HK1179637B

HK1179637B - Treatment for gastrointestinal disorders

Info

Publication number: HK1179637B
Application number: HK13106964.4A
Authority: HK
Inventors: Mark G. Currie; Daniel P. Zimmer; Angelika Fretzen; Marco Kessler; Brian M. Cali
Original assignee: Ironwood Pharmaceuticals, Inc.
Priority date: 2009-12-07
Filing date: 2010-12-07
Publication date: 2018-01-19

Description

Treatment for gastrointestinal disorders

Priority declaration

This application claims priority from U.S. provisional application 61/267,316 filed on 12, 7, 2009. The entire contents of the above application are incorporated herein by reference.

Technical Field

The present invention relates to peptides, compositions and methods for treating upper gastrointestinal disorders.

Sequence listing

A sequence listing entitled "IW 076_ st25. txt" (29.5 kilobytes) created at 11/30/2010 and filed electronically at the same time, which is incorporated by reference in its entirety.

Background

Functional Dyspepsia (FD) and Gastroparesis (GP) are upper Gastrointestinal (GI) disorders that are commonly characterized by symptoms including bloating, epigastric pain and/or burning, nausea, vomiting, and early satiety. Due to the lack of efficacy and poor safety profile of existing treatments, treatment regimens for FD and GP patients are very limited.

Dyspepsia is defined as The presence of one or more symptoms of dyspepsia (epigastric pain, burning, postprandial satiety and early satiety) believed to originate from The gastroduodenal area in The absence of any organ, systemic or metabolic disease that might explain The symptoms (see Drossman, D.A., eds., RomeIII: The functional gastric and gastric Disorders, third edition, McLean, VA: Degnon Associates, Inc., 2006). FD refers to dyspepsia with no structural explanation after standard medical studies (including upper endoscopy). Pathophysiological mechanisms that may be involved in FD include, among others, delayed gastric emptying, impaired gastric accommodation, hypersensitivity to gastric dilation, altered sensitivity of the duodenum to lipids or acids, and abnormal duodenal jejunal motility. Prolonged duodenal acid exposure is also observed in some FD and GP patients, and this exposure may slow gastric emptying and cause FD or GP-like symptoms. Dyspepsia is a common syndrome that causes about 30% of the cases observed by gastroenterologists, and FD represents about 60% of all these dyspepsia cases.

GP refers to a gastric motility abnormality characterized by delayed gastric emptying in the absence of mechanical obstruction. GP may be idiopathic or may be caused by a variety of conditions, including type I or type II diabetes, viral infections, scleroderma, neurological disorders such as Parkinson's disease, metabolic disorders such as hypothyroidism, post-operative ileus, and certain drugs, including narcotic analgesics, tricyclic antidepressants, and calcium channel blockers. Treatment of cancer, including chemotherapeutic drugs and radiation to the chest and abdomen, may also cause gastroparesis (temporary or permanent). The most common symptoms are nausea, vomiting, bloating, epigastric pain, weight loss and early satiety. Gastroparesis is a chronic condition that can lead to frequent hospitalizations, reduced quality of life and increased disability and, in severe cases, increased mortality. Severe, symptomatic GP is common in individuals with diabetes, affecting 5-10% of the total patient population of 100 million in the united states alone.

Conventional treatment regimens for FD and GP, as well as other upper gastrointestinal disorders, are of limited efficacy for many patients. Thus, there remains a need for new compounds and methods for treating FD, GP and other gastrointestinal disorders.

Summary of The Invention

The invention features peptides, compositions, and related methods for treating upper gastrointestinal disorders and conditions (e.g., dyspepsia, Gastroparesis (GP), post-operative gastrointestinal obstruction, functional esophageal disorders, functional gastroduodenal disorders, gastroesophageal reflux disease (GERD), or duodenal or gastric ulcers), as well as other conditions and disorders described herein. The composition is characterized by a peptide that activates guanylate cyclase C (GC-C) in the upper GI but activates GC-C much less or not at all in the lower GI. Without being bound by any particular theory, the peptides of the invention are useful because they can alleviate the symptoms of upper GI disorders (in whole or in part by increasing upper GI motility and/or reducing upper abdominal pain/discomfort and bloating) without causing significant effects (e.g., dose-limiting changes in bowel habits, including diarrhea) in the lower GI tract at dosage levels and dosing frequency sufficient to reduce upper GI symptoms.

One aspect of the invention provides a peptide comprising the amino acid sequence:

Xaa₁Xaa₂Xaa₃Xaa₄Cys₅Xaa₆Xaa₇Xaa₈Cys₉Asn₁₀Pro₁₁Ala₁₂Cys₁₃Xaa₁₄Gly₁₅Xaa₁₆Xaa₁₇(SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof; wherein

Xaa₁Is Asn, D-Asn, Gln, D-Gln, Pro, D-Pro, Ala, β -Ala, D-Ala, Val, D-Val, Gly, Thr, D-Thr, Asp, D-Asp, β -carboxylated Asp, Glu, D-Glu, gamma-carboxylated Glu, α -aminosuberic acid (Asu), α -aminoadipic acid (Aad) or α -aminopimelic acid (Apm);

Xaa₂is Asp, β -carboxylated Asp, Glu, gamma-carboxylated Glu, Asu, Aad, Apm or deleted;

Xaa₃is Asp, β -carboxylated Asp, Glu, gamma-carboxylated Glu, Asu, Aad, Apm or deleted;

Xaa₄cys or D-Cys;

Xaa₆asp or Glu;

Xaa₇tyr, Leu, Phe, or Ile;

Xaa₈cys or D-Cys;

Xaa₁₄is Thr, Ala or Phe;

Xaa₁₆cys or D-Cys; and

Xaa₁₇is Tyr, D-Tyr, or is absent;

wherein:

Xaa₁may be modified at its amino group with methyl, oxalic, malonic, succinic, glutaric, adipic, pimelic or suberic acid; and is

If Xaa₂And Xaa₃Are all absent, then Xaa₁Must be β -carboxylated Asp or gamma-carboxylated Glu, or Xaa₁Must be Asp, D-Asp, Glu, D-Glu, Asu, Aad or Apm, and must be modified at its amino group by oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid or suberic acid.

A second aspect of the invention provides a pharmaceutical composition comprising a peptide of the invention.

A third aspect of the invention provides a method for treating a gastrointestinal disorder, said method comprising administering a pharmaceutical composition according to the invention.

