6 wt%, 5 wt%, 4 wt%, 3 wt%, 2 wt%, 1 wt%, or 0.5 wt% compared to the weight of linaclotide.

[0065] In a further embodiment, the pharmaceutical composition comprises linaclotide and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] and the pharmaceutical composition contains about 0.01-9% by weight (e.g., about 0.01-8% by weight, about 0.01-7% by weight, about 0.01-6% by weight, about 0.01-5% by weight, about 0.01-4% by weight, about 0.01-3% by weight, about 0.01-2% by weight, about 0.01-2% by weight, about 0.01-1% by weight) of the peptide or a pharmaceutically acceptable salt thereof, relative to the weight of linaclotide.

[0066] In some embodiments, Cys 1In another exemplary embodiment, the -α-ketone peptide comprises less than about 15% by weight of the composition, less than about 10% by weight of the composition, less than about 7% by weight of the composition, less than about 5% by weight of the composition, less than about 4% by weight of the composition, less than about 3% by weight of the composition, less than about 2% by weight of the composition, or less than about 1% by weight of the composition. 1 In further exemplary embodiments, the Cys-α-ketone peptide comprises from about 0.01% to about 15% by weight of the composition, from about 0.05% to about 10% by weight of the composition, from about 0.05% to about 7% by weight of the composition, or from about 0.05% to about 5% by weight of the composition. 1 The -α-ketone peptide comprises about 0.5% to about 2% by weight of the composition.

[0067] In a further embodiment, linaclotide and Cys 1

[0010] A method of treating a gastrointestinal disorder in a patient in need thereof is provided, comprising administering a pharmaceutical composition comprising a .alpha.-ketone peptide.

[0068] In some embodiments, provided is a pharmaceutical composition comprising linaclotide and linaclotide trisulfide product.In one embodiment, linaclotide trisulfide product is formed by adding a single sulfur atom to one of the three disulfide cysteinyl bonds of linaclotide.Three possible structures of this product are shown below, but those skilled in the art will recognize that sulfur atom can be attached to any one of six cysteinyl sulfurs. [ka] [ka] [ka]

[0069] In another embodiment, there may be the addition of two or more sulfur atoms to linaclotide, increasing its molecular weight by 32 AU per added sulfur atom.

[0070] In some other embodiments, the linaclotide trisulfide product comprises less than about 15% by weight of the composition, less than about 10% by weight of the composition, less than about 7% by weight of the composition, less than about 5% by weight of the composition, less than 4% by weight of the composition, less than about 3% by weight of the composition, less than about 2% by weight of the composition, or less than about 1% by weight of the composition.

[0071] In other exemplary embodiments, the linaclotide trisulfide product comprises about 0.01% to about 15% by weight of the composition, about 0.05% to about 10% by weight of the composition, about 0.05% to about 7% by weight of the composition, or about 0.05% to about 5% by weight of the composition. In further exemplary embodiments, the linaclotide trisulfide product comprises about 0.5% to about 2% by weight of the composition.

[0072] In a further embodiment, there is provided a method of treating a gastrointestinal disorder in a patient in need thereof comprising administering a pharmaceutical composition comprising linaclotide and a linaclotide trisulfide product.

[0073] In other embodiments, the pharmaceutical composition consists essentially of a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] It contains the amino acid structure of

[0074] In other embodiments, the pharmaceutical composition consists essentially of a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is [ka] It contains the amino acid structure of

[0075] The term "consisting essentially of" and variations thereof, when used in reference to a composition, is used herein to mean that the composition comprises only the active peptide and other desired pharmaceutically inactive additives, excipients, and / or components (e.g., polymers, sterically hindered primary amines, cations, fillers, binders, carriers, excipients, diluents, disintegration additives, lubricants, solvents, dispersants, coating additives, absorption enhancing additives, controlled release additives, anti-caking additives, antimicrobial additives, preservatives, sweetening additives, colorants, flavoring agents, desiccants, plasticizers, dyes, etc.), and is free of other active pharmaceutical ingredient(s).

[0076] In some embodiments, linaclotide and Cys 1 -α-ketone peptide, i. The peptide is [ka] Peptides containing the amino acid structure of 1 -IMD") or a pharmaceutically acceptable salt thereof; ii. The peptide is [ka] Hydrolyzed peptides ("Asp") containing the amino acid structure 7 ") or a pharmaceutically acceptable salt thereof; iii. The peptide is [ka] Acetylated peptides ("Cys") containing the amino acid structure 1 -N-acetyl") or a pharmaceutically acceptable salt thereof, iv. The peptide is Cys Cys Glu Tyr Cys Cys Asn Pro a linaclotide trisulfide peptide or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence Ala Cys Thr Gly Cys Tyr, wherein an additional sulfur atom may be attached to any one of the six cysteinyl sulfurs; and v. The peptide [ka] Peptides ("Des-Tyr") containing the amino acid structure 14 and one or more peptides selected from the group consisting of:

[0077] In some embodiments, Cys 1 In other exemplary embodiments, the -α-ketone peptide comprises less than about 15% by weight of the composition, less than about 10% by weight of the composition, less than about 7% by weight of the composition, less than about 5% by weight of the composition, less than about 4% by weight of the composition, less than about 3% by weight of the composition, less than about 2% by weight of the composition, less than about 1.5% by weight of the composition, or less than about 1% by weight of the composition. 1 The -α-ketone peptide comprises from about 0.01% to about 15% by weight of the composition, from about 0.05% to about 10% by weight of the composition, from about 0.05% to about 7% by weight of the composition, or from about 0.05% to about 5% by weight of the composition.

[0078] In some embodiments, Cys 1 The -IMD peptide comprises less than about 15% by weight of the composition, less than about 10% by weight of the composition, less than about 7% by weight of the composition, less than about 5% by weight of the composition, less than about 4% by weight of the composition, less than about 3.5% by weight of the composition, less than about 3% by weight of the composition, less than about 2% by weight of the composition, or less than about 1% by weight of the composition. 1 The IMD peptide comprises from about 0.01% to about 15% by weight of the composition, from about 0.05% to about 10% by weight of the composition, from about 0.05% to about 7% by weight of the composition, or from about 0.05% to about 5% by weight of the composition.

