Compositions for colon cleansing and treatment of gastrointestinal disorders

Abstract

To provide peptides and compositions useful for treatment of gastrointestinal disorders or for colon cleansing.SOLUTION: The present invention provides compositions and methods of treating gastrointestinal disorders as well as pharmaceutical compositions for accomplishing the same. In some embodiments, these pharmaceutical compositions include oral dosage forms. The present invention features peptides, compositions and related methods for colon cleansing treatment as well as other conditions and disorders described herein. In one embodiment, the peptides may be used to prepare subjects for colonoscopy treatment. In some embodiments, the peptides or pharmaceutically acceptable salts may be used to prepare subjects for surgery, such as bowel surgery.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to peptides, compositions, and methods for colon cleansing and treatment of disorders of the gastrointestinal tract and other internal organs.

[0002] Priority claims This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 156,077, filed May 1, 2015, the entire contents of which are hereby incorporated by reference.

[0003] Sequence Listing This application incorporates by reference in its entirety the Sequence Listing entitled "IW154PCT1Sequence_ST25.txt" (45.1 kilobytes), created on April 28, 2016, and submitted electronically herewith. [Background technology]

[0004] background Approximately 15 million colonoscopies are performed in the United States each year, all of which require proper colon preparation. Inadequate bowel preparation has been reported in approximately 20% of colonoscopies. Inadequate bowel preparation can result in lower adenoma detection, longer procedure times, and shorter intervals between exams. As a result, there is a need for safe, effective, and well-tolerated colon preparations that allow a broad population of patients to reliably and effectively undergo colonoscopies and reduce their risk of colon cancer. Summary of the Invention [Means for solving the problem]

[0005] overview The present invention features peptides, compositions, and related methods for colon cleansing therapy, as well as other conditions and disorders described herein. In one embodiment, the peptides may be used to prepare a subject for colonoscopy therapy. In some embodiments, the peptides or pharmaceutically acceptable salts may be used to prepare a subject for surgery, such as bowel surgery. In other embodiments, the peptides may be used to treat colon cancer, hereditary nonpolyposis colorectal cancer (HNPCC), i.e., Lynch syndrome, gastroparesis (GP), polyps, pain, generalized abdominal pain, postoperative ileus, opioid-induced constipation, functional dyspepsia, diverticular disease, including but not limited to SUDD (symptomatic simple diverticular disease) and SCAD (segmental colitis associated with diverticulosis), diverticulosis, diarrhea-predominant irritable bowel syndrome, and irritable bowel syndrome (IBS). Pain, ulcerative colitis, ulcerative proctitis, Crohn's disease, inflammatory bowel disease (IBD), chronic or acute radiation proctopathy, rectal pain, chronic rectal neuralgia, transient rectal neuralgia, anal pain, chronic anal fissure, postoperative anal pain, overactive bladder syndrome, stress urinary incontinence, interstitial cystitis, bladder pain syndrome, colorectal cancer, cancer-related pain, diffuse pelvic pain, endometriosis, testicular pain, chronic prostatitis, prostatodynia, urethral syndrome, penile pain, perianal pain, and other gastrointestinal and visceral disorders may be treated.

[0006] One aspect of the present invention provides a peptide or a pharmaceutically acceptable salt thereof, said peptide having the amino acid sequence: [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK, Asn or absent; Xaa3 is Asn, Ser, or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile, BE, or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe), BK, or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 11 is Cys, Ag, or penicillamine (Pen); Xaa 12 is Cys, allylglycine (Ag), Hag, Cth, Dpr, or Val; Xaa 13 is Asn or Leu; Xaa 14 is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 16 is Cys, Ag, Pen, or Cth; Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, Ser, or Ala; Xaa 19 is Cys, Ag, or Pen; Xaa 20 is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, Dpr, or OH-Pro; and The peptide is located at Xaa7 and Xaa 12 , Xaa8 and Xaa 16 , and Xaa 11 and Xaa 19 Contains a covalent bond between

[0007] A second aspect of the present invention provides a pharmaceutical composition comprising a peptide of the present invention.

[0008] A third aspect of the present invention provides a method for treating gastrointestinal disorders, comprising administering a pharmaceutical composition according to the present invention. The pharmaceutical composition according to the present invention may be used as a preparation for colonoscopy or for treating gastrointestinal disorders and pain. In some embodiments, the composition is a solid oral composition.

[0009] The details of one or more embodiments of the invention are set forth in the accompanying description. [Brief explanation of the drawings]

[0010] [Figure 1] Activity and stability results for representative peptides are discussed. [Figure 2] 1 illustrates the results of cGMP accumulation in a T84 cell assay for the analysis of GC-C activity for SEQ ID NOs: 3-7. [Figure 3] 1 illustrates the results of cGMP accumulation in a T84 cell assay for the analysis of GC-C activity for SEQ ID NOs: 8-10. [Figure 4] 1 presents the results of cGMP accumulation in a T84 cell assay for analysis of GC-C activity for SEQ ID NOs: 12-13 and 23-27. [Figure 5] 1 illustrates the results of cGMP accumulation in a T84 cell assay for the analysis of GC-C activity for SEQ ID NOs: 28-34. [Figure 6] 1 illustrates the results of cGMP accumulation in a T84 cell assay for the analysis of GC-C activity for SEQ ID NOs: 35-37, 39, and 44. [Figure 7] 1 illustrates the results of cGMP accumulation in a T84 cell assay for the analysis of GC-C activity for SEQ ID NOs: 2 and 40-42. [Figure 8] 1 illustrates the results of a cGMP accumulation in T84 cell assay for analysis of GC-C activity for SEQ ID NO:43. [Figure 9] 1 illustrates the results of cGMP accumulation in a T84 cell assay for the analysis of GC-C activity for SEQ ID NOs: 44-47. [Figure 10] 1 illustrates the results of a cGMP accumulation in T84 cell assay for analysis of GC-C activity for SEQ ID NOs: 45, 48, and 51-52. [Figure 11] 1 illustrates the results of a cGMP accumulation in T84 cell assay for analysis of GC-C activity for SEQ ID NOs: 12, 13, 27, 47, and 53. [Figure 12] The results of the cGMP accumulation in T84 cell assay for analysis of GC-C activity for SEQ ID NO:47 are described, along with the original study, a repeat of the original study, and a second assay. [Figure 13] 1 illustrates the results of cGMP accumulation in a T84 cell assay for the analysis of GC-C activity for SEQ ID NOs: 54-59. [Figure 14] 1 illustrates the results of cGMP accumulation in a T84 cell assay for the analysis of GC-C activity for SEQ ID NOs: 60-64. [Figure 15] 1 illustrates the results of an in vivo rat duodenal loop assay for SEQ ID NOs: 62 and 47. [Figure 16] 1 illustrates the results of a rat duodenal loop capacity study for SEQ ID NOs: 62 and 47. [Figure 17]1 illustrates the results of a mouse gastrointestinal transit (mGIT) assay for SEQ ID NOs: 2 and 47 compared to ST core. [Figure 18] 1 illustrates the results of an in vivo ligated rat loop assay for SEQ ID NOs: 67 and 69. [Figure 19] 1 illustrates the results of an in vivo ligated rat loop capacitance study for SEQ ID NOs: 67 and 69. [Figure 20] 1 presents the results of an in vitro rat intestinal fluid (RIF) assay for SEQ ID NOs: 2, 26, 43, 47, and 53. [Figure 21] 1 presents the results of an in vitro RIF assay for SEQ ID NOs:60-66. [Figure 22] The results of an in vivo RIF assay for SEQ ID NOs: 47, 62, 67, and 69 are presented. [Figure 23] The results of various assays performed on representative peptides are presented. [Figure 24] 1 presents the results of cGMP accumulation in a T84 cell assay for SEQ ID NOs: 14-22. [Figure 25] 1 illustrates the results of a mouse gastrointestinal transit assay for SEQ ID NOs: 2, 7, and 11. [Figure 26] 1 illustrates the results of a mouse gastrointestinal transit assay for SEQ ID NO:47. [Figure 27] 1 illustrates the results of a mouse gastrointestinal transit assay for SEQ ID NOs: 62, 67, and 69. DETAILED DESCRIPTION OF THE INVENTION

[0011] These figures are provided as examples and are not intended to limit the scope of the invention.

[0012] Detailed Description Guanylate cyclase C (GC-C) is a transmembrane receptor located on the apical surface of epithelial cells in the stomach and intestine. The receptor possesses 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 leading to increased chloride and bicarbonate secretion into the intestinal lumen, increased luminal pH, decreased luminal sodium absorption, increased fluid secretion, and accelerated intestinal transit. cGMP secreted bidirectionally from the epithelium to the mucosa and lumen has also been shown to attenuate the firing of afferent C fibers, suggesting a potential mechanism for the observed analgesic effects of GC-C agonists on visceral pain.

[0013] Linaclotide, a peptide GC-C agonist orally administered and currently approved in the United States for the treatment of irritable bowel syndrome with constipation (IBS-c) and chronic idiopathic constipation (CIC), has numerous effects on lower GI physiology, including (1) reducing visceral pain, (2) reducing distension, and (3) increasing GI transit, which can lead to increased bowel movement frequency and improved stool consistency. Orally administered linaclotide acts locally by activating GC-C receptors on the luminal surface. Thus, results from clinical trials of linaclotide and preclinical studies conducted with linaclotide and related peptides suggest that GC-C peptide agonists may be used therapeutically. The peptides described and claimed herein can bind to and activate GC-C receptors, or can be characterized as GC-C peptide agonists.

[0014] definition As used herein, C12 is a C12 alkyl carboxylic acid, C14 is a C14 alkyl carboxylic acid, C16 is a C16 alkyl carboxylic acid, C18 is a C18 alkyl carboxylic acid, (4-F)Phe is 4-fluorophenylalanine, Cth is cystathionine, Ag is allylglycine, Hag is allylglycine with a reduced dicarba bond, Pent is pentenoic acid, Pen is penicillamine, Cha is cyclohexylalanine, Sar is sarcosine, OH-Pro is hydroxyproline, Nme-Tyr is N-methyltyrosine, 4-Mepip is 1-methyl-piperidine-4-carboxylic acid, Dpr is di-aminopropionic acid, BE is glutamic acid whose side chain carboxylic acid forms a peptide linkage, and BK is lysine whose side chain amine forms a peptide linkage. An example of a BE BK binding pattern is: [ka] It would be.