The details of one or more embodiments of the invention are set forth in the accompanying description.

Brief Description of Drawings

Figure 1 shows the dose response of exemplary peptides of the invention (including peptide 1 and peptide 17) in a T84 cell cGMP assay at pH 5 and pH 8.

The drawings are provided by way of example and are not intended to limit the scope of the invention.

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 has an extracellular ligand binding domain, a single transmembrane domain, and a C-terminal guanylyl cyclase domain. When the 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 triggers 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. In addition, cGMP (bi-directional secretion from the epithelium to the mucosa and lumen) has also been shown to inhibit afferent C fiber firing (C fiber firing), suggesting a possible mechanism for the observed analgesic effect of GC-C agonists on visceral pain.

Linaclotide (Linaclotide) is a peptide GC-C agonist administered orally and is currently used in clinical trials to treat irritable bowel syndrome with constipation (IBS-C) and Chronic Constipation (CC), which has many effects on lower GI physiology, including: (1) reduced visceral pain, (2) reduced bloating and (3) increased GI transport, which can lead to increased stool frequency and improved stool consistency. Orally administered linaclotide acts locally on the luminal surface by activating GC-C; no systemic detectable levels of linaclotide were observed after oral administration at therapeutic dose levels. Thus, the results from clinical trials of linaclotide and preclinical studies with linaclotide and related peptides suggest that GC-C peptide agonists may be therapeutically useful.

It would be useful to have a composition that could be used to alleviate upper GI disorders and symptoms (e.g., Functional Dyspepsia (FD) and Gastroparesis (GP)) without promoting significant effects on bowel habits that may result from GC-C stimulation in the lower portion of the GI tract. Such GC-C agonists will reduce the likelihood of low GI adverse events, including altered stool habits and diarrhea. The GC-C peptide agonists described herein are more active in the upper GI tract (e.g., stomach and duodenum) and less active in the lower GI tract. Such agonists would benefit patients with upper GI disorders (e.g., FD and GP) by: (1) reduce visceral pain through cGMP production and/or other mechanisms, (2) reduce bloating, (3) increase gastric emptying and/or upper small intestinal transit (e.g., duodenal transport), and (4) neutralize acid in the duodenum by promoting bicarbonate secretion. Importantly, due to their targeted activity in the upper GI, these agonists will be able to alleviate the symptoms of FD and GP without causing significant effects on stool habit (e.g., likely due to GC-C stimulation in the lower small intestine).

In one aspect, the present invention provides novel GC-C peptide agonists for use in the treatment of gastrointestinal disorders (particularly upper GI disorders such as FD and GP). The GC-C peptide agonists described herein have pH dependent GC-C agonist activity. In normal, healthy individuals, the pH of the GI tract varies along its length, being lowest in the stomach (pH 1.5-3.5), rising in the upper duodenum to about pH6 and increasing further in the lower small intestine (pH 7-8). The large intestine pH tends to vary from pH6 to pH 8. The GC-C agonist peptides described herein are more active at lower pH and less active at higher pH. Thus, GC-C agonist peptides are more active in the upper GI (including the esophagus, stomach, and upper small intestine (upper duodenum)), but less active as they pass through the rest of the small intestine. The exemplary GC-C agonist peptides described herein exhibit a 10 to 1000-fold reduced potency at pH 8 compared to their potency at pH 5, as determined by in vitro assays measuring the ability to bind to and/or stimulate GC-C. Some GC-C peptide agonists, such as those exemplified in peptides 1 and 2, exhibit little or no pH sensitivity with respect to GC-C binding and/or activation. In general, the peptides described in the present invention show that the addition of a specific N-terminal amino acid makes GC-C peptide agonist activity more sensitive to changes in pH.

In some embodiments, the present invention provides a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the following amino acid sequence

Xaa₄cys or D-Cys;

Xaa₆asp or Glu;

Xaa₇tyr, Leu, Phe, or Ile;

Xaa₈cys or D-Cys;

Xaa₁₄is Thr, Ala or Phe;

Xaa₁₆cys or D-Cys; and

Xaa₁₇is Tyr, D-Tyr, or is absent;

wherein:

In some embodiments, Xaa₁One or two hydrogen atoms on its amino group are modified with methyl, oxalic, malonic, succinic, glutaric, adipic, pimelic or suberic acid.

In some embodiments, when Xaa₂And Xaa₃Xaa when either or both are present₁It is not modified at its amino group.

In some embodiments, Xaa₂Asp or Glu. In other embodiments, Xaa₂Is Asp.

In some embodiments, Xaa₂And Xaa₃Are present. In some embodiments, Xaa₂Is present and Xaa₃Is absent. In some embodiments, Xaa₂And Xaa₃Are absent.

In some embodiments, Xaa₃Asp or Glu. In other embodiments, Xaa₃Is Asp.

In some embodiments, Xaa₂And Xaa₃One or both are present and Xaa₁Is Asn, D-Asn, Gln, D-Gln, Pro, D-Pro, Ala, β -Ala, D-Ala, Val, D-Val, Gly, Thr, D-Thr, Asp, D-Asp, β -carboxylated Asp, Glu, D-Glu, gamma-carboxylated Glu, Asu, Aad or apm₁Is Asn, D-Asn, Gln, D-Gln, Pro, D-Pro, Ala, β -Ala, D-Ala, Val, D-Val, Gly, Thr, D-Thr, Asp, D-Asp, Glu or D-Glu₁Asp, D-Asp, Glu or D-Glu.

In some embodiments, Xaa₄Cys is used.

In some embodiments, Xaa₆Is Glu.

In some embodiments, Xaa₇Tyr or Leu.

In some embodiments, Xaa₈Cys is used.

In some embodiments, Xaa₁₄Is Thr.

In some embodiments, Xaa₁₆Cys is used.

In some embodiments, Xaa₁₇Is Tyr.

In some embodiments, Xaa₁₇Is absent.

In some embodiments, the present invention provides a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide comprises the amino acid sequence Xaa₁Xaa₂Xaa₃Cys₄Cys₅Glu₆Xaa₇Cys₈Cys₉Asn₁₀Pro₁₁Ala₁₂Cys₁₃Thr₁₄Gly₁₅Cys₁₆Xaa₁₇(SEQ ID NO: 2); wherein

Xaa₁Is Asn, D-Asn, Gln, D-Gln, Pro, D-Pro, Ala, β -Ala, D-Ala, Val, D-Val, Gly, Thr, D-Thr, Asp, D-Asp, Glu or D-Glu;

Xaa₂asp or Glu;

Xaa₃asp, Glu or absent;

Xaa₇tyr or Leu; and

Xaa₁₇tyr or absent.