[0079] In some embodiments, the hydrolyzed peptide ("Asp 7 ") comprises less than about 15% by weight of the composition, less than about 10% by weight of the composition, less than about 7% by weight of the composition, less than about 5% by weight of the composition, less than about 4% by weight of the composition, less than about 3.5% by weight of the composition, less than about 3% by weight of the composition, less than about 2% by weight of the composition, or less than about 1% by weight of the composition. In other exemplary embodiments, hydrolyzed peptides ("Asp 7 ") comprises from about 0.01% to about 15% by weight of the composition, from about 0.05% to about 10% by weight of the composition, from about 0.05% to about 7% by weight of the composition, or from 0.05% to about 5% by weight of the drug of the composition.

[0080] In some embodiments, the acetylated peptide ("Cys 1 -N-Acetyl") comprises less than about 15% by weight of the composition, less than about 10% by weight of the composition, less than about 7% by weight of the composition, less than about 5% by weight of the composition, less than about 4% by weight of the composition, less than about 3.5% by weight of the composition, less than about 3% by weight of the composition, less than about 2% by weight of the composition, or less than about 1% by weight of the composition. In other exemplary embodiments, the acetylated peptide ("Cys 1 -N-Acetyl") comprises from about 0.01% to about 15% by weight of the composition, from about 0.05% to about 10% by weight of the composition, from about 0.05% to about 7% by weight of the composition, or from about 0.05% to about 5% by weight of the composition.

[0081] In some embodiments, the linaclotide trisulfide peptide comprises less than about 15% by weight of the composition, less than about 10% by weight of the composition, less than about 7% by weight of the composition, less than about 5% by weight of the composition, less than about 4% by weight of the composition, less than about 3.5% by weight of the composition, less than about 3% by weight of the composition, less than about 2% by weight of the composition, or less than about 1% by weight of the composition. In other exemplary embodiments, the linaclotide trisulfide peptide comprises between about 0.01% and about 15% by weight of the composition, between about 0.05% and about 10% by weight of the composition, between about 0.05% and about 7% by weight of the composition, or between about 0.05% and about 5% by weight of the composition.

[0082] In some embodiments, Des-Tyr14 The peptide comprises less than about 15% by weight of the composition, less than about 10% by weight of the composition, less than about 7% by weight of the composition, less than about 5% by weight of the composition, less than about 4% by weight of the composition, less than about 3.5% by weight of the composition, less than about 3% by weight of the composition, less than about 2% by weight of the composition, or less than about 1% by weight of the composition. 14 The peptide comprises from about 0.01% to about 15% by weight of the composition, from about 0.05% to about 10% by weight of the composition, from about 0.05% to about 7% by weight of the composition, or from about 0.05% to about 5% by weight of the composition.

[0083] In some embodiments, the pharmaceutical composition comprises linaclotide, Cys 1 -α-ketone peptides, and disulfide-linked multimers at any desired concentration.

[0084] In some embodiments, the composition comprises less than 10% by weight of multimer(s).

[0085] In some embodiments, the composition comprises less than 7% by weight of multimer(s).

[0086] In some embodiments, the composition comprises less than 6% by weight of multimer(s).

[0087] In some embodiments, the composition comprises less than 5% by weight of multimer(s).

[0088] In some embodiments, the composition comprises less than 4% by weight of multimer(s).

[0089] In some embodiments, the composition comprises less than 3% by weight of multimer(s).

[0090] In some embodiments, the composition comprises less than 2% by weight of multimer(s).

[0091] In some embodiments, the composition comprises less than 1% by weight of multimer(s).

[0092] The peptides described herein can be combined with any pharmaceutically acceptable carrier or vehicle, such as solvents, dispersing agents, coating agents, absorption enhancers, controlled-release agents, and one or more inert excipients, including starches, polyols, granulating agents, microcrystalline cellulose (e.g., Celphere, Celphere beads®), diluents, lubricants, binders, disintegrants, etc. If desired, tablet dosage forms of the disclosed compositions may be coated by standard aqueous or nonaqueous techniques.

[0093] Examples of excipients for use as pharmaceutically acceptable carriers and pharmaceutically acceptable inert carriers, as well as the additional ingredients mentioned above, include, but are not limited to, binders, fillers, disintegrants, lubricants, antimicrobial agents, and coating agents.

[0094] As used herein, the term "binder" refers to any pharmaceutically acceptable binder that can be used in the practice of the present invention. Examples of pharmaceutically acceptable binders include, but are not limited to, starch (e.g., corn starch, potato starch, and pregelatinized starch (e.g., STARCH 1500® and STARCH 1500 LM®, sold by Colorcon, Ltd., and other starches), maltodextrin, gelatin, natural and synthetic gums (e.g., gum acacia, powdered gum tragacanth, guar gum, etc.), cellulose or cellulose ethers and derivatives thereof (e.g., methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose (hypromellose), ethylcellulose, cellulose acetate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, carboxymethylcellulose, microcrystalline cellulose (e.g., FMC Corporation, Marcus AVICEL™ (e.g., AVICEL-PH-101™, -103™, and -105™) available from Hook, PA, USA, polyvinyl alcohol, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K30, povidone), cellulose ethers, and mixtures thereof.

[0095] As used herein, the term " filler " refers to any pharmaceutically acceptable filler that can be used in the implementation of the present invention.The example of pharmaceutically acceptable filler includes but is not limited to talc, calcium carbonate (for example, granules or powder), dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate (for example, granules or powder), microcrystalline cellulose (for example, Avicel PH101 or Celphere CP-305), powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch (for example, Starch 1500), pregelatinized starch, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, isomalt, raffinose, maltitol, melezitose, stachyose, lactitol, palatinit, xylitol, myoinositol, and mixtures thereof.

[0096] Examples of pharmaceutically acceptable fillers that may be used, in particular to coat the peptides, include, but are not limited to, talc, microcrystalline cellulose (e.g., Avicel PH101 or Celphere CP-305), fine cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, isomalt, dibasic calcium phosphate, raffinose, maltitol, melezitose, stachyose, lactitol, palatinite, xylitol, mannitol, myo-inositol, and mixtures thereof.

[0097] As used herein, the term "additive" refers to any pharmaceutically acceptable additive.Pharmaceutically acceptable additives include, but are not limited to, disintegrants, dispersion additives, lubricants, glidants, antioxidants, coating additives, diluents, surfactants, flavor additives, wetting agents, absorption-promoting additives, controlled-release additives, anti-caking additives, antimicrobial agents (e.g., preservatives), colorants, desiccants, plasticizers, and dyes.As used herein, the term "excipient" refers to any pharmaceutically acceptable additive, filler, binder, or drug.