[0015] Further examples of peptide bonds include, without limitation: I. Dicarba [ka] II. Hag-Hag [ka] III.Cth-C [ka] IV.C-Cth [ka] VD-Dpr (lactam bond) [ka] VI. [ka] [ka] others [ka] VII. Ag-Ag(2,-10) [ka] VIII. Hag-Hag (2, -10) [ka] IX.Cth-C(2,-10) [ka] XC-Cth(2,-10) [ka] XI.(2,-10) [ka] [ka] others [ka] XII. Ag-Ag(5,-13) [ka] XIII. Hag-Hag (5, -13) [ka] XIV.Cth-C(5,-13) [ka] XV.C-Cth(5,-13) [ka] XVI.(5,-13) [ka] [ka] others [ka] Includes:

[0016] peptide In one aspect, the present invention provides a peptide or a pharmaceutically acceptable salt thereof useful in the methods described herein, said peptide having the amino acid sequence: [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK, Asn or absent; Xaa3 is Asn, Ser, or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile, BE, or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe), BK, or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 11 is Cys, Ag, or penicillamine (Pen); Xaa 12 is Cys, allylglycine (Ag), Hag, Cth, Dpr, or Val; Xaa 13 is Asn or Leu; Xaa 14 is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 16 is Cys, Ag, Pen, or Cth; Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, Ser, or Ala; Xaa19 is Cys, Ag, or Pen; Xaa 20 is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, Dpr, or OH-Pro; and The peptide is located at Xaa7 and Xaa 12 , Xaa8 and Xaa 16 , and Xaa 11 and Xaa 19 Contains a covalent bond between

[0017] In some embodiments, the N-terminus of the peptide is acetylated or modified at the N-terminus to provide additional stability to the peptide. In other embodiments, the N-terminus is capped with pentenoic acid, biotin, 4-Mepip (1-methyl-4-carboxylic acid), a C12 alkyl carboxylic acid, a C14 alkyl carboxylic acid, a C16 alkyl carboxylic acid, or a C18 alkyl carboxylic acid. In some embodiments, the C=C double bond of the pentenoic acid can be cyclized with another C=C double bond to form a dicarba bond.

[0018] In some embodiments, the C-terminus of the peptide is amidated or modified at the C-terminus to provide additional stability to the peptide.

[0019] In yet another embodiment, the N-terminus of the peptide is acetylated or capped and the C-terminus is amidated.

[0020] In some embodiments, dicarba bonds (CH2-CH=CH-CH2) or other covalent bonds described herein between peptide residues can be useful in stabilizing peptides. Dicarba bonds and other covalent bonds described herein can, in some cases, provide greater stability to peptides than disulfide bonds. In some embodiments, dicarba bonds can be reduced (CH2-CH2-CH2-CH2).

[0021] In some embodiments, the enhanced stability of the peptide allows for storage at room temperature for extended periods of time.

[0022] In some embodiments, Xaa7 and Xaa 12 are both Ag, and the dicarba bonds are Ag7 and Ag 12 Located between Xaa7 and Xaa 12 are both Cys and the disulfide bond is between Cys7 and Cys 12 Located between Xaa8 and Xaa 16 are both Cys and the disulfide bond is between Cys8 and Cys 16 Presents between Xaa 11 and Xaa 19 are both Cys and the disulfide bond is Cys 11 and Cys 19 or any combination thereof.

[0023] In other embodiments, Xaa7 is Cth and Xaa 12 is Cys, and the bond is Cth7 and Cys 12 Xaa8 is Cth and Xaa 16 is Cys and the bond is Cth8 and Cys 16 or any combination thereof.

[0024] In some embodiments, Xaa7 is allylglycine or Cys.

[0025] In some embodiments, Xaa8 is Cys or cystathionine.

[0026] In some embodiments, Xaa9 is Glu.

[0027] In some embodiments, Xaa 10 is Leu.

[0028] In some embodiments, Xaa 12 is Cys or allylglycine.

[0029] In some embodiments, Xaa 14 is Val or Pro.

[0030] In some embodiments, Xaa 17 is Tyr or Thr.

[0031] In some embodiments, Xaa 20 is Tyr or absent.

[0032] In some embodiments, Xaa 21 is lacking.

[0033] In some embodiments, Xaa1 is absent; Xaa2 is absent; Xaa3 is absent; Xaa4 is absent; Xaa5 is absent; Xaa6 is absent; Xaa7 is Ag, Cys, or Cth; Xaa8 is Cys or Cth; Xaa9 is Glu; 10 is Leu;Xaa 12 is Ag or Cys; Xaa 14 is Val or Pro; Xaa 17 is Tyr or Thr; and Xaa 20 is Tyr or absent.

[0034] In some embodiments, Xaa 14 is not Pro.

[0035] In some embodiments, Xaa 17 is not Phe.

[0036] In some embodiments, a peptide or a pharmaceutically acceptable salt thereof is provided, wherein the peptide has the amino acid sequence: [ka] [ka] [ka] [ka] wherein Ac- indicates an acetylated N-terminus, Pent- indicates an N-terminus capped with pentenoic acid, biotin- indicates an N-terminus capped with biotin, 4-Mepip indicates an N-terminus capped with 4-Mepip (1-methyl-piperidine-4-carboxylic acid), C12- indicates an N-terminus capped with a C12 alkyl carboxylic acid, C14- indicates an N-terminus capped with a C14 alkyl carboxylic acid, C16- indicates an N-terminus capped with a C16 alkyl carboxylic acid, C18- indicates an N-terminus capped with a C18 alkyl carboxylic acid, H- indicates an unmodified N-terminus, -NH2 indicates an amidated C-terminus, and -COOH indicates an unmodified C-terminus.

[0037] In further embodiments, the dicarba bond between two Ag residues can be either a cis or trans isomer at the dicarba bond. As used herein, a cis isomer at the dicarba bond has both hydrogen atoms on the same side of the C=C double bond, and a trans isomer at the dicarba bond has hydrogen atoms on opposite sides of the C=C double bond.

[0038] In some embodiments, a peptide or a pharmaceutically acceptable salt thereof is provided, wherein the peptide comprises no more than 50, 40, 30, or 20 amino acids. In further embodiments, the peptide comprises no more than 19, 18, 17, 16, 15, or 14 amino acids.

[0039] In another aspect, the present invention provides a peptide or a pharmaceutically acceptable salt thereof, said peptide having the amino acid sequence [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK, Asn or absent; Xaa3 is Asn, Ser, or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile, BE, or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe), BK, or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 11 is Cys, Ag, or penicillamine (Pen); Xaa 12is Cys, allylglycine (Ag), Hag, Cth, Dpr, or Val; Xaa 13 is Asn or Leu; Xaa 14 is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 16 is Cys, Ag, Pen, or Cth; Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, Ser, or Ala; Xaa 19 is Cys, Ag, or Pen; Xaa 20 is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, Dpr, or OH-Pro; and The peptide is located at Xaa7 and Xaa 12 , Xaa8 and Xaa 16 , and Xaa 11 and Xaa 19 Contains a covalent bond between

[0040] In some embodiments, the N-terminus of the peptide is acetylated to provide additional stability to the peptide. In other embodiments, the N-terminus is capped with pentenoic acid, biotin, 4-Mepip (1-methyl-piperidine-4-carboxylic acid), a C12 alkyl carboxylic acid, a C14 alkyl carboxylic acid, a C16 alkyl carboxylic acid, or a C18 alkyl carboxylic acid. In some embodiments, the C=C double bond of the pentenoic acid can be cyclized with another C=C double bond to form a dicarba bond.

[0041] In some embodiments, the C-terminus of the peptide is amidated to provide additional stability to the peptide.

[0042] In some embodiments, Xaa7 and Xaa 12 are both Ag, and the dicarba bonds are Ag7 and Ag 12 Located between Xaa7 and Xaa 12 are both Cys and the disulfide bond is between Cys7 and Cys 12 Located between Xaa8 and Xaa 16 are both Cys and the disulfide bond is between Cys8 and Cys 16 Presents between Xaa 11 and Xaa 19 are both Cys and the disulfide bond is Cys 11 and Cys 19 or any combination thereof.

[0043] In some embodiments, Xaa7 is allylglycine or Cys.

[0044] In some embodiments, Xaa8 is Cys or cystathionine.

[0045] In some embodiments, Xaa9 is Glu.

[0046] In some embodiments, Xaa 10 is Leu.

[0047] In some embodiments, Xaa 12 is Cys or allylglycine.

[0048] In some embodiments, Xaa 14 is Val or Pro.

[0049] In some embodiments, Xaa 17 is Tyr or Thr.

[0050] In some embodiments, Xaa 20 is Tyr or absent.

[0051] In some embodiments, Xaa 21 is lacking.

[0052] In some embodiments, Xaa1 is absent; Xaa2 is absent; Xaa3 is absent; Xaa4 is absent; Xaa5 is absent; Xaa6 is absent; Xaa7 is Ag, Cys, or Cth; Xaa8 is Cys or Cth; Xaa9 is Glu; 10 is Leu;Xaa 12 is Ag or Cys; Xaa 14 is Val or Pro; Xaa 17 is Tyr or Thr; and Xaa 20 is Tyr or absent.

[0053] In some embodiments, a peptide or a pharmaceutically acceptable salt thereof is provided, wherein the peptide has the amino acid sequence: [ka] [ka] [ka] It consists of:

[0054] In another embodiment, the present invention provides a peptide or a pharmaceutically acceptable salt thereof useful in the methods described herein, said peptide having the amino acid sequence: [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK or absent; Xaa3 is Asn or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe) or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 12 is Cys, allylglycine (Ag), Hag, or Val; Xaa 14 is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, or Ala; Xaa 20 is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, or OH-Pro; and The peptide is located at Xaa7 and Xaa 12 , Xaa8 and Cys 16 , and Cys 11 and Cys 19 Contains a covalent bond between

[0055] In a further embodiment, the peptide or pharmaceutically acceptable salt thereof, said peptide has the amino acid sequence: [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK or absent; Xaa3 is Asn or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe) or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 12 is Cys, allylglycine (Ag), Hag, or Val; Xaa 14is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, or Ala; Xaa 20 is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, or OH-Pro; and wherein Xaa7 and Xaa 12 are both Ag, and the dicarba bonds are Ag7 and Ag 12 exists between; Xaa7 and Xaa 12 are both Cys and the disulfide bond is between Cys7 and Cys 12 exists between; Xaa8 is Cys and the disulfide bond is between Cys8 and Cys 16 exists between; Xaa7 is Cth and the bond is Cth7 and Cys 12 exists between; Xaa8 is Cth and the bond is Cth8 and Cys 16 exists between; Disulfide bond is Cys 11 and Cys 19 exists between; or Any combination of them.

[0056] In some embodiments, the peptide is isolated. In other embodiments, the peptide is purified.

[0057] In some embodiments, pharmaceutically acceptable salts of the peptides are provided. In some cases, the pharmaceutically acceptable salt is a chloride, acetate, phosphate, or sulfate salt.

[0058] In some embodiments, the N-terminus of the peptides described herein is acetylated or capped. This modification can provide the peptide with enhanced stability. In other embodiments, the N-terminus of the peptide is modified with an imidazolidinone derivative as a cysteine ​​residue.

[0059] The peptides disclosed herein can also be used for detection or colon cancer treatment.When using the peptide in detection situations, a linker is conjugated to the N-terminus.The linker can then be conjugated to a dye, or can be conjugated to a dye before binding to the peptide.Those skilled in the art will recognize that the dye conjugated to the peptide will be useful in detecting peptide-binding interactions.

[0060] Another embodiment includes conjugating the peptides disclosed herein to toxins via a linker. The toxins conjugated and coated with the peptides or pharmaceutically acceptable salts described herein will be useful as colon cancer treatments. Such formulations will provide systemic circulation for long-acting treatment. Those skilled in the art will recognize the use of the long-acting peptides or pharmaceutically acceptable salts described herein in the treatment of colon cancer.