In some embodiments, the present invention provides a peptide, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises:

Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：3)；

Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：4)；

Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly CysTyr(SEQ IDNO：5)；

Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly CysTyr(SEQ IDNO：6)；

Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：7)；

Asn Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：8)；

Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：9)；

Asn Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：10)；

Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：11)；

Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：12)；

β-Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQID NO：13)；

β-Ala Asp Asp Cys Cys GluTyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQID NO：14)；

Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：15)；

Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：16)；

β-Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：17)；

β-Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：18)；

Pro Asp Asp Cys CysGlu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：19)；

Pro Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：20)；

Pro Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：21)；

Pro Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：22)；

Thr Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：23)；

Thr Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：24)；

Thr Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：25)；

Thr Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：26)；

Gly Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：27)；

Gly Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：28)；

Gly Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：29)；

Gly Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：30)；

Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：31)；

Asp Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：32)；

Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：33)；

Asp Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：34)；

Glu Glu Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：35)；

Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：36)；

Glu Glu Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly CysTyr(SEQ IDNO：37)；

Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO：38)；

Glu Glu Glu Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：39)；

Glu Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：40)；

Glu Glu Glu Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：41)；

Glu Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：42)；

Glu Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：43)；

Glu Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：44)；

glu Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly CysTyr (SEQ ID NO: 45); or

Glu Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO：46)；

Wherein the C-terminal amino acid residue may be an L-amino acid residue or a D-amino acid residue, and if the N-terminal amino acid residue is Asn, Asp, Ala, Pro, Thr or Glu, the N-terminal residue may be an L-amino acid residue or a D-amino acid residue.

In other embodiments, the present invention provides a peptide, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises:

D-Asp Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：47)；

D-Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：48)；

D-Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：49)；

D-Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：50)；

Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys D-Tyr(SEQ ID NO：51)；

D-Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQID NO：52)；

Asn Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：53)；

Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly CysTyr(SEQ IDNO：54)；

Asp Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：55)；

Gly Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：56)；

Pro Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：57)；

Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：58)；

Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：59)；

Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：60)；

β-Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：61)；

Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly CysTyr(SEQ IDNO：62)；

Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：63)；

Pro Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：64)；

Gly Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：65)；

asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys (SEQ ID NO: 66); or

Glu Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：67)。

Xaa₁Asp₂Xaa₃Cys₄Cys₅Glu₆Xaa₇Cys₈Cys₉Asn₁₀Pro₁₁Ala₁₂Cys₁₃Thr₁₄Gly₁₅Cys₁₆Xaa₁₇(SEQ ID NO: 68); wherein

Xaa₃asp or absent;

Xaa₇tyr or Leu; and

Xaa₁₇tyr or absent.

In some embodiments, the peptide comprises the amino acid sequence:

Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：3)；

Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：4)；

Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly CysTyr(SEQ IDNO：5)；

Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly CysTyr(SEQ IDNO：6)；

Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：7)；

Asn Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：8)；

β-Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQID NO：14)；

Pro Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：19)；

Gly Asp Asp Cys Cys Glu Tyr Cys CysAsn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：30)；

Glu Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：43)；

Glu Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：44)；

In other embodiments, the peptide comprises no more than 50, 40, 30, or 20 amino acids. For example, the peptide comprises no more than 19, 18, 17, or 16 amino acids.

In some embodiments, the present invention provides a peptide, or a pharmaceutically acceptable salt thereof, wherein the peptide consists of the amino acid sequence of seq id no:

Xaa₁Is Asn, D-Asn, Gln, D-Gln, Pro, D-Pro, Ala, β-Ala, D-Ala, Val, D-Val, Gly, Thr, D-Thr, Asp, D-Asp, β -carboxylated Asp, Glu, D-Glu, gamma-carboxylated Glu, α -aminosuberic acid (Asu), α -aminoadipic acid (Aad), or α -aminopimelic acid (Apm);

Xaa₄cys or D-Cys;

Xaa₆asp or Glu;

Xaa₇tyr, Leu, Phe, or Ile;

Xaa₈cys or D-Cys;

Xaa₁₄is Thr, Ala or Phe;

Xaa₁₆cys or D-Cys; and

Xaa₁₇is Tyr, D-Tyr, or is absent;

wherein:

If Xaa₂And Xaa₃Are all absent, then Xaa₁Must be β -carboxylated Asp or gamma-carboxylated Glu, or Xaa₁Must be Asp, D-Asp, Glu, D-Glu, Asu, Aad, Apm, and must be modified at its amino group by oxalic, malonic or succinic acid, glutaric, adipic, pimelic or suberic acid.

In some embodiments, Xaa₂Asp or Glu. In other embodiments, Xaa₂Is Asp.

In some embodiments, Xaa₃Asp or Glu. In other embodiments, Xaa₃Is Asp.

In some embodiments, Xaa₄Cys is used.

In some embodiments, Xaa₆Is Glu.

In some embodiments, Xaa₇Tyr or Leu.

In some embodiments, Xaa₈Cys is used.

In some embodiments, Xaa₁₄Is Thr.

In some embodiments, Xaa₁₆Cys is used.

In some embodiments, Xaa₁₇Is Tyr.

In some embodiments, Xaa₁₇Is absent.

In some embodiments, the present invention provides a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide consists of the amino acid sequence Xaa₁Xaa₂Xaa₃Cys₄Cys₅Glu₆Xaa₇Cys₈Cys₉Asn₁₀Pro₁₁Ala₁₂Cys₁₃Thr₁₄Gly₁₅Cys₁₆Xaa₁₇(SEQ ID NO: 2); wherein

Xaa₂asp or Glu;

Xaa₃asp, Glu or absent;

Xaa₇tyr or Leu; and

Xaa₁₇tyr or absent.

Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：3)；

Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：4)；

Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly CysTyr(SEQ IDNO：5)；

Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly CysTyr(SEQ IDNO：6)；

Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：7)；

Asn Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：8)；

β-Ala Asp Asp Cys Cys GluTyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：18)；

Glu Glu Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：35)；

Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：36)；

Glu Glu Cys Cys Glu Leu Cys Cys AsnPro Ala Cys Thr Gly CysTyr(SEQ IDNO：37)；

Glu Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：43)；

Glu Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：44)；

In other embodiments, the present invention provides a peptide, or a pharmaceutically acceptable salt thereof, wherein the peptide consists of the amino acid sequence of seq id no:

Glu Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr(SEQ ID NO：67)；

In some embodiments, the present invention provides a peptide, or a pharmaceutically acceptable salt thereof, wherein the peptide consists of the amino acid sequence

Xaa₁Asp₂Xaa₃Cys₄Cys₅Glu₆Xaa₇Cys₈Cys₉Asn₁₀Pro₁₁Ala₁₂Cys₁₃Thr₁₄Gly₁₅Cys₁₆Xaa₁₇(SEQ ID NO. 68); wherein

Xaa₃asp or absent;

Xaa₇tyr or Leu; and

Xaa₁₇tyr or absent.

In some embodiments, the peptide consists of the amino acid sequence:

Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：3)；

Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：4)；

Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly CysTyr(SEQ IDNO：5)；

Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly CysTyr(SEQ IDNO：6)；

Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：7)；

Asn Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys(SEQ IDNO：8)；

Glu Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：43)；

Glu Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ IDNO：44)；

In some cases, the peptide is isolated. In other cases, the peptide is purified.

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

Variant peptides

In some cases, it may be desirable to treat a patient with a variant peptide that binds to and activates an intestinal GC-C receptor, but is less or more active than the non-variant form of the peptide. The reduced activity may be due to reduced affinity for the receptor or reduced ability to activate the receptor upon binding or reduced stability of the peptide. The increased activity may be due to increased affinity for the receptor or increased ability to activate the receptor upon binding or increased stability of the peptide.

In some peptides, one or both portions of one or both Cys residue pairs that normally form disulfide bonds may be substituted with homocysteine, penicillamine, 3-mercaptoproline (Kolodziej et al, 1996 Int J Pept Protein Res 48: 274); beta, beta-dimethylcysteine (Hunt et al, 1993Int J Pept Protein Res 42: 249) or diaminopropionic acid (Smith et al, 1978J MedChem 21: 117) to form alternative internal crosslinks at the sites of the normal disulfide bonds. In other embodiments, the disulfide bonds may be replaced by hydrocarbon crosslinks (Schaffeister et al, 2000J Amchem Soc 122: 5891, Patgiri et al, 2008Acc Chem Res 41: 1289, Henchey et al, 2008 Curr Opin Chem Biol 12: 692).

Preparation of peptides

In one embodiment, the peptides or pre-peptides of the invention may be recombinantly produced in any known protein expression system, including, without limitation, bacteria (e.g., e.coli) or bacillus subtilis), insect cell systems (e.g., drosophila Sf9 cell system), yeast cell systems (e.g., brewer's yeast (s.cerevisiae), saccharomyces cerevisiae (s.saccharomyces)), or filamentous fungal expression systems or animal cell expression systems (e.g., mammalian cell expression systems). The peptides or precursor peptides of the invention may also be chemically synthesized.

If the peptide or variant peptide is to be recombinantly produced (e.g., E.coli), the nucleic acid molecule encoding the peptide may also encode a leader sequence that allows secretion of the mature peptide from the cell. Thus, the sequence encoding the peptide may include, for example, the pre sequence and pro sequence of the native bacterial ST peptide. The secreted mature peptide can be purified from the culture medium.

The sequences encoding the peptides described herein can be inserted into vectors capable of delivering and maintaining nucleic acid molecules in bacterial cells. The DNA molecule may be inserted into an autonomously replicating vector (suitable vectors include, for example, pGEM3Z and pcDNA3, as well as derivatives thereof). The vector nucleic acid may be bacterial or bacteriophage DNA such as bacteriophage lambda or M13 and derivatives thereof. Construction of vectors containing the nucleic acids described herein can be performed after transformation of a host cell, such as a bacterium. Suitable bacterial hosts include, but are not limited to, E.coli, Bacillus subtilis, Pseudomonas (Pseudomonas) and Salmonella (Salmonella). In addition to the coding nucleic acid molecules, the gene constructs also comprise elements which allow expression, such as promoters and regulatory sequences. The expression vector may contain transcriptional control sequences that control the initiation of transcription, such as promoter, enhancer, operator, and repressor sequences. A variety of transcriptional control sequences are well known to those skilled in the art. The expression vector can also include translational regulatory sequences (e.g., untranslated 5 'sequences, untranslated 3' sequences, or internal ribosome entry sites). The vector is capable of autonomous replication or it may integrate into the host DNA to ensure stability during peptide production.

The protein coding sequence comprising the peptides described herein may also be fused to a nucleic acid encoding a peptide affinity tag (e.g., glutathione S-transferase (GST), maltose E binding protein, protein A, FLAG tag, hexahistidine, myc tag, or influenza HA tag) to facilitate purification. Affinity tag or reporter fusion the reading frame of the peptide of interest is linked to the reading frame of the gene encoding the affinity tag thus producing a translational fusion. Expression of the fusion gene results in translation of a single peptide comprising the target peptide and the affinity tag. In some cases where an affinity tag is used, the DNA sequence encoding the protease recognition site will be fused between the affinity tag and the reading frame of the target peptide.

Suitable for the preparation of the peptide and variants described herein in protein expression systems other than bacteria in immature and mature forms, and to the technicians in this field are familiar with the gene constructs and methods, can also be used in biological systems for the preparation of peptides.

In other embodiments, peptides containing amino acids not normally incorporated by the translational machinery and the amino acids described above (e.g., β -carboxylated Asp, γ -carboxylated Glu, Asu, Aad, and Apm) can be recombinantly made by tRNA modification methods. Methods for modifying tRNA's, including but not limited to modifying the anticodon, amino acid attachment site, and/or acceptor stem to allow for unnatural and/or arbitrary amino acid incorporation, are known in the art (biochem. Biophys. Res. Comm. (2008) 372: 480-485; chem. Biol. (2009) 16: 323-36; nat. Methods (2007) 4: 239-44; nat. Rev. mol. cell Biol. (2006) 7: 775-82; Methods (2005) 36: 227-238; Methods (2005) 36: 270-278; Annu. Rev. biochem. (2004) 73: 147-176; Nuc. acids Res. (2004) 32: 2004 6200-6211; Proc. Natl. Acad. Sci. USA (2003) 100: 6353; Roya 6357: 422) 422: 422-.