[0098] The compositions of the present invention can also optionally include other therapeutic ingredients, anti-caking agents, preservatives, sweeteners, colorants, flavoring agents, desiccants, plasticizers, dyes, glidants, anti-adhesive agents, anti-static agents, surfactants (wetting agents), antioxidants, film coating agents, etc. Any such optional ingredients must be compatible with the compounds described herein to ensure the stability of the formulation. The compositions may optionally contain other additives, including, for example, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, raffinose, maltitol, melezitose, stachyose, lactitol, palatinit, starch, xylitol, mannitol, myo-inositol, etc., and hydrates thereof, as well as amino acids, such as alanine, glycine, and betaine, and peptides and proteins, such as egg white.

[0099] The compositions can include, for example, various additional solvents, dispersants, coating agents, absorption-enhancing additives, controlled-release additives, and one or more inert additives (including, for example, starches, polyols, granulating additives, microcrystalline cellulose, diluents, lubricants, binders, disintegration additives, etc.). If desired, tablet dosage forms of the disclosed compositions can be coated by standard aqueous or non-aqueous techniques. The compositions can also include, for example, anti-caking additives, preservatives, sweetening additives, colorants, flavoring agents, desiccants, plasticizers, dyes, etc.

[0100] Suitable disintegrants include, for example, agar, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, povidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pregelatinized starch, clays, other algins, other celluloses, gums, and mixtures thereof.

[0101] Suitable lubricants include, for example, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, syloid silica gel (AEROSIL 200, W.R. Grace Co., Baltimore, MD USA), agglomerated aerosol of synthetic silica (Evonik Degussa Co., Plano, TX USA), pyrogenic silicon dioxide (CAB-O-SIL, Cabot Co., Boston, MA USA), and mixtures thereof.

[0102] Suitable glidants include, for example, leucine, colloidal silicon dioxide, magnesium trisilicate, powdered cellulose, starch, talc, and tricalcium phosphate.

[0103] Suitable anti-caking additives include, for example, calcium silicate, magnesium silicate, silicon dioxide, colloidal silicon dioxide, talc, and mixtures thereof.

[0104] For example, suitable antimicrobial additives that may be used as preservatives for peptide compositions include, for example, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride, cresol, chlorobutanol, dehydroacetic acid, ethylparaben, methylparaben, phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate, potassium sorbate, propylparaben, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid, thimerosal, thymo, and mixtures thereof.

[0105] Suitable antioxidants include, for example, BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), vitamin E, propyl gallate, ascorbic acid and its salts or esters, tocopherol and its esters, alpha-lipoic acid, and beta-carotene.

[0106] Suitable coating additives include, for example, sodium carboxymethylcellulose, cellulose acetate phthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, methylcellulose, polyethylene glycol, polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline wax, and mixtures thereof. Suitable protective coating agents include Aquacoat (e.g., Aquacoat Ethylcellulose Aquaeous Dispersion, 15% by weight, FMC Biopolymer, ECD-30), Eudragit (e.g., Eudragit E PO PE-EL, Roehm Pharma Polymers), and Opadry (e.g., Opadry AMB Aqueous Dispersion, 20% by weight, Colorcon).

[0107] In certain embodiments, suitable excipients for peptide compositions include one or more of sucrose, talc, magnesium stearate, crospovidone, or BHA.

[0108] The compositions of the present invention may also contain, but are not limited to, L-histidine, Pluronic®, poloxamers (such as Lutrol® and Poloxamer 188), ascorbic acid, glutathione, permeation enhancers (e.g., lipids, sodium cholate, acylcarnitines, salicylates, bile salt mixtures, fatty acid micelles, chelating agents, fatty acids, surfactants, medium chain glycerides), protease inhibitors (e.g., soybean trypsin inhibitor, organic acids), pH-lowering agents and absorption enhancers effective to enhance bioavailability (including, but not limited to, those described in U.S. Pat. Nos. 6,086,918 and 5,912,014), chewable tablet materials (dextrose, fructose, lactose monohydrate, lactose and aspartame, lactose and cellulose, maltodextrin ... cellulose, mannitol, microcrystalline cellulose and guar gum, crystalline sorbitol, etc.; parenterals (mannitol and povidone, etc.); plasticizers (dibutyl sebacate, coating plasticizers, polyvinyl acetate phthalate, etc.); powdered lubricants (glyceryl behenate, etc.); soft gelatin capsules (sorbitol special solutions, etc.); coating spheres (sugar spheres, etc.); spheronizing agents (glyceryl behenate and microcrystalline cellulose, etc.); suspending / gelling agents (carrageenan, gellan gum, mannitol, microcrystalline cellulose, povidone, sodium starch glycolate, xanthan gum, etc.); sweeteners (aspartame, aspartame and lactose, glucose, fructose, honey, maltodextrin, maltose, mannitol, molasses, crystalline sorbitol, sorbitol special solutions, sucrose, etc.);Wet granulating agents (calcium carbonate, lactose anhydrous, lactose monohydrate, maltodextrin, mannitol, microcrystalline cellulose, povidone, starch, etc.), caramel, sodium carboxymethylcellulose, cherry cream flavor and cherry flavor, anhydrous citric acid, citric acid, powdered sugar, D&C Red No. 33, D&C Yellow No. 10 Aluminum Lake, edetate disodium, 15% ethyl alcohol, FD&C Yellow No. 6 Aluminum Lake, FD&C Blue No. 1 Aluminum Lake, FD&C Blue No. 1, FD&C Blue No. 2 Aluminum Lake, FD&C Green No. 3, FD&C Red No. 40, FD&C Yellow No. 6 Aluminum Lake, FD&C Yellow No. 6, FD&C Yellow No. 10 May contain other excipients, drugs, and categories thereof, including: glycerol palmitostearate, glyceryl monostearate, indigo carmine, lecithin, mannitol, methyl and propylparabens, monoammonium glycyrrhizinate, natural and artificial orange flavor, pharmaceutical glaze, poloxamer 188, polydextrose, polysorbate 20, polysorbate 80, polyvidone, pregelatinized corn starch, pregelatinized starch, red iron oxide, sodium saccharin, sodium carboxymethyl ether, sodium chloride, sodium citrate, sodium phosphate, strawberry flavor, synthetic black iron oxide, synthetic red iron oxide, titanium dioxide, and white wax;

[0109] In some embodiments, a peptide described herein and Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K. + , Na + , or Al 3+ , combinations thereof, and / or one or more agents selected from sterically hindered primary amines.