[0061] Peptide production In one embodiment, the peptides or precursor peptides of the present invention can be recombinantly produced in any known protein expression system, including, but not limited to, bacteria (e.g., E. coli or Bacillus subtilis), insect cell systems (e.g., Drosophila Sf9 cell systems), yeast cell systems (e.g., S. cerevisiae, S. saccharomyces), or filamentous fungal expression systems, or animal cell expression systems (e.g., mammalian cell expression systems). When the peptide or variant peptide is to be produced recombinantly, for example, 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 heat-stable enterotoxin (ST) peptide. The secreted mature peptide can be purified from the culture medium.

[0062] The sequences encoding the peptides described herein can be inserted into vectors capable of delivering and maintaining the nucleic acid molecule in bacterial cells. The DNA molecule can be inserted into an autonomously replicating vector (suitable vectors include, for example, pGEM3Z and pcDNA3 and their derivatives). The vector nucleic acid can be bacterial or bacteriophage DNA, such as bacteriophage λ or M13 and their 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, E. coli, B. subtilis, Pseudomonas, and Salmonella. In addition to the coding nucleic acid molecule, the genetic construct also includes elements enabling expression, such as promoters and regulatory sequences. Expression vectors can contain transcriptional control sequences that control transcription initiation, such as promoter, enhancer, operator, and repressor sequences. A variety of transcriptional control sequences are well known to those skilled in the art. An expression vector may also include 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 it may integrate into host DNA to ensure stability during peptide production.

[0063] Protein coding sequences containing the peptides described herein can also be fused to nucleic acids encoding peptide affinity tags to facilitate purification, such as glutathione S-transferase (GST), maltose E-binding protein, protein A, FLAG tags, hexa-histidine, myc tags, or influenza HA tags. Fusion of the affinity tag or reporter links the reading frame of the peptide of interest with the reading frame of the gene encoding the affinity tag, thereby generating a translational fusion. Expression of the fused 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 frames for the affinity tag and the peptide of interest.

[0064] Genetic constructs and methods suitable for producing immature and mature forms of the peptides and variants described herein in protein expression systems other than bacteria and well known to those skilled in the art may also be used to produce the peptides in biological systems.

[0065] In some embodiments, peptides can be produced chemically. Peptides can be synthesized by several different methods, including solution-phase and solid-phase synthesis using traditional BOC or FMOC protection. For example, peptides can be synthesized on 2-chlorotrityl chloride or Wang resin using sequential amino acid coupling. A variety of protecting groups can be used, including, but not limited to, the following: 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); trityl (β-amid functionality of asparagine side chain and phenolic group of tyrosine, if present); trityl or tert-butyldimethylsilyl (hydroxyl group of serine, if present), and tert-butyloxycarbonyl (N-terminus before subsequent side chain modification). Coupling, deprotection, and cleavage can be achieved by various methods. In some embodiments, coupling is 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% trifluoroacetic acid, 5% phenol, 5% thioanisole, 2.5% 1,2-ethanediol, 3% water, 2% dimethyl sulfide, 1.5% ammonium iodide w / w). The peptide can be isolated by various methods. In one embodiment, after removal of trifluoroacetic acid and other volatile substances, the peptide can be precipitated using an organic solvent. Disulfide bonds between Cys residues can be formed using various methods. In one embodiment, 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 various methods, including, but not limited to, reverse-phase chromatography, and lyophilized.

[0066] The peptides may be prepared, isolated, or used in their free base form or as a pharmaceutically acceptable salt thereof. Examples of salts include, but are not limited to, acetate, chloride, sulfate, and phosphate salts of the peptides.

[0067] Compositions of peptides and GC-C receptor agonists In another aspect, compositions are provided in which peptides, alone or in combination, can be combined with any pharmaceutically acceptable carrier or vehicle. In some embodiments, the peptide or a pharmaceutically acceptable salt thereof can be formulated into a pharmaceutically acceptable composition. In other embodiments, the peptide or a pharmaceutically acceptable salt thereof can be formulated into a non-pharmaceutically acceptable composition. The peptide can be combined with a material that does not produce adverse, allergic, or otherwise undesirable reactions when administered to a patient. The carrier or vehicle used can include solvents, dispersants, coatings, absorption enhancers, controlled-release agents, and one or more inert excipients, including starch, polyols, granulating agents, microcrystalline cellulose (e.g., celphere, Celphere beads®), diluents, lubricants, binders, disintegrants, etc. If desired, tablets of the disclosed compositions can be coated by standard aqueous or nonaqueous techniques.

[0068] Examples of pharmaceutically acceptable carriers and pharmaceutically acceptable inert carriers and excipients for use as the aforementioned additional ingredients include, but are not limited to, binders, fillers, disintegrants, lubricants, antimicrobial agents, and coating agents.

[0069] As used herein, the term "binder" refers to any pharmaceutically acceptable binder that may be used in the practice of the present invention. Examples of pharmaceutically acceptable binders include, but are not limited to, starches (e.g., corn starch, potato starch, and pregelatinized starches (e.g., STARCH 1500® and STARCH 1500 LM® sold by Colorcon, Ltd., as well as other starches), maltodextrin, gelatin; natural and synthetic gums such as acacia, powdered tragacanth, and guar gum; cellulose and its derivatives (e.g., methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose (hypromellose), ethylcellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethylcellulose, carboxymethylcellulose, powdered cellulose, microfine cellulose, microcrystalline cellulose (e.g., FMC Corporation, Marcus AVICEL™, such as AVICEL-PH-101™, -103™, and -105™ sold by Hook, PA, USA, polyvinyl alcohol, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K30), and mixtures thereof.

[0070] Examples of binders that may be particularly useful in pharmaceutical compositions include polyvinyl alcohol, polyvinylpyrrolidone (povidone), starch, maltodextrin, or cellulose ethers (such as methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose).

[0071] As used herein, the term "filler" refers to any pharmaceutically acceptable filler that can be used in the practice of the present invention.Examples of pharmaceutically acceptable fillers include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate (e.g., granules or powder), microcrystalline cellulose (e.g., Avicel PH101 or Celphere CP-305), microfine cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch (e.g., Starch 1500), pregelatinized starch, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, isomalt, raffinose, maltitol, melezitose, stachyose, lactitol, palatinit, xylitol, myoinositol, and mixtures thereof.

[0072] Examples of pharmaceutically acceptable fillers that may be particularly used to coat the peptides include, but are not limited to, talc, microcrystalline cellulose (e.g., Avicel PH101 or Celphere CP-305), powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, isomalt, dicalcium phosphate, raffinose, maltitol, melezitose, stachyose, lactitol, palatinite, xylitol, mannitol, myo-inositol, and mixtures thereof.

[0073] 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, humectants, absorption-promoting additives, controlled-release additives, anti-caking additives, antimicrobial agents (e.g., preservatives), colorants, desiccants, plasticizers, and dyes. As used herein, "excipient" refers to any pharmaceutically acceptable additive, filler, binder, or active substance.

[0074] The compositions of the present invention may optionally contain other therapeutic ingredients, anticaking agents, preservatives, sweeteners, colorants, flavorings, desiccants, plasticizers, pigments, lubricants, antiadhesives, antistatic 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 contain other additives as required, including, for example, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, raffinose, maltitol, melezitose, stachyose, lactitol, palatinit, starch, xylitol, mannitol, myo-inositol, etc., and their hydrates, as well as amino acids such as alanine, glycine, and betaine, and peptides and proteins, such as egg white.

[0075] The compositions may include, for example, various additional solvents, dispersants, coatings, 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, and the like. If desired, tablets of the disclosed compositions may be coated by standard aqueous or nonaqueous techniques. The compositions may also include, for example, anti-caking additives, preservatives, sweetening additives, colorants, flavorings, desiccants, plasticizers, dyes, and the like.

[0076] Suitable disintegrants include, for example, agar-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.

[0077] Suitable lubricating oils 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), coagulated aerosol of synthetic silica (Evonik Degussa Co., Plano, TX USA), calcined silicon dioxide (CAB-O-SIL, Cabot Co., Boston, MA USA), and mixtures thereof.

[0078] Suitable lubricants include, for example, leucine, colloidal silicon dioxide, magnesium trisilicate, powdered cellulose, starch, talc, and tribasic calcium phosphate.

[0079] Suitable anti-caking additives include, for example, calcium silicate, magnesium silicate, silicon dioxide, colloidal silicon dioxide, talc, and mixtures thereof.

[0080] Suitable antimicrobial additives that may be used, for example, 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, thimersol, thymo, and mixtures thereof.

[0081] 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, α-lipoic acid, and β-carotene.

[0082] 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 coatings include Aquacoat (e.g., Aquacoat ethylcellulose aqueous dispersion, 15% w / w, FMC Biopolymer, ECD-30), Eudragit (e.g., Eudragit E PO PE-EL, Roehm Pharma Polymers), and Opadry (e.g., Opadry AMB dispersion, 20% w / w, Colorcon).

[0083] In certain embodiments, suitable additives for peptide compositions include one or more of sucrose, talc, magnesium stearate, crospovidone, or BHA.

[0084] The compositions of the present invention may contain L-histidine, Pluronic®, poloxamers (such as Lutrol® and Poloxamer 188), ascorbic acid, glutathione, permeability enhancers (e.g., lipids, sodium cholate, acylcarnitines, salicylates, mixed bile salts, 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 in promoting bioavailability (see U.S. Pat. No. 6,086,918 and U.S. Pat. No. 6,086,918). 5912014), materials for chewable tablets (such as dextrose, fructose, lactose monohydrate, lactose and aspartame, lactose and cellulose, maltodextrin, maltose, mannitol, microcrystalline cellulose and guar gum, sorbitol crystals); parenterals (such as mannitol and povidone); plasticizers (such as dibutyl sebacate, plasticizers for coatings, polyvinyl acetate phthalate); powdered lubricants (such as glyceryl behenate); soft gelatin capsules (such as sorbitol special solutions); spheres for coatings (such as sugar spheres); spheronizing agents (such as glyceryl behenate and microcrystalline cellulose); suspending / gelling agents (such as carrageenan, gellan gum, mannitol, microcrystalline cellulose sweeteners (such as aspartame, aspartame and lactose, dextrose, fructose, honey, maltodextrin, maltose, mannitol, molasses, sorbitol crystals, sorbitol special solution, sucrose); wet granulating agents (such as calcium carbonate, lactose anhydrous, lactose monohydrate, maltodextrin, mannitol, microcrystalline cellulose, povidone, starch), caramel, sodium carboxymethylcellulose, cherry cream flavor and cherry flavor, anhydrous citric acid, citric acid, powdered sugar, D&C Red No. 33, D&C Yellow #10 Aluminum Lake, edetate disodium, ethyl alcohol 15%, FD&C Yellow No. 6 Aluminum Lake, FD&C Blue #1 Aluminum Lake, FD&C Blue No.It may also contain other excipients, agents, and classes thereof, including, but not limited to, 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, 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, iron oxide red, sodium saccharin, sodium carboxymethyl ether, sodium chloride, sodium citrate, sodium phosphate, strawberry flavor, synthetic iron oxide black, synthetic iron oxide red, titanium dioxide, and white wax.