In some embodiments, the peptide may be prepared chemically. Peptides can be synthesized by a number of different methods, including solution and solid phase synthesis using traditional BOC or FMOC protection. For example, peptides can be synthesized using sequential amino acid couplings on 2-chlorotrityl or Wang resins. The following protecting groups may be used: fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl (alpha-amino, N-terminal); trityl or tert-butyl (thiol group of Cys); tert-butyl (gamma-carboxy of glutamic acid and hydroxy of threonine, if present); and trityl (the β -amide function of the asparagine side chain and the phenolic group of tyrosine, if present). The coupling can be carried out using DIC and HOBt in the presence of a tertiary amine, and the peptide can be deprotected and cleaved from the solid support using mixture K (trifluoroacetic acid 81%, phenol 5%, thioanisole 5%, 1, 2-ethanedithiol 2.5%, water 3%, dimethylsulfide 2%, ammonium iodide 1.5% w/w). After removal of trifluoroacetic acid and other volatiles, the peptide can be precipitated using an organic solvent. Disulfide bonds between Cys residues can be formed using dimethylsulfoxide (Tam et al, (1991) J.am.chem.Soc.113: 6657-62) or using an air oxidation strategy. The resulting peptide can be purified by reverse phase chromatography and lyophilized.

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

Peptide composition and GC-C receptor agonist

In another aspect, the provided compositions can be prepared by combining the peptides (alone or in combination) with any pharmaceutically acceptable carrier or vehicle. The peptides may be combined with materials that do not produce harmful, allergic, or otherwise undesirable reactions when administered to a patient. The carrier or medium employed may include solvents, dispersants, coatings, absorption promoters, controlled release agents and one or more inert excipients including starches, polyols, granulating agents, microfine celluloses, microcrystalline celluloses (e.g., Celphere)) Diluents, lubricants, binders, disintegrants, etc.), and the like. Of the disclosed compositions, if desiredThe tablet dosage may be coated by standard aqueous or anhydrous techniques.

Examples of excipients and other ingredients described above that serve as pharmaceutically acceptable carriers and pharmaceutically acceptable inert carriers include, but are not limited to, binders, fillers, disintegrants, lubricants, antimicrobial agents, and coating agents.

The term "binder" as used herein refers to any pharmaceutically acceptable binder that can be used in the practice of the present invention. Examples of pharmaceutically acceptable binders include, without limitation, starches (e.g., corn STARCH, potato STARCH, and pregelatinized STARCH (e.g., STARCH)And STARCH1500Sold by Colorcon, ltd.) and other starches), maltodextrin, gelatin, natural and synthetic gums such as acacia, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose (hypromellose), ethylcellulose, cellulose acetate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, carboxymethylcellulose, powdered cellulose, micronized cellulose, microcrystalline cellulose (e.g., AVICEL @)^TME.g. AVICEL-PH-101^TM、-103^TMAnd-105^TMSold by FMCCorporation, Marcus Hook, PA, USA)), polyvinyl alcohol, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K30), and mixtures thereof.

Examples of binders that can be used in particular in the pharmaceutical composition include polyvinyl alcohol, polyvinylpyrrolidone (povidone), starch, maltodextrin or cellulose ethers such as methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose and hydroxypropyl methyl cellulose.

The term "filler" as used herein refers to any pharmaceutically acceptable filler that may be used in the practice of the present invention. Examples of pharmaceutically acceptable fillers include, without limitation, 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), microcrystalline cellulose, powdered cellulose, dextrates (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, palatinite, xylitol, inositol, and mixtures thereof.

Examples of pharmaceutically acceptable fillers that may be particularly useful for coating the peptide include, without limitation, 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, dibasic calcium phosphate, raffinose, maltitol, melezitose, stachyose, lactitol, palatinite, xylitol, mannitol, inositol, and mixtures thereof.

The term "additive" as used herein refers to any pharmaceutically acceptable additive. Pharmaceutically acceptable additives include, without limitation, disintegrants, dispersing additives, lubricants, glidants, antioxidants, coating additives, diluents, surfactants, flavoring additives, wetting agents, absorption-promoting additives, controlled-release additives, anti-caking additives, antimicrobial agents (e.g., preservatives), colorants, drying agents, plasticizers, and dyes. As used herein, an "excipient" is any pharmaceutically acceptable additive, filler, binder or agent.

The compositions of the present invention may also optionally include other therapeutic ingredients, anti-caking agents, preservatives, sweeteners, colorants, flavorants, drying agents, plasticizers, dyes, glidants, anti-adherents, antistatic agents, surfactants (wetting agents), antioxidants, film coating agents, and the like. Any of these optional ingredients must be compatible with the compounds described herein to ensure stability of the formulation. The composition may contain other additives as desired, including, for example, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, raffinose, maltitol, melezitose, stachyose, lactitol, palatinite, starch, xylitol, mannitol, inositol, and the like, and hydrates thereof, as well as amino acids such as alanine, glycine, and betaine, and peptides and proteins such as albumin.

The composition may include, for example, various other solvents, dispersants, coatings, absorption-promoting additives, controlled-release additives, and one or more inert additives (including, for example, starches, polyols, granulation additives, microcrystalline cellulose, diluents, lubricants, binders, disintegration additives, and the like), and the like. If desired, tablet doses of the disclosed compositions can be coated by standard aqueous or anhydrous techniques. The composition may also include, for example, anti-caking additives, preservatives, sweetening additives, coloring agents, flavoring agents, drying agents, plasticizers, dyes, and the like.

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

Suitable lubricants include, for example, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerol, 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 dodecanoate, agar, syloid silica gel (AEROSIL 200, w.r.grace co., Baltimore, MD USA), condensation aerosols of synthetic silica (Evonik Degussa co., Plano, TX USA), fumed silica (CAB-O-SIL, cabotco., Boston, MAUSA), and mixtures thereof.

Suitable glidants include, for example, leucine, colloidal silicon dioxide, magnesium trisilicate, powdered cellulose, starch, talc and calcium phosphate.

Suitable anti-caking additives include, for example, calcium silicate, magnesium silicate, silica, colloidal silica, talc, and mixtures thereof.

Suitable antimicrobial additives that may be used (e.g., as preservatives for peptide compositions) include, for example, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butyl paraben, cetylpyridinium chloride, cresol, chlorobutanol, dehydroacetic acid, ethyl paraben, methyl paraben, phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate, potassium sorbate, propyl paraben, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid, thimerosal (thimerosol), thymo, and mixtures thereof.

Suitable antioxidants include, for example, BHA (butylhydroxyanisole), BHT (butylhydroxytoluene), vitamin E, propyl gallate, ascorbic acid and salts or esters thereof, tocopherol and esters thereof, alpha-lipoic acid, and beta-carotene.