[0110] In a further embodiment, the agent is Mg 2+ , Ca 2+ , or Zn 2+or a combination thereof. In some embodiments, the cations are provided as, but not limited to, magnesium acetate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium acetate, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganese acetate, manganese chloride, manganese phosphate, manganese sulfate, potassium acetate, potassium chloride, potassium phosphate, potassium sulfate, sodium acetate, sodium chloride, sodium phosphate, sodium sulfate, aluminum acetate, aluminum chloride, aluminum phosphate, or aluminum sulfate. In further embodiments, the cations are provided as magnesium chloride, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, manganese chloride, potassium chloride, sodium chloride, or aluminum chloride. In other embodiments, the cations are provided as calcium chloride, magnesium chloride, or zinc acetate.

[0111] In another embodiment, the agent is a sterically hindered primary amine.

[0112] In a further embodiment, the sterically hindered primary amine is an amino acid.

[0113] In a further embodiment, the amino acid is a naturally occurring amino acid.

[0114] In a further embodiment, the naturally occurring amino acid is selected from the group consisting of histidine, phenylalanine, alanine, glutamic acid, aspartic acid, glutamine, leucine, methionine, asparagine, tyrosine, threonine, isoleucine, tryptophan, glycine, and valine, and further, the naturally occurring amino acid is leucine, isoleucine, alanine, or methionine.

[0115] In a further embodiment, the naturally occurring amino acid is leucine. In another embodiment, the sterically hindered primary amine is an unnatural amino acid (e.g., 1-aminocyclohexanecarboxylic acid, lanthanine, or theanine).

[0116] In a further embodiment, the sterically hindered primary amine is cyclohexylamine, 2-methylbutylamine, or a polymeric amine (eg, chitosan).

[0117] In another embodiment, one or more sterically hindered primary amines may be used in the composition.

[0118] In some cases, the sterically hindered primary amine has the formula [ka] wherein R1, R2, and R3 are independently selected from H, C(O)OH, C1-C6 alkyl, C1-C6 alkyl ether, C1-C6 alkyl thioether, C1-C6 alkyl carboxylic acid, C1-C6 alkyl carboxylamide, and alkylaryl, any of which groups may be mono- or polysubstituted with halogen or amino, provided that no more than two of R1, R2, and R3 are H.

[0119] In another embodiment, no more than one of R 1 , R 2 , and R 3 is H.

[0120] In another embodiment, a pharmaceutically acceptable carrier, a peptide, and Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K. + , Na + , or Al 3+ and a sterically hindered primary amine.

[0121] In one embodiment, the cation is Mg 2+ , Ca 2+ , or Zn 2+ , or a mixture thereof.

[0122] In a further embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable binder and / or a pharmaceutically acceptable glidant, lubricant, or an excipient that acts as both a glidant and a lubricant, and / or an antioxidant.

[0123] In some embodiments, the pharmaceutical composition is applied to a carrier.

[0124] In some embodiments, the carrier is a filler.

[0125] In some cases, the molar ratio of cation:sterically hindered primary amine:peptide in the aqueous solution applied to the carrier is 5-100:5-50:1. In some cases, the molar ratio of cation:sterically hindered primary amine may be 2:1 or greater (e.g., 5:1-2:1). Thus, in some cases, the molar ratio of cation:sterically hindered primary amine:peptide applied to the carrier is 100:50:1, 100:30:1, 80:40:1, 80:30:1, 80:20:1, 60:30:1, 60:20:1, 50:30:1, 50:20:1, 40:20:1, 20:20:1, 10:10:1, 10:5:1, or 5:10:1. If a binder, such as methylcellulose, is present in the GC-C agonist peptide solution applied to the carrier, it may be present at 0.5% to 2.5% by weight (e.g., 0.7% to 1.7%, or 0.7% to 1%, or 1.5%, or 0.7%).

[0126] In a further embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable binder or excipient, and / or a pharmaceutically acceptable glidant, lubricant, or excipient that acts as both a glidant and a lubricant, and / or an antioxidant.

[0127] Suitable pharmaceutical compositions according to the present invention generally contain an amount of active compound(s) together with a pharmaceutically acceptable diluent or excipient, such as a sterile aqueous solution, to give a wide range of final concentrations depending on the intended use. Preparation techniques, such as those exemplified by Remington's Pharmaceutical Sciences (18th Edition, Mack Publishing Company, 1995), are generally well known in the art.

[0128] The compositions described herein may be administered systemically or locally, for example, orally (e.g., using capsules, powders, solutions, suspensions, tablets, sublingual formulations, etc.), by inhalation (e.g., using aerosols, gases, inhalers, nebulizers, etc.), auricularly (e.g., using ear drops), topically (e.g., using creams, gels, liniments, lotions, ointments, pastes, transdermal patches, etc.), ophthalmically (e.g., using eye drops, eye drop gels, eye drop ointments), rectally (e.g., using an enema or suppository), nasally, buccally, vaginally (e.g., using a vaginal douche, intrauterine device, vaginal suppository, vaginal ring or tablet, etc.), via an implantable reservoir, etc., or parenterally, depending on the severity and type of disease being treated. The term "parenteral" as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally, or intravenously.

[0129] For the treatment of gastrointestinal disorders, the peptides described herein are preferably administered orally, for example, as tablets, capsules, sachets containing pellets of a predetermined amount of active ingredient, gels, pastes, syrups, boluses, electuaries, slurries, powders, lyophilized powders, granules, solutions or suspensions in aqueous or non-aqueous solutions, oil-in-water or water-in-oil emulsions, liposomal formulations (see, e.g., European Patent No. EP 736299), or any other form. Orally administered compositions can include binders, lubricants, inert diluents, lubricating, surfactant, or dispersing agents, flavoring agents, and humectants. Orally administered formulations, such as tablets, can optionally be coated or scored and formulated to provide sustained, delayed, or controlled release of the active ingredient therein.

[0130] The peptides can be co-administered with other agents used to treat gastrointestinal disorders, including, but not limited to, the agents described herein.