[0085] In some embodiments, a peptide described herein and Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K. + , Na + , or Al 3+ In a further embodiment, the agent is a pharmaceutical composition comprising one or more stabilizers selected from the group consisting of Mg, Mg(II), ... 2+ , Ca 2+ , or Zn 2+or a combination thereof. In some embodiments, the cation is provided as, without limitation, 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 cation is 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 cation is provided as calcium chloride, magnesium chloride, or zinc acetate.

[0086] In another embodiment, the stabilizer is a sterically hindered primary amine. In a further embodiment, the sterically hindered primary amine is an amino acid. In yet a further embodiment, the amino acid is a naturally occurring amino acid. In yet 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 still further, the naturally occurring amino acid is leucine, isoleucine, alanine, or methionine. In another embodiment, the sterically hindered primary amine is a non-naturally occurring amino acid (e.g., 1-aminocyclohexanecarboxylic acid). In a further embodiment, the sterically hindered primary amine is a polymeric amine such as cyclohexylamine, 2-methylbutylamine, or chitosan. In another embodiment, one or more sterically hindered primary amines may be used in the composition.

[0087] 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, wherein any group may be singly or multiply substituted with halogen or amino, and with the proviso that no more than two of R1, R2, and R3 are H. In another embodiment, no more than one of R1, R2, and R3 is H.

[0088] In another embodiment, a pharmaceutically acceptable carrier, a peptide, Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K. + , Na + , or Al 3+ In one embodiment, the cation is selected from Mg 2+ , Ca 2+ , or Zn 2+ or a mixture thereof. In further embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable binder and / or a pharmaceutically acceptable glidant, lubricant, or an additive that acts as both a glidant and a lubricant, and / or an antioxidant. In some embodiments, the pharmaceutical composition is applied to a carrier. In some embodiments, the carrier is a filler.

[0089] 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 can be equal to or greater than 2:1 (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%).

[0090] Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K. + , Na + , and Al 3+ Cations selected from the group consisting of have been found to be useful in suppressing the formation of oxidation products of GC-C receptor agonist polypeptides during storage. Sterically hindered primary amines have also been found to be useful in suppressing the formation of formaldehyde imine adducts ("formaldehyde imine products") of GC-C receptor agonist polypeptides during storage. Thus, Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K. + , Na + , or Al 3+ Cations selected from, for example, Zn 2+ , Mg 2+ , and Ca 2+GC-C receptor agonist polypeptide formulations containing a divalent cation selected from the group consisting of hydroxyl groups, hydroxypropyl groups, hydroxypropyl groups, and / or sterically hindered primary amines, such as amino acids, have a shelf life (as measured by chromatographic purity and / or by weight / weight assay) sufficient for manufacturing, storing, and distributing the drug. Furthermore, the presence of sterically hindered amines alone may increase the formation of hydrolysis products of the GC-C receptor agonist polypeptide during storage, whereas the presence of hydroxypropyl groups alone may increase the formation of hydrolysis products of the GC-C receptor agonist polypeptide during storage, such as leucine and Ca. 2+ Combinations of, but not limited to, a sterically hindered primary amine with a cation suppress the formation of hydrolysis products of the GC-C receptor agonist polypeptide, as well as oxidation products of the GC-C receptor agonist polypeptide, during storage, leading to even greater overall stability as determined by weight / weight assay and / or chromatographic purity.

[0091] In further embodiments, 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.

[0092] Suitable pharmaceutical compositions according to the present invention generally contain an amount of active compound together with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give a range of final concentrations depending on the intended use. Techniques for preparation are generally well known in the art, as exemplified by Remington's Pharmaceutical Sciences (18th Edition, Mack Publishing Company, 1995).

[0093] For the treatment of gastrointestinal disorders, the peptides described herein may be administered orally or rectally, for example, as tablets, capsules, sachets containing a predetermined amount of active ingredient pellets, gels, pastes, syrups, boluses, electuaries, slurries, powders, lyophilized powders, granules, as solutions or suspensions in aqueous or non-aqueous liquids; as oil-in-water or water-in-oil liquid emulsions; in liposomal formulations (see, e.g., EP 736299); or in some other form. Orally administered compositions may contain binders, lubricants, inert diluents, lubricants, surfactants, or dispersing agents, flavoring agents, and humectants. Orally administered formulations, such as tablets, may optionally be coated or scored and formulated to provide sustained, delayed, or controlled release of the active ingredient therein. The peptides may be co-administered with other agents used to treat gastrointestinal disorders, including, but not limited to, those described herein.

[0094] In another embodiment, suitable pharmaceutical compositions may contain one or more other therapeutic agents.Such therapeutic agents include, but are not limited to, analgesics; antisecretory agents, including proton pump inhibitors, acid pump antagonists, H2 receptor antagonists; PDE5 inhibitors; ODC inhibitors; GABA-B antagonists; bile acid sequestrants; prokinetic and functional modulators; antidepressants; antibiotics; antiemetics; opioids; and mucosal protectants.

[0095] Treatment methods In various embodiments, the peptide or a pharmaceutically acceptable salt thereof may be useful in a method for colon cleansing treatment or for treating gastrointestinal disorders. In some embodiments, the peptide or a pharmaceutically acceptable salt thereof may be useful in a method for cleansing the colon before colonoscopy or surgical procedures. In further embodiments, the peptide may be used to prepare an object for colonoscopy treatment. In some embodiments, the peptide or a pharmaceutically acceptable salt thereof may be used to prepare an object for surgery, such as intestinal surgery. In other embodiments, the peptides are used to treat gastrointestinal disorders, visceral disorders, colon cancer, hereditary nonpolyposis colorectal cancer (HNPCC), i.e., Lynch syndrome, gastroparesis (GP), polyps, pain, generalized abdominal pain, postoperative ileus, opioid-induced constipation, functional dyspepsia, diverticular disease, including but not limited to SUDD (symptomatic simple diverticular disease) and SCAD (segmental colitis associated with diverticulosis), diverticulosis, diarrhea-predominant irritable bowel syndrome, irritable bowel syndrome (IBS), and inflammatory bowel syndrome (IGS). The present invention may treat pain associated with colon cancer (BS), ulcerative colitis, ulcerative proctitis, Crohn's disease, inflammatory bowel disease (IBD), chronic or acute radiation proctopathy, rectal pain, chronic rectal neuralgia, transient rectal neuralgia, anal pain, chronic anal fissure, postoperative anal pain, overactive bladder syndrome, stress urinary incontinence, interstitial cystitis, bladder pain syndrome, colorectal cancer, cancer-associated pain, generalized pelvic pain, endometriosis, testicular pain, chronic prostatitis, prostatodynia, urinary tract syndrome, penile pain, perianal pain, and other gastrointestinal and visceral disorders. In some embodiments of the invention, methods of treatment are provided for gastrointestinal disorders. In one embodiment, the gastrointestinal disorder is colon cancer or polyps. In another embodiment, the gastrointestinal disorder is hereditary nonpolyposis colorectal cancer (HNPCC), i.e., Lynch syndrome. In another embodiment, the gastrointestinal disorder is gastrointestinal pain. In a further embodiment, the gastrointestinal disorder is visceral or abdominal pain, or pain associated with cancer. In another embodiment, the gastrointestinal disorder is rectal cancer. In another embodiment, the gastrointestinal disorder is functional dyspepsia.

[0096] In one embodiment, there is provided a method for cleansing the colon of a subject in preparation for a colonoscopy procedure, comprising administering to the subject an effective dose of a colon cleansing composition comprising a pharmaceutically acceptable excipient, diluent, or carrier, and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide or the pharmaceutically acceptable salt thereof has the amino acid sequence: [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK, Asn or absent; Xaa3 is Asn, Ser, or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile, BE, or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe), BK, or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 11 is Cys, Ag, or penicillamine (Pen); Xaa 12 is Cys, allylglycine (Ag), Hag, Cth, Dpr, or Val; Xaa 13 is Asn or Leu; Xaa 14is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 16 is Cys, Ag, Pen, or Cth; Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, Ser, or Ala; Xaa 19 is Cys, Ag, or Pen; Xaa 20 is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, Dpr, or OH-Pro, and the peptide is 12 , Xaa8 and Xaa 16 , and Xaa 11 and Xaa 19 Contains a covalent bond between

[0097] In another embodiment, a method is provided for cleansing the colon of a subject in preparation for a colonoscopy procedure, comprising administering to the subject an effective dose of a colon cleansing composition comprising a pharmaceutically acceptable excipient, diluent, or carrier, and a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide or the pharmaceutically acceptable salt thereof has the amino acid sequence: [ka] [ka] [ka] [ka] Includes.

[0098] In one embodiment, the peptides and compositions described herein provide a treatment to prepare a patient prior to a colonoscopy.

[0099] In some embodiments, the peptides and pharmaceutically acceptable salts described herein can be used as a method for colon cleansing in preparation for a colonoscopy procedure. In some embodiments, the method for colon cleansing includes administering an effective first dose, such as 5 μg to 100 mg of the peptide or a pharmaceutically acceptable salt. The following morning, an effective second dose, such as 5 μg to 100 mg of the peptide or a pharmaceutically acceptable salt, is administered to substantially cleanse the colon. In other embodiments, the peptide or a pharmaceutically acceptable salt thereof is administered in a single dose of 5 μg to 200 mg.

[0100] The peptides and pharmaceutically acceptable salts described herein may be used alone or in combination therapy to treat, prevent, or reduce pain associated with gastrointestinal disorders, cancer, generalized pelvic pain, bladder pain, overactive bladder, endometriosis, testicular pain, chronic prostatitis, prostatodynia, urinary tract syndrome, penile pain, and visceral pain associated with perianal pain, or another disorder described herein.

[0101] The peptides and pharmaceutically acceptable salts described herein can be administered in combination with other active substances.For example, the peptides can be administered together with an analgesic peptide, a soluble guanylate cyclase (sGC) stimulator or a compound.The analgesic peptide or compound can be covalently attached to the peptides described herein, or it can be a separate active substance that is administered together or sequentially with the peptides described herein in combination therapy. The peptides and pharmaceutically acceptable salts described herein may also be administered in combination with other agents used to treat GI disorders, including anticholinergics, including, but not limited to, antidepressants, prokinetic or prokinetic agents, antiemetics, antibiotics, proton pump inhibitors, acid blockers (e.g., histamine H2 receptor antagonists), acid pump antagonists, PDE5 inhibitors, ODC inhibitors, GABA-B agonists, bile acid sequestrants, COX-2 inhibitors, NSAIDS, corticosteroids, opioids, beta-3 adrenergic receptor agonists; muscarinic receptor antagonists, tricyclic antidepressants, and mucosal protectants.

[0102] In other embodiments, peptide therapeutic combinations include other nonsteroidal anti-inflammatory drugs (NSAIDS), including celecoxib, Sulidac, and isomers, as well as phosphodiesterase (PDE) inhibitors and ornithine decarboxylase (ODC) inhibitors (e.g., d,l-α-difluoromethylornithine DFMO) for idiopathic and Lynch syndrome colon polyps; mesalamine or 5-aminosalicylic acid (5-asa), or the steroid, budesonide, for inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); the opioid, tramadol, and tramadol isomers and analogs, or eluxalodine for chronic pain, including cancer pain.