Suitable coating additives include, for example, sodium carboxymethylcellulose, cellulose acetate phthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropyl methylcellulose 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 Aquaeouous 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).

In certain embodiments, suitable additives for the peptide composition include one or more of sucrose, talc, magnesium stearate, crospovidone, or BHA.

The compositions of the present invention may also include other excipients, agents, and types thereof including, but not limited to, L-histidine,Poloxamers (e.g. Poloxamers)And Poloxamer 188), ascorbic acid, glutathione, permeability enhancers (e.g., lipids, sodium cholate, acylcarnitines, salicylates, mixed bile salts, fatty acid micelles, chelators, fatty acids, surfactants, medium chain glycerides), protease inhibitors (e.g., soybean trypsin inhibitor, organic acids), pH-lowering agents, and absorption enhancers effective to promote bioavailability (including but not limited to those described in U.S. patent nos. 6,086,918 and 5,912,014), materials for chewable tablets (such as glucose, fructose, lactose monohydrate, lactose and aspartame, lactose and cellulose, maltodextrin, maltose, mannitol, microcrystalline cellulose and guar gum, sorbitol crystals); parenteral drugs (such as mannitol and povidone); plasticizers (such as dibutyl sebacate, plasticizers for coatings, polyvinyl acetate phthalate); powder lubricants (e.g., glyceryl behenate); soft gelatin capsules (e.g., sorbitol special solutions); pellets for coating (e.g. sugar pellets); spheronizing agents (such as glyceryl behenate and microcrystalline cellulose); suspending/gelling agents (e.g., carrageenan, gellan gum, mannitol, microcrystalline cellulose, povidone, sodium starch glycolate, xanthan gum); sweeteners (e.g., aspartame and lactose, glucose, fructose, honey, maltodextrin, maltose, mannitol, molasses, sorbitol crystals, sorbitol specific solutions, sucrose); wet granulation agent (such as calcium carbonate, anhydrous lactose, lactose monohydrate, maltodextrin, mannitol, microcrystalline cellulose, povidone, starch), caramel, sodium carboxymethylcellulose, cherry butter flavor and cherry flavor, anhydrous citric acid, lemonAcid, sugar powder (confectioner's sugar), D&C red No. 33, D&C yellow #10 aluminum lake, edetate disodium, ethanol 15%, FD&C yellow No.6 aluminum lake, FD&C blue #1 aluminum lake, FD&C blue No. 1, FD&Aluminum C blue No. 2 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, glyceryl palmitostearate, glyceryl monostearate, indigo carmine, lecithin, mannitol, methyl and propyl parabens, monoammonium glycyrrhizinate, natural and artificial orange flavor, medicinal glaze, poloxamer 188, polydextrose, polysorbate 20, polysorbate 80, povidone, pregelatinized corn starch, pregelatinized starch, red iron oxide, sodium saccharin, carboxymethyl ether sodium, sodium chloride, sodium citrate, sodium phosphate, strawberry flavor, synthetic black iron oxide, synthetic red iron oxide, titanium dioxide, and white wax.

In some embodiments, provided are compositions comprising a peptide described herein and one or more selected from Mg²⁺、Ca²⁺、Zn² ⁺、Mn²⁺、K⁺、Na⁺Or Al³⁺Combinations thereof and/or stabilizers for sterically hindered primary amines. In other embodiments, the agent is Mg²⁺、Ca²⁺Or Zn²⁺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 other embodiments, the cation is provided as magnesium 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.

In another embodiment, the stabilizer is a sterically hindered primary amine. In yet another embodiment, the sterically hindered primary amine is an amino acid. In yet another embodiment, the amino acid is a natural amino acid. In yet another embodiment, the natural 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; still further, the natural amino acid is leucine, isoleucine, alanine, or methionine. In yet another embodiment, the natural amino acid is leucine. In another embodiment, the sterically hindered primary amine is a non-natural amino acid (e.g., 1-aminocyclohexane carboxylic acid, lanthanine, or theanine). In yet another embodiment, the sterically hindered primary amine is cyclohexylamine, 2-methylbutylamine, or a polymeric amine such as chitosan. In another embodiment, one or more sterically hindered primary amines may be used in the composition.

In some cases, the sterically hindered primary amine has the formula: ,wherein R is₁、R₂And R₃Independently selected from: H. c (O) OH, C₁-C₆Alkyl radical, C₁-C₆Alkyl ethers, C₁-C₆Alkyl sulfides, C₁-C₆Alkyl carboxylic acid, C₁-C₆Alkylcarboxamides and alkylaryl groups, any of which may be mono-or polysubstituted by halogen or amino, and with the proviso that R is₁、R₂And R₃No more than two of which are H. In another embodiment, R₁、R₂And R₃No more than one of which is H.

In other embodiments, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier, a peptide selected from Mg²⁺、Ca²⁺、Zn²⁺、Mn²⁺、K⁺、Na⁺Or Al³⁺Pharmaceutical composition of a cationic and sterically hindered primary amine or mixtures thereof. In one embodiment, the cation is Mg²⁺、Ca²⁺Or Zn²⁺Or mixtures thereof. In yet another embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable binder and/or a pharmaceutically acceptable glidant, lubricant or 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.

In some cases, the molar ratio of cation to sterically hindered primary amine to peptide in the aqueous solution applied to the support is from 5-100: 5 to 50: 1. In some cases, the molar ratio of cationic to sterically hindered primary amine can be equal to or greater than 2: 1 (e.g., between 5: 1 and 2: 1). Thus, in some cases, the molar ratio of cation to sterically hindered primary amine to peptide applied to the support 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: 1, 10: 1, 10: 5: 1, or 5: 10: 1. When a binder (e.g., methylcellulose) is present in the GC-C agonist peptide solution applied to the support, it may be present at 0.5 wt% to 2.5 wt% (e.g., 0.7% to 1.7% or 0.7% to 1%, 1.5% or 0.7%).

In yet another embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable binder or additive, and/or a pharmaceutically acceptable glidant, lubricant, or additive that acts as both a glidant and a lubricant and/or an antioxidant.

Suitable pharmaceutical compositions according to the present invention will generally comprise an amount of the active compound(s) in association with a pharmaceutically acceptable diluent or excipient (e.g., sterile aqueous solution) to achieve the final concentration range (depending on the intended use). Preparation techniques are generally well known in the art, as exemplified by Remington's Pharmaceutical Sciences (18 th edition, Mack publishing company, 1995).