[0131] In another embodiment, suitable pharmaceutical compositions may contain one or more other therapeutic agents, including, but not limited to, analgesics, antisecretory agents including proton pump inhibitors, acid pump antagonists, and H2 receptor antagonists, PDE5 inhibitors, GABA-B antagonists, bile acid sequestrants, prokinetic and prokinetic agents, antidepressants, antibiotics, antiemetics, and mucosal protectants.

[0132] Treatment method In various embodiments, the peptides and compositions described herein are useful for treating gastrointestinal disorders in a patient.

[0133] In some embodiments, the digestive disorder is selected from the group consisting of irritable bowel syndrome (IBS), constipation, functional gastrointestinal disorder, gastroesophageal reflux disease, functional heartburn, dyspepsia, visceral pain, gastroparesis, chronic intestinal pseudo-obstruction, colonic pseudo-obstruction, Crohn's disease, ulcerative colitis, and inflammatory bowel disease.

[0134] In a further embodiment, the digestive disorder is constipation.Constipation can be chronic constipation, idiopathic constipation, postoperative ileus, or caused by the use of opiates.Clinically accepted criteria for defining constipation include stool frequency, stool consistency, and ease of defecation.One common definition of constipation is having fewer than three bowel movements per week.Other definitions include abnormally hard stools or defecation requiring excessive straining (Schiller 2001, Aliment Pharmacol Ther 15:749-763).Constipation can be idiopathic (functional constipation or slow colonic transit constipation) or secondary to other causes, including neurological, metabolic, or endocrine disorders. These diseases include diabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcemia, multiple sclerosis, Parkinson's disease, spinal cord injury, neurofibromatosis, autonomic neuropathy, Chagas' disease, Hirschsprung's disease, and cystic fibrosis. Constipation can also be the result of surgery (postoperative ileus) or can result from the use of medications such as analgesics (such as opioids), antihypertensives, anticonvulsants, antidepressants, antispasmodics, and antipsychotics.

[0135] In another embodiment, the digestive disorder is irritable bowel syndrome (IBS). The irritable bowel syndrome can be constipation-predominant irritable bowel syndrome (c-IBS), diarrhea-predominant irritable bowel syndrome (d-IBS), or alternating forms of irritable bowel syndrome (a-IBS).

[0136] In another embodiment, the gastrointestinal disorder is dyspepsia.

[0137] In another embodiment, the gastrointestinal disorder is gastroparesis, which can be selected from idiopathic, diabetic, or post-operative gastroparesis.

[0138] In yet another embodiment, the gastrointestinal disorder is chronic intestinal pseudo-obstruction.

[0139] In another embodiment, the gastrointestinal disorder is Crohn's disease.

[0140] In some embodiments, the gastrointestinal disease is ulcerative colitis.

[0141] In some embodiments, the gastrointestinal disease is inflammatory bowel disease.

[0142] In another embodiment, the gastrointestinal disorder is visceral pain.

[0143] In a further embodiment, the invention features a method for alleviating gastrointestinal or visceral pain in a patient, comprising administering to the patient a pharmaceutical composition comprising a peptide described herein. The peptide agonists described herein can be used alone or in combination therapy to treat, prevent, or alleviate visceral pain associated with a gastrointestinal disorder or pain associated with another disorder.

[0144] In another embodiment, the invention features a method for treating inflammation of the gastrointestinal tract, including inflammation associated with a gastrointestinal disease or infection or any other condition, comprising administering to a patient a pharmaceutical composition comprising a purified peptide described herein.

[0145] In another embodiment, the invention features a method for treating a gastrointestinal disorder, comprising administering an agonist of intestinal guanylate cyclase (GC-C) receptor either orally, via rectal suppository, or parenterally.

[0146] In yet another embodiment, the invention features a method for treating a gastrointestinal disorder, comprising administering an agonist of the intestinal guanylate cyclase (GC-C) receptor.

[0147] In another embodiment, the invention features a method for increasing guanylate cyclase C (GC-C) receptor activity in a biological sample, tissue (e.g., intestinal mucosa), or cell (e.g., a cell bearing a GC-A receptor), or in a whole organism.

[0148] In another aspect, the invention features a method of increasing levels of cyclic guanosine 3'-monophosphate (cGMP) in a biological sample, tissue (e.g., intestinal mucosa), or cell (e.g., a cell bearing a GC-A receptor), or in a whole organism, by contacting the sample, tissue, or organism with a peptide described herein.

[0149] The peptide GC-C receptor agonists described herein can be administered in combination with other drugs.For example, the peptide can be administered together with an analgesic peptide or compound.The analgesic peptide or compound can be covalently attached to the peptide described herein, or can be a separate drug that is administered together or sequentially with the peptide described herein in combination therapy.The peptides described herein can also be administered in combination with other drugs used to treat GI disorders, including antidepressants, prokinetic or prokinetic agents, antiemetics, antibiotics, proton pump inhibitors, acid blockers (e.g., histamine H2 receptor antagonists), acid pump antagonists, PDE5 inhibitors, GABA-B agonists, bile acid sequestrants, and mucosal protectants.

[0150] In some embodiments, useful analgesics that may be used with the peptides described herein include Ca channel blockers (e.g., ziconotide), 5HT receptor antagonists (e.g., 5HT3, 5HT4, and 5HT1 receptor antagonists), 5HT4 agonists (e.g., tegaserod [Zelnorm®], mosapride, zacopride, cisapride, renzapride, prucalopride [Resolor®], BIMU1 and BIMU2). benzimidazolone derivatives such as 8, and lirexapride), 5HT1 agonists (e.g., sumatriptan and buspirone), opioid receptor agonists (e.g., loperamide, fedotozine, enkephalin pentapeptides, morphine, diphenyloxylate, furafamide, trimebutine, and fentanyl), CCK receptor agonists (e.g., loxiglumide and dexloxiglumide), NK1 receptor antagonists (e.g., aprepitant, vofopitant, ezlopitant, R-673 (Hoffmann-La Roche Ltd), SR-48968 and SR-14033 (Sanofi Synthelabo), CP-122,721 (Pfizer, Inc.), GW679769 (Glaxo Smith Kline), and TAK-637 (Takeda / Abbot)), NK2 receptor antagonists (e.g., nepadutant, saredutant, GW597599 (Glaxo Smith Kline), SR-144190 (Sanofi-Synthelabo), and UK-290795 (Pfizer Inc)), NK3 receptor antagonists (e.g., osanetant (SR-142801; Sanofi-Synthelabo), SR-241586, and talnetant), norepinephrine-serotonin reuptake inhibitors (NSRIs) (e.g., milnacipran), mixed and selective dopamine receptor antagonists (e.g., metoclopramide, itopride, domperidone), vanilloid and cannabinoid receptor agonists, sialorphin, and sialorphin-related peptides. Various classes of analgesics have been described in the literature.