[0103] In some embodiments, useful analgesics that can 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®), benzimidazolone derivatives such as mosapride, metoclopramide, zacopride, cisapride, renzapride, BIMU1 and BIMU8, and lirexapride), 5HT 1 agonists (e.g., sumatriptan and buspirone), opioid receptor agonists (e.g., loperamide, fedotozine, enkephalin pentapeptides, morphine, diphenyloxylate, flakefamide, 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), vanilloid and cannabanoid receptor agonists, sialorphin and sialorphin-related peptides. Various classes of analgesics have been described in the literature.

[0104] In some embodiments, one or more other therapeutic agents can be used in combination with the peptide described herein.Such agents include antidepressants, prokinetic or prokinetic agents, antiemetics, antibiotics, proton pump inhibitors, acid blockers (e.g., histamine H2 receptor antagonists), acid pump antagonists, PDE5 inhibitors, ODC inhibitors, GABA-B agonists, bile acid sequestrants, opioids, and mucosal protectants.

[0105] Examples of antidepressants include, without limitation, tricyclic antidepressants such as amitriptyline (Elavil®), desipramine (Norpramin®), imipramine (Tofranil®), amoxapine (Asendin®), nortriptyline; selective serotonin reuptake inhibitors (SSRIs) such as paroxetine (Paxil®), fluoxetine (Prozac®), sertraline (Zoloft®), and citralopram (Celexa®); and others such as doxepin (Sinequan®) and trazodone (Desyrel®).

[0106] Examples of prokinetic agents 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 antiemetics include, but are not limited to, prochlorperazine.

[0107] Examples of antibiotics that can be used include those that can be used to treat Heliobacter 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.

[0108] 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).

[0109] Examples of PDE5 inhibitors include, but are not limited to, avanafil, lodenafil, mirodenafil, sildenafil citrate, tadalafil, vardenafil, and udenafil. 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)alkylamines. Examples of mucosal protectants include, but are not limited to, sucralfate, teprenone, polaprezinc, cetraxate, and bismuth subsalicylate.

[0110] Combination therapy can be achieved by administering two or more active agents, each of which is formulated and administered separately, such as the peptide or pharmaceutically acceptable salt described herein and another therapeutic peptide or compound, or by administering two or more active agents in a single formulation.Other combinations are also encompassed by combination therapy.For example, two active agents can be formulated together and administered in conjunction with a separate formulation containing a third active agent.Two or more active agents in combination therapy can be administered simultaneously, but they do not have to be.For example, the administration of a first active agent (or combination of active agents) can precede the administration of a second active agent (or combination of active agents) by several minutes, hours, days, or weeks. Therefore, two or more active agents can be administered within a few 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, 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.Although it is often desirable that two or more active agents used in combination therapy are present in patient's body at the same time, this does not have to be the case.

[0111] Dosage The dosage range for adults may be generally 5 μg to 100 mg per day, orally or rectally, for the peptides and pharmaceutically acceptable salts described herein. Tablets, capsules, or other presentation forms provided in discrete units may conveniently contain an amount of the compounds described herein effective at such dosages or as multiples thereof, e.g., a unit containing 25 μg to 2 mg or approximately 100 μg to 1 mg. The exact amount of compound prescribed to a patient is the responsibility of the attending physician. However, the dosage employed will depend on several factors, including the patient's age and sex, the exact disorder being treated, and its severity.

[0112] In various embodiments, the dosage unit is administered with food any time of day, without food any time of day, with food after an overnight fast (e.g., with breakfast), or before bed after a low-fat snack. In one particular embodiment, the dosage unit is administered before or after food consumption (e.g., a meal). In a further embodiment, the dosage unit is administered approximately 15 minutes to 1 hour before food consumption. 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 equivalent.

[0113] In some embodiments, compositions containing the peptides described herein are provided in divided doses, which are administered the night before and the day of colonoscopy. In other embodiments, the dosage is provided as a single dose the night before, the day before, or the day of colonoscopy.

[0114] In the combination therapy embodiment of the present invention, the exact amount of each of the two or more active ingredients in the dosage unit depends on the desired dosage of each component.Therefore, it may be useful to create a dosage unit that, when administered according to a specific dosage schedule (e.g., a dosage schedule specifying a certain number of units and a specific timing of administration), will deliver the same dosage of each component as would be administered if the patient were treated with only a single component.In other situations, it may be desirable to create a dosage unit that will deliver a dosage of one or more components that is lower than what would be administered if the patient were treated with only a single component.Finally, it may be desirable to create a dosage unit that will deliver a dosage of one or more components that is higher than what would be administered if the patient were treated with only a single component.

[0115] Pharmaceutical compositions may contain additional ingredients, including but not limited to the active ingredients and excipients described herein. In certain embodiments, one or more therapeutic agents in a dosage unit may be in a long-term or controlled-release formulation, and additional therapeutic agents may not be in a long-term release formulation. For example, a peptide or agonist described herein may be in a controlled-release or long-term release formulation in the same dosage unit, along with another agent that may or may not be in a controlled-release or long-term release formulation. Thus, in certain embodiments, it may be desirable to provide immediate release of one or more of the agents described herein and controlled release of one or more other agents.

[0116] The present invention has been described with reference to certain exemplary embodiments thereof. However, it will be readily apparent to those skilled in the art that the present invention can be employed in specific forms other than those of the exemplary embodiments described above. This can be done without departing from the spirit of the invention. The exemplary embodiments are merely illustrative and should not be considered limiting in any way. The scope of the present invention is defined by the appended claims and their equivalents, rather than by the preceding description. [Example]

[0117] Example 1: cGMP accumulation in T84 cells for analysis of GC-C activity For cGMP assay, 2.0 x 10 5T84 cells at 1000 cells / mL were grown overnight in a 96-well tissue culture plate. The next day, T84 cells were washed twice with 200 μL of DMEM + 20 mM MES (pH 5), DMEM + 50 mM sodium bicarbonate (pH 8), or DMEM without additives for pH 7. These buffers contain no serum. After the second wash, cells were incubated with 180 μL of 1 mM isobutylmethylxanthine (IBMX) in either pH 5, 7, or 8 buffer for 10 minutes at 37°C to inhibit any phosphodiesterase activity. Peptides were then diluted to a 10x concentration in either pH 5, 7, or 8 buffer. 20 μL of peptide solution was diluted with T84 cells to a final volume of 200 μL, resulting in a 1x concentration of each peptide. An 11-point curve analysis was performed for each peptide with final peptide concentrations tested in each assay of 10000, 3000, 1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1 nM.

[0118] No peptide control was used to determine endogenous levels of cGMP. The peptides were incubated at 37°C for 30 minutes. After 30 minutes, the supernatant was removed and the cells were lysed with 200 μL of 0.1 M HCl. The cells were lysed on ice for 30 minutes. After 30 minutes, the lysate was removed with a pipette and placed in a 96-well HPLC plate and spun at 10,000×G for 10 minutes to remove any cell debris. The supernatant from the previous spin was removed and placed in a fresh 96-well HPLC plate. The samples were diluted with an equal volume of 1 M ammonium acetate (pH 7) to neutralize the samples for better chromatography. A 2× cGMP standard curve was prepared with 0.1 M HCl and then diluted with an equal volume of 1 M ammonium acetate, with the following final concentrations in ng / mL: 1000, 500, 250, 125, 50, 25, 5, 2.5, and 0.5. The ST core peptide (hereafter referred to as ST core) has the amino acid sequence: [ka] It has.

[0119] The cGMP concentration was determined from each sample using the LC / MS conditions and calculated standard curve in Table 4. The EC 50 Values ​​were calculated. Results for selected peptides can be found in Figures 1-14, 23, and 24. [Table 1]

[0120] Example 2: GC-C binding assay All GC-C couplings were performed in a final volume of 200 μL of medium at pH 5, 7, or 8. pH 5 medium was prepared using DMEM, 0.5% BSA, and 20 mM 2-(N-morpholino)ethanesulfonic acid (MES). pH 7 medium was prepared using DMEM, 0.5% BSA. pH 8 medium was prepared using DMEM, 0.5% BSA with 20 mM sodium bicarbonate.

[0121] T84 cells were used at 250,000 cells per reaction. Cells were grown to confluence in a T-150 flask using DMEM-F12 50 / 50 medium, 5 mM L-glutamine, and 5% FBS. Cells were scraped using DMEM and 0.5% BSA and counted to determine how much volume to add to give 250,000 cells per reaction in a final volume of 200 μL. Next, 200,000 CPM of I125-STp, cold peptide competitor, and then T84 cells were added per reaction to initiate the reaction. The samples were then incubated at 37°C for 1 hour. After 1 hour, the entire sample was added to a pre-blocked GF-C plate and aspirated. Each well was then washed twice with 200 μL of cold PBS. The filter plate bottom was removed, and the 96-well plate was placed at 50°C to dry. After drying, 100 μL of scintillation fluid was added to each well and gently vortexed before counting. Results for selected peptides can be seen in Figures 1 and 23.

[0122] Example 3: Rat Intestinal Fluid (RIF) In Vitro Metabolic Incubation Rat intestinal fluid was obtained by adding PBS to ligated rat jejunal loops for 30 minutes. The fluid was then collected, pooled, and kept on ice before centrifugation at 4°C. The supernatant was removed and flash-frozen. 60 μM of peptide (100 μg / mL) was then added to the rat intestinal fluid along with PBS and 0.5% BSA. Control incubations were performed in PBS. 50 μL aliquots from all samples were then taken in duplicate at 0, 10, 30, and 60 minutes and stopped with 12% trichloroacetic acid containing an internal standard.

[0123] Samples were spun and the supernatant removed for analysis by LC-MS using the calculated accurate mass of each peptide (or predicted metabolite) to generate extracted ion chromatograms. Relative response factors (analyte peak area / internal standard peak area) for each sample were used to construct percent remaining relative to time = 0. Results for selected peptides can be found in Figures 1, 20, 21, and 23.

[0124] Example 4: In vivo ligated rat loop First, 60 μM peptide was prepared in 200 μL of PBS. This solution was injected into ligated rat duodenal loops approximately 3-5 cm in length. Three animals were used per peptide per time point. At 30 and 60 minutes, the loops were excised, measured, and weighed. The fluid was then collected in Eppendorf tubes and flash-frozen, and the loops were reweighed to determine their empty weight. The samples were then thawed and spun, and the supernatant was removed from each sample. A 50 μL aliquot was then removed and added to 12% trichloroacetic acid containing an internal standard.

[0125] Analysis was performed by LC-MS using the calculated accurate mass of each peptide (or predicted metabolite) to generate extracted ion chromatograms. The relative response factor (analyte peak area / internal standard peak area) for each sample was used to construct percent remaining relative to time=0. The amount of fluid secretion during incubation was determined by the formula (fluid secretion=(full loop-empty loop) / length). This formula was used to calculate how much fluid entered the loop at each time point. Results for selected peptides can be found in Figures 15, 16, 18, 19, 22, and 23.