For the treatment of gastrointestinal disorders, the peptides described herein are preferably administered orally, e.g., as tablets, capsules, sachets containing a predetermined amount of particles of the active ingredient, gels, ointments, syrups, pills, electuaries, slurries, powders, lyophilized powders, sachets of particles, as a solution or suspension in an aqueous liquid or a non-aqueous liquid; as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, by a liposome formulation (see, e.g., EP 736299) or in some other form. Compositions for oral administration may include binders, lubricants, inert diluents, lubricants, surfactants or dispersants, flavoring agents, and humectants. Formulations for oral administration, such as tablets, may optionally be coated or labeled and may be formulated so as to provide long, sustained or controlled release of the active ingredient therein. The peptides may be administered with other agents useful in the treatment of gastrointestinal disorders including, but not limited to, the agents described herein.

In another aspect, a suitable pharmaceutical composition may comprise one or more additional therapeutic agents. These therapeutic agents include, but are not limited to, analgesics; antisecretory agents including proton pump inhibitors, acid pump antagonists, H2 receptor antagonists; a PDE5 inhibitor; GABA-B antagonists; a bile acid sequestrant; a prokinetic agent; an antidepressant; (ii) an antibiotic; a vomit-stopping agent; and a mucosa-protecting agent.

Method of treatment

In some embodiments of the invention, methods of treatment for gastrointestinal disorders are provided.

In some embodiments, the gastrointestinal disorder is an upper GI disorder. In yet another embodiment, the disorder is GP, post-operative gastrointestinal obstruction, functional esophageal disorder, functional gastroduodenal disorder, gastroesophageal reflux disease (GERD), celiac disease, mucositis, or a duodenal or gastric ulcer.

In some embodiments, the gastrointestinal disorder is GP. In other embodiments, the GP is an idiopathic, diabetic, or post-operative GP.

In some embodiments, the gastrointestinal disorder is post-operative gastrointestinal obstruction.

In some embodiments, the gastrointestinal disorder is a functional esophageal disorder.

In some embodiments, the functional esophageal disorder is functional heartburn, presumed esophageal-derived functional chest pain, functional dysphagia, or globus hystericus.

In some embodiments, the gastrointestinal disorder is a functional gastroduodenal disorder.

In some embodiments, the functional gastroduodenal disorder is FD, belching, nausea or vomiting disorder, or rumination syndrome. In yet another embodiment, the functional gastroduodenal disorder is FD. In some embodiments, the FD is postprandial discomfort syndrome or epigastric pain syndrome. In some embodiments, the eructation disorder is a gas regurgitation disorder or undefined excessive eructation. In some embodiments, the nausea or vomiting disorder is chronic idiopathic nausea, functional vomiting, or periodic vomiting syndrome.

In some embodiments, the gastrointestinal disorder is gastroesophageal reflux disease (GERD).

In some embodiments, the gastrointestinal disorder is celiac disease.

In some embodiments, the gastrointestinal disorder is mucositis.

In some embodiments, the gastrointestinal disorder is a duodenal or gastric ulcer.

The peptides and agonists described herein may be used alone or in a combination therapy to treat, prevent or reduce visceral pain associated with a gastrointestinal disorder or pain associated with another disorder as described herein.

The GC-C receptor agonists described herein may be administered in combination with other agents. For example, the peptide may be administered with an analgesic peptide or compound. The analgesic peptide or compound may be covalently linked to a peptide described herein, or it may be a separate agent administered in combination therapy together or sequentially with a peptide described herein. The GC-C receptor agonists described herein may also be administered in combination with other agents useful for treating upper GI disorders, including antidepressants, prokinetic agents, antiemetics, antibiotics, proton pump inhibitors, acid blockers (e.g., histamine H2 receptor antagonists), acid pump antagonists, PDE5 inhibitors, GABA-B agonists, bile acid sequestrants, and mucoprotectants.

In some embodiments, useful analgesics that may be used with the peptides described herein include Ca channel blockers (e.g., ziconotide), 5HT receptor antagonists (e.g., 5HT3, 5HT4, and 5HT1 receptor antagonists), 5HT4 agonists (e.g., tegaserod)Mosapride (mosapride), zacopride (zacopride), cisapride (cisapride), renzapride (renzapride), prucalopride (prucalopride)Benzimidazolone derivatives such as BIMU 1 and BIMU 8, and lisapride (lirexapride)), 5HT1 agonists (e.g., sumatriptan (sumatriptan) and buspirone (buspirone)), opioid receptor agonists (e.g., loperamide (loperamide), fedotozine (fedotozine), enkephalin pentapeptide, morphine, diphenyl oxalate, frafamine (frakeffamide), trimebutine (trimebutine) and fentanyl (fentanyl)), CCK receptor agonists (e.g., cloglutethimide (loxyglumide) and dexcloglutethimide (dexloxygride)), NK 56 receptor antagonists (e.g., aprepitant), voritant (voritant), eprotant (ezetitanat), R-673 (Hoffmann-Lache), SR-481408 and Klebsiella (elhoffol), SR-6735, Klebs-673, Klebs-6735), Klebs-6735 (Klebs-3, Klebs-3 (Klebs-3, Klebsiella (e), opioid receptor antagonists such as E, Klebs-3, Klebs-, nepadutant (nepadutant), saredutant, GW597599(Glaxo Smith Kline), SR-144190(Sanofi-Synthelabo) and UK-290795(Pfizer Inc)), NK3 receptor antagonists (e.g., osanetant (S.osnanetant)) (S.R-142801; sanofi-synthiabo), SR-241586 and talnetant (talnetant), noradrenaline-serotonin reuptake inhibitors (NSRI) (e.g., milnacipran), mixed and selective dopamine receptor antagonists (e.g., metoclopramide, itopride, domperidone), capsaicin and cannabinoid receptor agonists, sialorphin and sialorphin related peptides. Various classes of analgesics are described in the literature.

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

Examples of antidepressants include, without limitation, tricyclic antidepressants such as amitriptyline (amitriptyline)Desipramine (desipramine)Imipramine (imipramine)Amoxicapine (amoxapine)Nortriptyline (nortriptyline); selective serotonin reuptake inhibitors (SSRI's) such as paroxetine (parooxetine)Fluoxetine (fluooxetine)Sertraline (sertraline)And citalopram (citalopram)And others such as doxepin (doxepin)And trazodone (trazodone)

Examples of prokinetic agents include, without limitation, itopride (itopride), octreotide (octreotide), bethanecol (bethanecol), metoclopramideDomperidone derivativesErythromycin (and derivatives thereof) and cisaprideExamples of antiemetic agents include, but are not limited to, prochlorperazine.