[0151] In some embodiments, one or more other therapeutic agents may be used in combination with the peptides described herein.Such agents include antidepressants, prokinetic or functional modifiers, antiemetics, antibiotics, proton pump inhibitors, acid blockers (e.g., histamine H2 receptor antagonists), acid pump antagonists, PDE5 inhibitors, GABA-B agonists, bile acid sequestrants, and mucosal protectants.

[0152] Examples of antidepressants include, but are not limited to, tricyclic antidepressants such as amitriptyline (Elavil®), desipramine (Norpramin®), imipramine (Tofranil®), amoxapine (Asendin®), nortriptyline, and the like; selective serotonin reuptake inhibitors (SSRIs) such as paroxetine (Paxil®), fluoxetine (Prozac®), sertraline (Zoloft®), and citralopram (Celexa®), and the like; and other antidepressants such as doxepin (Sinequan®) and trazodone (Desyrel®).

[0153] Examples of prokinetic and prokinetic agents include, but are not limited to, itopride, octreotide, bethanechol, metoclopramide (Reglan®), domperidone (Motilium®), erythromycin (and its derivatives), and cisapride (Propulsid®). Examples of antiemetic agents include, but are not limited to, prochlorperazine.

[0154] Examples of antibiotics that can be used include those that can be used to treat H. pylori infections, such as amoxicillin, tetracycline, metronidazole, or clarithromycin. Other antibiotics, such as erythromycin and its derivatives, can also be used in combination with the peptides described herein.

[0155] Examples of proton pump inhibitors include, but are not limited to, omeprazole (Prilosec®), esomeprazole (Nexium®), lansoprazole (Prevacid®), pantoprazole (Protonix®), and rabeprazole (Aciphex®). Examples of H2 receptor blockers include, but are not limited to, cimetidine, ranitidine, famotidine, and nizatidine. Examples of acid pump antagonists include, but are not limited to, revaprazan, CS-526 (J. Pharmacol. Exp. Ther. (2007) 323:308-317), PF-03716556 (J. Pharmacol. Exp. Ther. (2009) 328(2):671-9), and YH1885 (Drug Metab. Dispos. (2001) 29(1):54-9).

[0156] Examples of PDE5 inhibitors include, but are not limited to, avanafil, lodenafil, mirodenafil, sildenafil citrate, tadalafil, vardenafil, and udenafil. Examples of GABA-B agonists include, but are not limited to, baclofen and XP19986 (CAS Registry Number 847353-30-4). Examples of bile acid sequestrants include, but are not limited to, GT102-279, cholestyramine, colesevelam, colesevelam hydrochloride, ursodeoxycholic acid, colestipol, colestilan, sevelamer, polydiallylamine cross-linked with epichlorohydrin, dialkylaminoalkyl derivatives of cross-linked dextran, and N-(cycloalkyl)alkylamine. Examples of mucosal protectants include, but are not limited to, sucralfate, teprenone, polaprezinc, cetraxate, and bismuth subsalicylate.

[0157] Combination therapy can be achieved by administering two or more drugs, each formulated and administered separately, for example, a peptide described herein and another therapeutic peptide or compound, or by administering two or more drugs as a single formulation. Other combinations are also included in combination therapy. For example, two drugs can be formulated together and administered together with another formulation containing a third drug. In combination therapy, two or more drugs can be administered simultaneously, but this does not have to be the case. For example, the administration of a first drug (or drug combination) can precede the administration of a second drug (or drug combination) by minutes, hours, days, or weeks. Thus, two or more drugs can be administered within minutes of each other, or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days of each other, or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of each other. In some cases, even longer intervals are possible. While it is often desirable for the two or more agents used in combination therapy to be present in the patient's body at the same time, this need not be the case.

[0158] Dosage The dosage range for adult humans may generally be 5 μg to 100 mg of the peptides described herein administered orally per day. Tablets, capsules, or other presentation forms provided in individual units may conveniently contain units containing an amount of the compounds described herein effective at such dosages or as a multiple of such dosages, for example, 25 μg to 2 mg, or about 100 μg to 1 mg. The exact amount of compound prescribed to a patient is the responsibility of the attending physician. However, the dosage used will depend on numerous factors, including the patient's age and sex, the specific disease being treated, and its severity.

[0159] In other embodiments, the dose is 50 μg, 67.5 μg, 100 μg, 133 μg, 145 μg, 150 μg, 200 μg, 266 μg, 290 μg, 300 μg, 400 μg, 500 μg, or 600 μg orally per day.

[0160] In various embodiments, the dosage unit is administered with food at any time of day, without food at any time of day, with food after an overnight fast (e.g., with breakfast), or at bedtime after a low-fat snack.

[0161] In one particular embodiment, the dosage unit is administered before or after food intake (eg, a meal).

[0162] In a further embodiment, the dosage unit is administered about 15 minutes to 1 hour before food intake.

[0163] In various embodiments, the dosage unit is administered once daily, twice daily, three times daily, four times daily, five times daily, or six times daily. In certain embodiments, the dosage unit and the daily dose are equal.

[0164] The precise amount of each of the two or more active ingredients in a dosage unit will depend on the desired dosage of each component. As such, it may be useful to create a dosage unit that, when administered according to a specific dosing regimen (e.g., a regimen specifying a particular number of units and a specific timing for administration), delivers the same dosage of each component as would be delivered if the patient were treated with only a single component.

[0165] In other situations, it may be desirable to create a dosage unit that delivers a smaller dose of one or more components than would be administered if the patient were treated with only a single component.

[0166] Finally, it may be desirable to create a dosage unit that delivers a larger dose of one or more of the components than would be administered if the patient were treated with only a single component.