[0126] Example 5: Preparation of dicarbapeptides Dicarbapeptides were made using standard Fmoc- / Trt / Otbu-protected amino acids. Dicarbapeptides were synthesized on a Protein Technologies Symphony X® with amino acids dissolved in dimethylformamide (DMF) at a concentration of 0.5 M. Fmoc-Cys(Trt)-OH was dissolved in a solution containing 0.5 M oxyma pure in DMF. HCTU was then dissolved in NMP to 0.5 M, and 1.0 M DIPEA in NMP was also used. DIC was dissolved in NMP to 0.5 M. Fmoc deprotection was performed using a solution containing 20% ​​piperidine in DMF.

[0127] Fmoc-Cys(Trt)-O-Wang resin (0.2 mmol, 0.29 mmol / g) was treated with 20% piperidine in DMF (3 × 6 mL, 3 min, 1 × 6 mL, 10 min). The resulting resin was washed with DMF (6 × 10 mL, 30 s). A solution containing Fmoc-Xaa-OH, HCTU, and DIPEA in NMP, premixed for 30 s, was added, and the mixture was stirred for 30 min. The resin was filtered and washed once with DMF. A second solution containing Fmoc-Xaa-OH, HCTU, and DIPEA in NMP, premixed for 30 s, was added, and the mixture was stirred again for 30 min. In the case of Fmoc-Cys(Trt)-OH, the amino acid / oxyma solution was mixed with DIC in NMP for 5 min, added to the resin, and stirred for 45 min. The resin was filtered and washed once with DMF. The resulting resin was filtered and washed with DMF (6 × 10 mL). The material was subjected to the protocol described above, and the peptide was elongated to the entire sequence. To dry the resin before metathesis, the resin was washed with CHCl (3 × 10 mL) and hexane (3 × 10 mL) and dried under vacuum overnight. The material was subjected to the protocol described above, and the peptide was elongated to macrocyclization.

[0128] The following procedure was used to perform metathesis on the peptide. To 500 mg (0.1 mmol) of the protected, resin-bound peptide was added a 6 mg / mL solution of HGII in 5 mL of 4:1 1,2-dichloroethane-0.4 M LiCl in DMA. The resulting suspension was heated to 160 °C in a microwave oven at 200 W for 5 min. The suspension was cooled, filtered, and washed with dichloromethane (3 × 10 mL) and NMP (3 × 10 mL). A small portion was cleaved to determine whether the metathesis was complete. The resin was soaked overnight in a 10% DMSO in NMP solution (10 mL) to scavenge excess HGII catalyst. The resulting resin was washed with NMP (3 × 10 mL) and treated with 20% piperidine in DMF (3 × 15 mL, 10 min). The resin was washed with DMF (5 x 10 mL), CH2Cl2 (3 x 10 mL), and hexane (3 x 10 mL) and dried under reduced pressure.

[0129] To cleave the peptide from the resin, the resin was treated with a solution (20 mL) containing 90:5:5 TFA-TIPS-HO. After 2 hours, the resin was filtered and washed with TFA (3 mL) and concentrated 50%. Chilled (-78°C) ether was added to the solution (50 mL), and the resulting mixture was centrifuged at 3500 rpm for 10 minutes. The ether was decanted, and the solid was subjected to two additional washes with chilled (-78°C) ether and centrifuged. The resulting solid was dried under reduced pressure, dissolved in 1:1 HO-ACN, frozen, and lyophilized.

[0130] The peptide was purified on a Waters Autopure® system using 0.1% TFA in water and 0.1% TFA in acetonitrile on a Waters PST C18 RP column (250 × 30 mm, 10μ, 130Å) at a flow rate of 40 mL / min. A linear gradient of 5 to 45% acetonitrile over 40 or 60 min was used. Fractions containing the desired product were pooled and oxidized.

[0131] The following procedure was used to oxidize cysteine ​​residues for disulfide bond formation: 5 mL of DMSO was added to a solution containing 100 mL of 0.05 N NH4HCO3 (pH ∼8) in 9:1 water-acetonitrile. After 72 h, oxidation appeared complete by HPLC, and the material was acidified with acetic acid, frozen, and lyophilized. The peptide was purified on a Waters Autopure system using 0.1% TFA in water and acetonitrile on a Waters PST C18 RP column (250 × 30 mm, 10 μm, 130 Å) at a flow rate of 40 mL / min. A linear gradient was used from 5 to 40% acetonitrile over 60 min. Fractions containing the desired product were pooled and lyophilized.

[0132] Example 6: Preparation of cystathionine-containing peptides Materials were synthesized on a Protein Technologies Symphony X® with amino acids dissolved in DMF at 0.5 M. Fmoc-Cys(Trt)-OH was dissolved in a solution containing 0.5 M oxyma pure in DMF. HCTU was dissolved in NMP at 0.5 M, and 1.0 M DIPEA in NMP was also used. DIC was dissolved in NMP at 0.5 M. Fmoc deprotection was performed using a solution containing 20% ​​piperidine in DMF.

[0133] To couple the peptide to the resin, Rink amide resin (0.2 mmol, 0.24 mmol / g) was treated with 20% piperidine in DMF (3 × 6 mL, 3 min, 1 × 6 mL, 10 min). The resulting resin was washed with DMF (6 × 10 mL, 30 s). A solution containing Fmoc-Xaa-OH, HCTU, and DIPEA in NMP, premixed for 30 s, was added, and the mixture was stirred for 30 min. The resin was filtered and washed once with NMP. A second solution containing Fmoc-Xaa-OH, HCTU, and DIPEA in NMP, premixed for 30 s, was added, and the mixture was stirred again for 30 min. In the case of Fmoc-Cys(Trt)-OH, the amino acid / oxyma pure solution was mixed with DIC in NMP for 5 min, added to the resin, and stirred for 45 min. The resin was filtered and washed once with DMF. The resulting resin was filtered and washed with DMF (6 x 10 mL). The material was subjected to the protocol described above and the peptide was elongated to macrocyclization.

[0134] To couple the diamino acid, the resin was treated with 20% piperidine in DMF (3 × 6 mL, 3 min, 1 × 6 mL, 10 min). The resulting resin was washed with DMF (6 × 10 mL, 30 s). A solution containing alloc-HCys((Fmoc-Ala-OH)-3-yl)-all (227 mg, 0.4 mmol), PyAOP (209 mg, 0.4 mmol), and DIPEA (139 μL, 125 mg, 0.8 mmol) in 5 mL of NMP was added to the resin. After 90 min, the resin was filtered and washed with DMF (6 × 10 mL). The peptide was then elongated using the protocol described above.

[0135] To perform allyl-Alloc deprotection, the resin (0.2 mmol) was suspended in 10 mL of DMF, and a solution containing Pd(PPh3)4 (300 mg, 0.26 mmol) in 10 mL of CHCl2 was added, followed by 0.25 mL (2 mmol) of phenylsilane. The resulting mixture was shaken for 2 h in the absence of light. A small sample was cleaved to ensure complete deprotection. The resulting resin was filtered and washed with CHCl2 (3 × 10 mL) and DMF (3 × 10 mL). The resin was treated with a solution containing 0.5% sodium diethyldithiocarbamate in DMF (10 mL, 4 × 15 min) and washed with DMF (3 × 10 mL).

[0136] To perform macrocyclization, the resin was treated with 20% piperidine in DMF (2 x 5 min, 1 x 10 min, 15 mL) and washed with DMF (6 x 15 mL). A solution containing 521 mg (1 mmol) of PyAOP in 15 mL of DMF was added, and after 1 min, 0.35 mL (2 mmol) of DIPEA was added and shaken for 60 min. A small sample was taken for analysis. The resulting resin was washed with DMF (3 x 15 mL) and returned to the SymphonyX to complete the synthesis.

[0137] To cleave the peptide from the resin, the resin was treated with a solution (20 mL) containing 90:5:3:2 TFA-TIPS-DODT-HO. After 2 h, the resin was filtered and washed with TFA (3 mL) and concentrated 50%. Chilled (-78 °C) ether was added to the solution (50 mL), and the resulting mixture was centrifuged at 3500 rpm for 10 min. The ether was decanted, and the solid was subjected to two additional washes with chilled (-78 °C) ether and centrifuged. The resulting solid was dried under vacuum, dissolved in 1:1 HO-ACN, frozen, and lyophilized. The peptide was then purified on a Waters PST C18 RP column (250 × 30 mm, 10 μm, 130 Å) at a flow rate of 40 mL / min using a Waters autopure system with 0.1% TFA in water and 0.1% TFA in acetonitrile. A linear gradient of 5 to 45% acetonitrile over 40 or 60 minutes was used. Fractions containing the desired product were pooled and oxidized.

[0138] Oxidation was carried out by adding 5 mL of DMSO to a solution containing 100 mL of 0.05 N NH4HCO3 (pH ∼8) in 9:1 water-acetonitrile. After 72 h, oxidation appeared complete by HPLC, and the material was acidified with acetic acid, frozen, and lyophilized. The peptide was purified on a Waters Autopure® system using 0.1% TFA in water and 0.1% TFA in acetonitrile on a Waters PST C18 RP column (250 × 30 mm, 10 μm, 130 Å) at a flow rate of 40 mL / min. A linear gradient from 5 to 40% acetonitrile over 60 min was used. Fractions containing the desired product were pooled and lyophilized.

[0139] Example 7: Production of lactam bond-containing peptides Peptides were synthesized using standard Fmoc- / Trt / Otbu-protected amino acids. Materials were synthesized on a Protein Technologies Symphony X® with amino acids dissolved in DMF at 0.5 M. Fmoc-Cys(Trt)-OH was dissolved in a solution containing 0.5 M oxyma pure in DMF. HCTU was dissolved in NMP at 0.5 M, and 1.0 M DIPEA in NMP was also used. DIC was dissolved in NMP at 0.5 M. Fmoc deprotection was performed using a solution containing 20% ​​piperidine in DMF. Unusual amino acids were coupled manually as described below.

[0140] To incorporate Fmoc-Dpr(ivDde)-OH, a solution containing 533 mg (1.0 mmol) of Fmoc-Dpr(ivDde)-OH, 521 mg (1.0 mmol) of PyAOP, and 348 μL (258 mg, 2.0 mmol) of DIPEA in 6 mL of DMF was added to the deprotected resin (0.2 mmol). After 90 min, the resin was filtered and washed with DMF (3 × 10 mL), CHCl (3 × 10 mL), and again with DMF (3 × 10 mL). The resin was returned to the synthesizer and the synthesis continued.

[0141] To incorporate Fmoc-Asp(ODmab)-OH, a solution containing 667 mg (1.0 mmol) of Fmoc-Asp(ODmab)-OH, 521 mg (1.0 mmol) of PyAOP, and 348 μL (258 mg, 2.0 mmol) of DIPEA in 6 mL of DMF was added to the deprotected resin (0.2 mmol). After 90 min, the resin was filtered and washed with DMF (3 × 10 mL), CHCl (3 × 10 mL), and again with DMF (3 × 10 mL). For final deprotection, the resin was returned to the synthesizer.