[0167] Pharmaceutical compositions can contain additional components, including but not limited to excipients as described herein.In certain embodiments, one or more therapeutic agents of a dosage unit can be in a sustained release formulation or a controlled release formulation, and additional therapeutic agents may not be in a sustained release formulation.For example, the peptide or agonist described herein can be in a sustained release formulation or a controlled release formulation in the same dosage unit as another drug that may or may not be in a controlled release formulation or a sustained release formulation.Therefore, in certain embodiments, it may be desirable to provide immediate release of one or more of the drugs described herein and controlled release of one or more other drugs.

[0168] The present invention has been described with reference to specific exemplary embodiments thereof. However, those skilled in the art will readily appreciate that it is possible to embody the present invention in specific forms other than those of the exemplary embodiments described above. This can be done without departing from the spirit of the present invention. The exemplary embodiments are illustrative only and should not be construed as limiting in any way. The scope of the present invention is defined not by the foregoing description, but rather by the appended claims and their equivalents. [Example]

[0169] The GC-C agonist peptides or pharmaceutically acceptable salts thereof as described herein were prepared by American Peptide Company (Sunnyvale, CA) by solid-phase chemical synthesis and native folding (air oxidation). In some cases, the peptides were modified after synthesis as described herein. Cys 1For example, the -α-ketone peptide was synthesized by adding 1.5 L of methanol / dimethylformamide (9:1 v / v) and 28.8 g of 3,5-di-tert-butyl-1,2-benzoquinone (10 equivalents) to 20 g (13.1 mmol) of linaclotide and stirring at room temperature for 1 hour. Schiff base formation was monitored by HPLC. After all the linaclotide was consumed, 17 L of 0.1 M HCl was added, and the reaction mixture was stirred for 2 days. The reaction mixture was filtered, extracted twice with dichloromethane, and the resulting aqueous solution was applied to a preparative-scale reverse-phase C-18 HPLC column to isolate Cys. 1 The HPLC column used for preparative HPLC was a 2-inch diameter C column equilibrated with mobile phase A (0.05% acetic acid in water). 18 The column was washed with mobile phase A at a flux of 100 mL / min to remove unbound material, and peptide-related material was eluted with a linear gradient of mobile phase B (acetonitrile) from 10% to 40% over 60 min. Fractions containing Cys 1-ketone were pooled, followed by removal of the solvent by lyophilization.

[0170] Example 1: cGMP accumulation in T84 cells for analysis of GC-C activity Human colon cancer cell line T84 cells were obtained from ATCC (P / N CCL-248) and cultured in T-150 flasks to a confluency of 60-70%. The monolayer was detached using trypsin and plated onto a 96-well tissue culture plate (Costar, P / N 3596) at 2.0 × 10 5 Cells were seeded at a cell density of 100 cells / well and grown overnight in a 5% carbon dioxide environment using 2 mM Dulbecco's modified Eagle's medium (DMEM) / nutrient mixture F-12 (50 / 50) supplemented with 5% fetal bovine serum (FBS) and L-glutamine (Mediatech, P / N 10-092-CV, 35-0150CV, and 25-005-Cl, respectively).

[0171] After overnight incubation, 2.0 × 10 5 96-well plates seeded with cells / well were cultured in 0.2 mL of DMEM (Mediatech, P / N The cells were washed twice with 10-013-CV (Figure 1). To inhibit any phosphodiesterase activity, the cells were incubated with 0.180 mL of 1 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma P / N I5879) in DMEM for 10 min at 37 °C. A standard curve ranging from 0.1 to 10,000 nM (final concentration) was prepared for each test substance using a Hamilton Microlab Robot (Model STARlet). GC-C activity assays were performed by incubating 0.02 mL of each standard substance with 0.180 mL of 1 mM IBMX in DMEM for 30 min at 37 °C in a 96-well plate. After incubation, the supernatant was removed, and the cells were lysed with 0.1 M cold hydrochloric acid (HCl) for 30 min on ice. A volume of 175 μL per well of each lysate was transferred to a new 96-well plate (Waters, P / N 186002481) and centrifuged at 1,000 × g for 10 minutes to remove any cellular debris. The resulting supernatant was transferred in 90 μL aliquots to a new 96-well plate and neutralized to pH 7 with 90 μL of 1 M ammonium acetate. The centrifuged and neutralized T84 cell lysates were analyzed using liquid chromatography with tandem mass spectrometry detection (LC / MS / MS). The concentration of guanosine 3',5'-cyclic monophosphate (cGMP) in each cell lysate sample was quantified using the method outlined in Table 1. A calibration curve in 0.1 M HCl was prepared using cyclic GMP (P / N G6129) purchased from Sigma. Each standard was neutralized with an equal volume of ammonium acetate to generate a cGMP calibration curve ranging from 1 to 1,024 nM (final concentration).

[0172] The cyclic GMP concentration of each sample was measured using LC / MS conditions (Table 1 below), and a standard curve was calculated. Analyte peak areas were used to generate a linear calibration curve weighted by 1 / x2, which was used to estimate the cGMP concentration in each sample. GraphPad Prism Version 5.01 (GraphPad Software, San Diego, CA) was used to calculate the 50% effective concentration (EC ) for each test substance. 50) values were generated. To determine whether differences in EC50 values were statistically significant, the average activity curves of each formulation degradation product were compared to the linaclotide control using an F-test in GraphPad software. For these comparisons, p-values were determined, with values of 0.05 or greater indicating significant differences in the activity of the GC-C agonists. [Table 1]

[0173] Example 2: Relative binding affinities of exemplary peptides to the GC-C receptor in T84 cells Linaclotide and Cys(III)-binding peptides were identified for the guanylate cyclase-C receptor (GC-C) using a competitive binding assay in which the peptides compete with porcine heat-stable enterotoxin (pSTa), a known GC-C agonist, for binding sites on the cell surface GC-C receptor on human colonic epithelial cells (T84). 1 The relative binding affinity of the α-ketones was measured. 125 The peptides were radiolabeled with I to enable measurement of their receptor binding. Various concentrations of each peptide (0.1–3,000 nM) were added to Dulbecco's modified Eagle's medium (DMEM), 0.5% bovine serum albumin (BSA), 2.0 × 10 5 T84 cells, and 170 pM [ 125 Competitive binding assays were performed by adding 0.20 mL of [I]-pSTa (200,000 cpm) to a reaction mixture containing 3,000 nM of [I]-pSTa. After 60 minutes of incubation at 37°C, the reaction mixture was applied to glass fiber filters by vacuum filtration to isolate receptor-bound material. The concentration of bound radioligand captured on the filters was then determined by scintillation counting. For each peptide, the reaction with the lowest amount of competitor was used to determine maximum specific binding of the radioligand. 3,000 nM of [I]-pSTa was added to a reaction containing each test peptide. 125 The nonspecific binding of [I]-pSTa was measured, and the data were used to construct binding curves for competing radioligands, and IC 50 and K. i Linaclotide and Cys measured by 1The relative binding affinities of the -α-ketones were measured.

[0174] Linaclotide and Cys 1 Both α-ketones bind to the cell surface GC-C receptor on T84 cells. 125 The inhibitory constant (K i Their relative binding affinities as measured by ) were as follows: K of linaclotide i = 3.9 ± 1.6 nM, and Cys 1 K for -α-ketones i =5.2±0.9nM (Figure 1).

[0175] Example 3: cGMP response in T84 cells induced by exemplary peptides The activity of guanylate cyclase-C receptor (GC-C) agonists, linaclotide and Cys, in T84 cells 1 -α-Ketones were tested as follows: Approximately 200,000 T84 cells / well were first incubated with 1 mM 3-isobutyl-1-methylxanthine (IBMX) in 0.18 mL of Dulbecco's modified Eagle's medium (DMEM) in each well of a 96-well plate at 37°C for 10 minutes. Each peptide was diluted to final concentrations ranging from 0.1 to 10,000 nM, and 0.02 mL of each dilution was added in duplicate to the 96-well plate containing T84 cells, resulting in a final volume of 0.2 mL per well. The peptide reactions were incubated at 37°C for 30 minutes. After incubation, the supernatant was removed and discarded, and the cells were lysed with 0.1 M cold hydrochloric acid (HCl) on ice for 30 minutes. Cell debris was removed by centrifugation, and the concentration of guanosine 3',5'-cyclic monophosphate (cGMP) in each lysate was measured using liquid chromatography with tandem mass spectrometry. The data were used to construct dose-response curves and determine the 50% effective concentration (EC 50 ) was calculated.

[0176] Linaclotide and Cys 1Linaclotide and Cys-α-ketone exhibited GC-C agonist activity in T84 cells, as measured by an increase in intracellular cGMP (Figure 2). 1 EC of -α-ketones 50 The values were 315±105 nM and 352±55 nM, respectively. 1 Comparison of the dose-response curve of linaclotide with that of α-ketones revealed that EC 50 The values were shown to be not statistically different (p=0.8884).

[0177] Example 4: Determination of Content and Purity of Exemplary Peptides Cys 1 -α-ketone peptides Linaclotide formulations (as described in U.S. Patent No. 2010 / 0048489, incorporated herein by reference) were compressed by incubating 20 g of formulation beads containing 1 mg of linaclotide sprayed onto 224 mg of Avicel beads at 40°C and 75% relative humidity for 2 months. Peptide-related materials were extracted from the beads by gentle agitation with 20 mL of 0.1 N HCl in a vortex mixer for 1 hour at room temperature. The resulting suspension was centrifuged at 1,000 x g for 5 minutes to pellet the beads, and the supernatant containing the extracted peptide was lyophilized. The dried sample was reconstituted in 2.5 mL of 0.1 N HCl, and Cys-a-ketone was isolated and purified by preparative HPLC using the following method: a YMC Pro™ C18 column (dimensions: 3.0 x 150 mm, 3.5 um, 120 Å; Waters Corp., Milford, MA) or equivalent maintained at 40°C. Mobile phase A (MPA) consisted of 98:2 water / acetonitrile containing 0.1% trifluoroacetic acid, and mobile phase B (MPB) consisted of 5:95 water / acetonitrile containing 0.1% trifluoroacetic acid. Peptide elution was achieved with a 12-minute gradient from 82% to 78% MPA and 18% to 22% MPB, followed by a 1-minute ramp to 50% MPA and 50% MPB, a 3-minute hold at 50% MPA and 50% MPB, followed by a 7-minute wash at 82% MPA and 18% MPB. The flow rate was 0.6 mL / min, and detection was achieved by UV at 220 nm.

[0178] Fractions were collected manually and Cys 1 Fractions containing the -α-ketone were pooled and lyophilized. The dried residue was reconstituted in 1.6 mL of water to a final concentration of 0.5 mg / mL and stored frozen at -20°C. Cys purified by analytical HPLC using the method described below. 1 An aliquot of the -α-ketone was tested and determined to be 90.7% pure.

[0179] The content and purity of the peptides of the present invention were determined using an Agilent Series 1100 analyzer using Chemstation Rev A.09.03 software or its equivalent. The peptides were determined by reversed-phase gradient liquid chromatography using an LC system. A YMC Pro™ C18 column (dimensions: 3.0 x 150 mm, 3.5 μm, 120 Å; Waters Corp., Milford, MA) or equivalent was used and maintained at 40°C. Mobile phase A (MPA) consisted of water containing 0.1% trifluoroacetic acid, and mobile phase B (MPB) consisted of 95% acetonitrile: 5% water containing 0.1% trifluoroacetic acid. Peptide elution was achieved with a gradient of 0% MPB for 4 minutes, followed by 10% MPB for 5 minutes, 23% MPB for 34 minutes, 34% MPB for 6 minutes, and 80% MPB for 10 minutes. The column was re-equilibrated by returning to 0% MPB in 1 minute, followed by a 7-minute hold at 100% MPB. The flow rate was 0.6 mL / min, and detection was achieved by UV at 220 nm.

[0180] Linaclotide and Cys by RP-HPLC 1 An example of the analysis of the -α-ketone product is shown in Figure 3

[0181] The content of purified peptides was measured by determining the peptide concentration in the prepared samples against similarly prepared external peptide standards. Other embodiments

[0182] All publications and patents mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If the meaning of a term in any patent or publication incorporated by reference conflicts with the meaning of the term used in this disclosure, the meaning of the term in this disclosure is intended to control. Furthermore, the foregoing discussion discloses and describes only exemplary embodiments of the present invention. Those skilled in the art will readily recognize from such considerations and the accompanying drawings and claims that various modifications and changes can be made without departing from the spirit and scope of the present invention, as defined in the following claims.

Claims

[Claim 1] A peptide or method as described in the specification.

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