[0142] To Boc-protect the N-terminus, the resin was treated with 218 mg (1.0 mmol) of di-tert-butyl dicarbonate in 5 mL of DMF. After 4 h, a small sample (approximately 10 mg) was removed and acetylated with 10 μL of acetic anhydride and 30 μL of DIPEA in DMF to check for completion. The resulting resin was washed with DMF (5 × 10 mL) and treated with 100 μL of acetic anhydride and 300 μL of DIPEA in DMF for 50 min. The resulting resin was washed with DMF (6 × 10 mL).

[0143] To remove ivDDE and Dmab, the protected resin was treated with a solution of 2% hydrazine monohydrate in DMF (5 × 5 mL, 5 min), and the resulting resin was washed with DMF (6 × 10 mL).

[0144] To form the lactam, the resin was treated with 521 mg (1.0 mmol) of PyAOP and 348 μL (258 mg, 2.0 mmol) of DIPEA in 5 mL of DMF. The resulting mixture was heated to 100° C. in a microwave oven (200 W) for 10 min. The resulting resin was cooled, filtered, and washed with DMF (6 × 10 mL) and CHCl (6 × 10 mL), and dried under reduced pressure.

[0145] To cleave the peptide from the resin, the resin was treated with a solution (20 mL) containing 90:5:3:2 TFA-TIPS-DODT-HO. After 2 hours, the resin was filtered and washed with TFA (3 mL) and concentrated 50%. Chilled (-78°C) ether was added to the solution (50 mL), and the resulting mixture was centrifuged at 3500 rpm for 10 minutes. The ether was decanted, and the solid was subjected to two additional washes / centrifugations with chilled (-78°C) ether. The resulting solid was dried under reduced pressure, dissolved in 1:1 HO-ACN, frozen, and lyophilized.

[0146] To oxidize the peptide, the crude peptide (280 mg) was dissolved in water and the mixture was treated with solid NH4HCO3 to adjust the pH to >7. 20 mL of acetonitrile was then added, followed by 10 mL of DMSO. After 24 h, the oxidation appeared complete by HPLC, and the material was acidified to pH ∼2 with trifluoroacetic acid, filtered, frozen, and lyophilized.

[0147] The peptide was purified on a Waters Autopure® system using 0.1% TFA in water and 0.1% TFA in acetonitrile on a Waters PST C18 RP column (250 × 30 mm, 5μ, 130 Å) at a flow rate of 40 mL / min. A linear gradient from 5% 0.1% TFA in acetonitrile to 25% 0.1% TFA in acetonitrile over 40 min was used. Fractions containing the desired product were pooled, frozen, and lyophilized. The material was subjected to a second pass purification.

[0148] The peptide was purified on a Waters Autopure® system using 0.1% TFA in water and 0.1% TFA in acetonitrile on a Waters PST C18 RP column (250 x 19 mm, 5 u, 130 A) at a flow rate of 20 mL / min. A linear gradient from 15% 0.1% TFA in acetonitrile to 25% 0.1% TFA in acetonitrile over 60 min was used. Fractions containing the desired product were pooled, frozen, and lyophilized to give a colorless solid.

[0149] Example 8: Gastrointestinal transit in mice The purpose of the assay was to test the effect of guanylate cyclase C agonist peptides on in vivo gastrointestinal transit in mice. Orally administered guanylate cyclase C agonists were demonstrated to increase the % distance traveled by a charcoal meal in mice.

[0150] For the assay, female CD-1 mice (n=10 per group) weighing 25-30 g were fasted overnight and given ad libitum access to water. Activated charcoal (20 g; 100 mesh; Sigma catalog #242276) was suspended in 200 mL of gum arabic (100 mg / mL) and stirred for at least 1 hour. Test peptides were prepared in 20 mM Tris pH 6.9 vehicle.

[0151] Test peptides and vehicle were administered in a 200 μL volume by oral gavage. Seven minutes after test peptide administration, 200 μL of charcoal / gum arabic suspension was administered by oral gavage. 15 minutes later, mice were sacrificed by CO2 overdose. The gastrointestinal tract was removed from the esophagus to the cecum. The total length of the small intestine was measured from the pyloric junction to the ileocecal junction. The distance traveled by charcoal was measured from the pyloric junction to the front line of the charcoal. The % traveled distance was determined as (distance traveled by charcoal / total length of small intestine) × 100. Data were entered into the GraphPad Prism software program and analyzed by ANOVA with Bonferroni multiple comparison post-hoc test. The GraphPad Prism software package was used to plot the data and calculate EDs. 50 The results for selected peptides can be found in Figures 17, 25, 26, and 27.

[0152] Other embodiments 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. In the unlikely event that the meaning of a term in any of the patents or publications 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 description merely discloses and describes exemplary embodiments of the present invention. Those skilled in the art will readily recognize from such description and from the accompanying drawings and claims that various changes, modifications, and variations can be made therein without departing from the spirit and scope of the present invention, as defined in the following claims. The present invention provides, for example, the following items. (Item 1) A peptide or a pharmaceutically acceptable salt thereof, said peptide having the amino acid sequence [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK, Asn or absent; Xaa3 is Asn, Ser, or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile, BE, or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe), BK, or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 11 is Cys, Ag, or penicillamine (Pen); Xaa 12 is Cys, allylglycine (Ag), Hag, Cth, Dpr, or Val; Xaa 13 is Asn or Leu; Xaa 14 is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 16 is Cys, Ag, Pen, or Cth; Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, Ser, or Ala; Xaa 19 is Cys, Ag, or Pen; Xaa 20 is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, Dpr, or OH-Pro; and The peptide is located at Xaa7 and Xaa 12 , Xaa8 and Xaa 16 , and Xaa 11 and Xaa 19 or a pharmaceutically acceptable salt thereof, wherein the peptide contains a covalent bond between: (Item 2) 2. The peptide or pharmaceutically acceptable salt thereof according to item 1, wherein the N-terminus is capped with acetic acid, pentenoic acid, biotin, 4-Mepip, a C12 alkyl carboxylic acid, a C14 alkyl carboxylic acid, a C16 alkyl carboxylic acid, or a C18 alkyl carboxylic acid. (Item 3) 3. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 1 or 2, wherein the C-terminus is amidated. (Item 4) Xaa7 and Xaa 12 are both Ag, and the dicarba bonds are Ag7 and Ag 12 exists between; Xaa7 and Xaa 12 are both Cys and the disulfide bond is between Cys7 and Cys 12 exists between; Xaa8 and Xaa 16 are both Cys and the disulfide bond is between Cys8 and Cys 16 exists between; Xaa7 is Cth and Xaa 12 is Cys, and the bond is Cth7 and Cys 12 exists between; Xaa8 is Cth and Xaa 16 is Cys and the bond is Cth8 and Cys 16 exists between; Xaa 11 and Xaa 19 is Cys and the disulfide bond is Cys 11 and Cys 19 exists between; or Any combination of these 4. The peptide according to any one of items 1 to 3, or a pharmaceutically acceptable salt thereof. (Item 5) 4. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 1 to 3, wherein Xaa7 is allylglycine, Cth, or Cys. (Item 6) 4. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 1 to 3, wherein Xaa8 is Cys or cystathionine. (Item 7) 7. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 1 to 6, wherein Xaa9 is Glu. (Item 8) Xaa 10 8. The peptide according to any one of items 1 to 7, or a pharmaceutically acceptable salt thereof, wherein is Leu. (Item 9) Xaa 12 is Cys or allylglycine, or a pharmaceutically acceptable salt thereof according to any one of items 1 to 8. (Item 10) Xaa 14 10. The peptide according to any one of items 1 to 9, or a pharmaceutically acceptable salt thereof, wherein is Val or Pro. (Item 11) Xaa 17 11. The peptide according to any one of items 1 to 10, or a pharmaceutically acceptable salt thereof, wherein is Tyr or Thr. (Item 12) Xaa 20 is Tyr or is absent, or a pharmaceutically acceptable salt thereof. (Item 13) Xaa 21 13. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 1 to 12, wherein (Item 14) Xaa1 is absent; Xaa2 is absent; Xaa3 is absent; Xaa4 is absent; Xaa5 is absent; Xaa6 is absent; Xaa7 is Ag, Cys, or Cth; Xaa8 is Cys or Cth; Xaa9 is Glu; Xaa10 is Leu; Xaa 12 is Ag or Cys; Xaa 14 is Val or Pro; Xaa 17 is Tyr or Thr; and Xaa 20 is Tyr or absent Item 1. The peptide according to item 1 or a pharmaceutically acceptable salt thereof. (Item 15) The peptide has the amino acid sequence: [ka] 14. The peptide according to item 13, or a pharmaceutically acceptable salt thereof, comprising: (Item 16) The peptide has the amino acid sequence: [ka] [ka] [ka] [ka] 2. The peptide according to item 1, or a pharmaceutically acceptable salt thereof, comprising: (Item 17) A peptide or a pharmaceutically acceptable salt thereof, said peptide having the amino acid sequence [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK, Asn or absent; Xaa3 is Asn, Ser, or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile, BE, or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe), BK, or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 11 is Cys, Ag, or penicillamine (Pen); Xaa 12 is Cys, allylglycine (Ag), Hag, Cth, Dpr, or Val; Xaa 13 is Asn or Leu; Xaa 14 is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 16 is Cys, Ag, Pen, or Cth; Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, Ser, or Ala; Xaa 19 is Cys, Ag, or Pen; Xaa 20is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, Dpr, or OH-Pro; and The peptide is located at Xaa7 and Xaa 12 , Xaa8 and Xaa 16 , and Xaa 11 and Xaa 19 or a pharmaceutically acceptable salt thereof, wherein the peptide contains a covalent bond between: (Item 18) Item 18. The peptide or pharmaceutically acceptable salt thereof according to Item 17, wherein the N-terminus is capped with acetic acid, pentenoic acid, biotin, 4-Mepip, a C12 alkyl carboxylic acid, a C14 alkyl carboxylic acid, a C16 alkyl carboxylic acid, or a C18 alkyl carboxylic acid. (Item 19) 19. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 17 or 18, wherein the C-terminus is amidated. (Item 20) Xaa7 and Xaa 12 are both Ag, and the dicarba bonds are Ag7 and Ag 12 exists between; Xaa7 and Xaa 12 are both Cys and the disulfide bond is between Cys7 and Cys 12 exists between; Xaa8 and Xaa 16 are both Cys and the disulfide bond is between Cys8 and Cys 16 exists between; Xaa7 is Cth and Xaa 12 is Cys, and the bond is Cth7 and Cys 12 exists between; Xaa8 is Cth and Xaa16 is Cys and the bond is Cth8 and Cys 16 exists between; Xaa 11 and Xaa 19 is Cys and the disulfide bond is Cys 11 and Cys 19 exists between; or Any combination of these 20. The peptide according to any one of items 17 to 19, or a pharmaceutically acceptable salt thereof. (Item 21) 20. The peptide or pharmaceutically acceptable salt thereof according to any one of items 17 to 19, wherein Xaa7 is allylglycine or Cys. (Item 22) 20. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 17 to 19, wherein Xaa8 is Cys or cystathionine. (Item 23) 23. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 17 to 22, wherein Xaa9 is Glu. (Item 24) Xaa 10 24. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 17 to 23, wherein is Leu. (Item 25) Xaa 12 25. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 17 to 24, wherein is Cys or allylglycine. (Item 26) Xaa 14 26. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 17 to 25, wherein is Val or Pro. (Item 27) Xaa 17 27. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 17 to 26, wherein is Tyr or Thr. (Item 28) Xaa 2028. The peptide or pharmaceutically acceptable salt thereof according to any one of items 17 to 27, wherein is Tyr or is absent. (Item 29) Xaa 21 29. The peptide or a pharmaceutically acceptable salt thereof according to any one of items 17 to 28, wherein (Item 30) Xaa1 is absent; Xaa2 is absent; Xaa3 is absent; Xaa4 is absent; Xaa5 is absent; Xaa6 is absent; Xaa7 is Ag, Cys, or Cth; Xaa8 is Cys or Cth; Xaa9 is Glu; Xaa 10 is Leu; Xaa 12 is Ag or Cys; Xaa 14 is Val or Pro; Xaa 17 is Tyr or Thr; and Xaa 20 is Tyr or absent Item 18. The peptide according to item 17, or a pharmaceutically acceptable salt thereof. (Item 31) The peptide has the amino acid sequence [ka] 31. The peptide according to item 30, or a pharmaceutically acceptable salt thereof, consisting of: (Item 32) The peptide has the amino acid sequence: [ka] [ka] [ka] [ka] 18. The peptide according to item 17, or a pharmaceutically acceptable salt thereof, consisting of: (Item 33) A peptide or a pharmaceutically acceptable salt thereof, said peptide having the amino acid sequence: [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK or absent; Xaa3 is Asn or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe) or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 12 is Cys, allylglycine (Ag), Hag, or Val; Xaa 14is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, or Ala; Xaa 20 is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, or OH-Pro; and The peptide is located at Xaa7 and Xaa 12 , Xaa8 and Cys 16 , and Cys 11 and Cys 19 or a pharmaceutically acceptable salt thereof, wherein the peptide contains a covalent bond between: (Item 34) 34. The peptide or pharmaceutically acceptable salt thereof according to item 33, wherein the N-terminus is capped with acetic acid, pentenoic acid, 4-Mepip, a C12 alkyl carboxylic acid, a C14 alkyl carboxylic acid, a C16 alkyl carboxylic acid, or a C18 alkyl carboxylic acid. (Item 35) 34. The peptide or a pharmaceutically acceptable salt thereof according to Item 33, wherein the C-terminus is amidated. (Item 36) A pharmaceutical composition comprising a peptide or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient, diluent, or carrier, wherein said peptide or pharmaceutically acceptable salt thereof has the amino acid sequence: [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK, Asn or absent; Xaa3 is Asn, Ser, or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile, BE, or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe), BK, or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), 4-pentenoic acid (Pent), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 11 is Cys, Ag, or penicillamine (Pen); Xaa 12 is Cys, allylglycine (Ag), Hag, Cth, Dpr, or Val; Xaa 13 is Asn or Leu; Xaa 14 is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 16 is Cys, Ag, Pen, or Cth; Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, Ser, or Ala; Xaa 19 is Cys, Ag, or Pen; Xaa20 is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, Dpr, or OH-Pro; and The peptide is located at Xaa7 and Xaa 12 , Xaa8 and Xaa 16 , and Xaa 11 and Xaa 19 The pharmaceutical composition comprising a covalent bond between: (Item 37) The peptide or a pharmaceutically acceptable salt thereof has the amino acid sequence: [ka] [ka] [ka] [ka] 37. The pharmaceutical composition according to item 36, comprising: (Item 38) The peptide or a pharmaceutically acceptable salt thereof has the amino acid sequence: [ka] 37. The pharmaceutical composition according to item 36, comprising: (Item 39) 39. The pharmaceutical composition of any one of items 36 to 38, wherein the N-terminus of the peptide is capped with acetic acid, pentenoic acid, 4-Mepip, a C12 alkyl carboxylic acid, a C14 alkyl carboxylic acid, a C16 alkyl carboxylic acid, or a C18 alkyl carboxylic acid; the C-terminus of the peptide is amidated; or any combination thereof. (Item 40) 40. The pharmaceutical composition according to any one of items 36 to 39, wherein the pharmaceutical composition is in a solid dosage form. (Item 41) 41. The pharmaceutical composition according to item 40, wherein the pharmaceutical composition is formulated as an oral solid dosage form. (Item 42) 1. A method of cleansing the colon of a subject in preparation for a colonoscopy procedure comprising administering to the subject an effective dose of a colon cleansing composition, said colon cleansing composition comprising a pharmaceutically acceptable excipient, diluent, or carrier, and a peptide or a pharmaceutically acceptable salt thereof, said peptide or a pharmaceutically acceptable salt thereof having the amino acid sequence: [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK, Asn or absent; Xaa3 is Asn, Ser, or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile, BE, or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe), BK, or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), 4-pentenoic acid (Pent), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 11 is Cys, Ag, or penicillamine (Pen); Xaa 12 is Cys, allylglycine (Ag), Hag, Cth, Dpr, or Val; Xaa 13 is Asn or Leu; Xaa 14 is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 16 is Cys, Ag, Pen, or Cth; Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, Ser, or Ala; Xaa 19 is Cys, Ag, or Pen; Xaa 20 is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, Dpr, or OH-Pro; and The peptide is located at Xaa7 and Xaa 12 , Xaa8 and Xaa 16 , and Xaa 11 and Xaa 19The method according to any one of claims 1 to 4, wherein the covalent bond between (Item 43) The method comprises: a. orally or rectally administering to the subject a first effective dose of a colon cleansing composition; and b. administering to the subject a second effective dose of a colon cleansing composition to substantially cleanse the colon of the subject. Item 43. The method of item 42, further comprising: (Item 44) 44. The method of claim 43, wherein the effective second dose is administered the morning after the effective first dose. (Item 45) The colon cleansing composition comprises the amino acid sequence: [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof. (Item 46) The colon cleansing composition comprises the amino acid sequence: [ka] or a pharmaceutically acceptable salt thereof. (Item 47) 36. A method for producing the peptide according to any one of Items 1 to 35, comprising providing a cell harboring a nucleic acid molecule encoding the polypeptide, culturing the cell under conditions in which the peptide is expressed, and isolating the expressed peptide. (Item 48) 36. A method for producing the peptide according to any one of items 1 to 35, comprising chemically synthesizing the peptide and purifying the synthesized peptide. (Item 49) 36. A method for treating a gastrointestinal or visceral disorder, comprising administering to a subject a peptide according to any one of items 1 to 35. (Item 50) 36. The method of claim 35, further comprising administering to a subject a peptide according to any one of items 1 to 35, the method ... A method of treating a disorder selected from: rectal pain, ulcerative colitis, ulcerative proctitis, Crohn's disease, inflammatory bowel disease (IBD), chronic or acute radiation proctopathy, rectal pain, chronic rectal neuralgia, transient rectal neuralgia, anal pain, chronic anal fissure, postoperative anal pain, overactive bladder syndrome, stress urinary incontinence, interstitial cystitis, bladder pain syndrome, colorectal cancer, cancer-related pain, diffuse pelvic pain, endometriosis, testicular pain, chronic prostatitis, prostatodynia, urethral syndrome, penile pain, perianal pain, and other gastrointestinal and visceral disorders. (Item 51) 52. The method of claim 51, further comprising administering to the subject a pharmaceutical composition comprising an active ingredient selected from an opioid, tramadol, a beta-3 adrenergic receptor agonist, an anticholinergic agent, and a tricyclic antidepressant. (Item 52) 1. A pharmaceutical composition for use in cleansing the colon in a subject in preparation for a colonoscopy procedure, said pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent, or carrier, and a peptide or a pharmaceutically acceptable salt thereof, said peptide or a pharmaceutically acceptable salt thereof having the amino acid sequence: [ka] or a pharmaceutically acceptable salt thereof; Xaa1 is BE or absent; Xaa2 is BK, Asn or absent; Xaa3 is Asn, Ser, or absent; Xaa4 is Ser or absent; Xaa5 is Ser, Asn, Ile, BE, or absent; Xaa6 is Tyr, Asp, 4-fluorophenylalanine ((4-F)Phe), BK, or absent; Xaa7 is Cys, cystathionine (Cth), allylglycine (Ag), 4-pentenoic acid (Pent), Hag, or Asp; Xaa8 is Cys, cystathionine (Cth), penicillamine (Pen), or allylglycine (Ag); Xaa9 is Glu, Asp, Ser, Thr, or Gln; Xaa 10 is Leu, cyclohexylalanine (Cha), Phe, or 4-fluorophenylalanine ((4-F)Phe); Xaa 11 is Cys, Ag, or penicillamine (Pen); Xaa 12 is Cys, allylglycine (Ag), Hag, Cth, Dpr, or Val; Xaa 13 is Asn or Leu; Xaa 14 is Pro, Val, sarcosine (Sar), Leu, or hydroxyproline (OH-Pro); Xaa 16 is Cys, Ag, Pen, or Cth; Xaa 17 is Tyr, Thr, cyclohexylalanine (Cha), 4-fluorophenylalanine ((4-F)Phe), Phe, Ser, or Ala; Xaa19 is Cys, Ag, or Pen; Xaa 20 is Tyr, Leu, 4-fluorophenylalanine ((4-F)Phe), cyclohexylalanine (Cha), D-Tyr, N-methylTyr (Nme-Tyr) or absent; Xaa 21 is absent or Asn; wherein at least one Xaa is BE, BK, (4-F)Phe, Cth, Ag, Hag, Pen, Cha, Sar, Dpr, or OH-Pro; and The peptide is located at Xaa7 and Xaa 12 , Xaa8 and Xaa 16 , and Xaa 11 and Xaa 19 The pharmaceutical composition comprising a covalent bond between: (Item 53) Colon cancer, hereditary nonpolyposis colorectal cancer (HNPCC), Lynch syndrome, gastroparesis (GP), polyps, pain, generalized abdominal pain, postoperative ileus, opioid-induced constipation, functional dyspepsia, diverticular disease including but not limited to SUDD (symptomatic simple diverticular disease) and SCAD (segmental colitis associated with diverticulosis), diverticulosis, diarrhea-predominant irritable bowel syndrome, pain associated with irritable bowel syndrome (IBS), ulcerative colitis, ulcerative proctitis, Crohn's disease, inflammatory bowel disease (IBD), chronic or acute radiation proctopathy 36. A pharmaceutical composition for use in treating a disorder selected from: colorectal cancer, rectal pain, chronic rectal neuralgia, transient rectal neuralgia, anal pain, chronic anal fissure, postoperative anal pain, overactive bladder syndrome, stress urinary incontinence, interstitial cystitis, bladder pain syndrome, colorectal cancer, cancer-associated pain, diffuse pelvic pain, endometriosis, testicular pain, chronic prostatitis, prostatodynia, urinary tract syndrome, penile pain, perianal pain, and other gastrointestinal and visceral disorders, said pharmaceutical composition comprising the peptide of any one of items 1 to 35.

Claims

[Claim 1] A composition, etc., as described in the specification.

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