Examples of antibiotics that may be used include those that may be used to treat helicobacter pylori (helicobacter pylori) infections, such as amoxicillin (amoxicillin), tetracycline, metronidazole (metronidazole) or clarithromycin (clarithromycin). Other antibiotics such as erythromycin and derivatives thereof may also be used in combination with the peptides described herein.

Examples of proton pump inhibitors include, but are not limited to, omeprazole (omeprazole)Esomeprazole (esomeprazole)Lansoprazole (lansoprazole)Pantoprazole (pantoprazole)And rabeprazole (rabeprazole)Examples of H2 receptor blockers include, but are not limited to, cimetidine (cimetidine), ranitidine (ranitidine), famotidine (famotidine), and nizatidine (nizatidine). Examples of acid pump antagonists include, without limitation, revaprazan, CS-526(j. pharmacol. exp. ther. (2007) 323: 308-.

Examples of PDE5 inhibitors include, without limitation, avanafil (avanafil), lotdenafil (lodenafil), milodenafil (mirodenafil), sildenafil (sildenafil) citrate, tadalafil (tadalafil), vardenafil (vardenafil), and udenafil (udenafil). GABA-B agonists include, but are not limited to baclofen (baclofen) and XP19986(CAS registry number 847353-30-4). Examples of bile acid sequestrants include, without limitation, GT102-279, cholestyramine (cholestyramine), colesevelam (colesevelam), colesevelam hydrochloride, ursodeoxycholic acid, colestipol (colestipol), colestilan (colestilan), sevelam (sevelamer), polydiallylamine crosslinked with epichlorohydrin, dialkylaminoalkyl derivatives of crosslinked dextran, and N- (cycloalkyl) alkylamines. Examples of mucosal protectants include, without limitation, sucralfate (Carafate), teprenone (teprenone), polaprezinc (polaprezinc), cetrarate (cetraxate), and bismuth salicylate (bismuth subsalicylate).

Combination therapy may be achieved by administering two or more agents, such as a GC-C receptor agonist and another therapeutic peptide or compound described herein, each formulated and administered separately, or by administering two or more agents in a single formulation. Combination therapy also encompasses other compositions. For example, two agents may be formulated together and administered with a separate formulation containing a third agent. While two or more agents in a combination therapy may be administered simultaneously, it is not necessary. For example, the administration of the first agent (or combination of agents) can precede the administration of the second agent (or combination of agents) by minutes, hours, days, or weeks. Thus, the two or more agents may be within minutes of each other; 1. within 2, 3, 6,9, 12, 15, 18, or 24 hours; 1. within 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days; or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some cases even longer intervals are possible. While in many cases it is desirable that two or more agents used in combination therapy be present in the patient at the same time, this is not necessary.

Dosage form

The dosage range for adults may typically be 5 μ g to 100 mg/day for oral administration of the GC-C peptide agonist described herein. Tablets, capsules, or other forms of presentation provided in discrete units may conveniently contain an amount of a compound described herein effective at these dosages or as a plurality of the same (for example) units containing from 25 μ g to 2mg or from about 100 μ g to 1 mg. The precise amount of compound prescribed to the patient should be in accordance with the attending physician. However, the dosage employed will depend upon a number of factors including the age and sex of the patient, the precise condition being treated and its severity.

In various embodiments, the dosage units are administered as follows: with food administration at any time of day, without food administration at any time of day, after an overnight fast with food administration (e.g., with breakfast), after a low fat snack before sleep. In a particular embodiment, the dosage unit is administered before or after a meal (e.g., a meal). In yet another embodiment, the dosage unit is administered about 15 minutes to 1 hour prior to food intake. 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 daily dose are equivalent.

In the combination therapy embodiments of the present invention, the precise amount of each of the two or more active ingredients in a dosage unit will depend upon the desired dosage of each component. Thus, it may be useful to manufacture dosage units that, when administered according to a particular dosage schedule (e.g., a dosage schedule that specifies certain units and particular administration times), will deliver the same dose of the various components as would be administered if the patient were being treated with only a single component. In other cases, it may be desirable to manufacture a dosage unit that will deliver a dose of one or more components that is less than the dose administered if the patient is being treated with only a single component. Finally, it may be desirable to manufacture a dosage unit that will deliver a dose of one or more components that is greater than the dose administered if the patient is being treated with only a single component.

The pharmaceutical composition may include other ingredients, including but not limited to the active ingredients and excipients described herein. In certain embodiments, the one or more therapeutic agents of the dosage unit may be present in a sustained or controlled release formulation and the other therapeutic agent may not be present in the sustained release formulation. For example, a peptide or agonist described herein can be present in a controlled or sustained release formulation in the same dosage unit as another agent that may or may not be in the controlled or sustained release formulation. Thus, in certain embodiments, it may be desirable to provide for immediate release of one or more of the agents described herein, as well as controlled release of one or more other agents.

The invention has been described with reference to certain exemplary embodiments thereof. However, it will be readily apparent to those skilled in the art that it is possible to embody the invention in specific forms other than those of the exemplary embodiments described above. This can be accomplished without departing from the spirit of the invention. The exemplary embodiments are merely illustrative and should not be considered restrictive in any way. The scope of the invention is defined by the appended claims and equivalents thereof, rather than by the foregoing description.

Examples

GC-C agonist peptides as described herein or pharmaceutically acceptable salts thereof are prepared by solid phase chemical synthesis and native folding (air oxidation) from American Peptide Company (Sunnyvale, CA). The peptides and their sequences are shown below (where the amino acid sequence is the standard one-letter code and "dD" refers to D-Asp, "dA" refers to D-Ala, "dY" refers to D-Tyr, "dN" refers to D-Asn and "β a" refers to β -Ala):

peptide name	Amino acid sequence	ID
			Peptide 1	CCELCCNPACTGCY	(SEQ ID NO：69)
Peptide 2	CCEFCCNPACTGCY	(SEQ ID NO：70)
			Peptide 3	NDDCCEYCCNPACTGCY	(SEQ ID NO：53)
Peptide 4	DDCCEYCCNPACTGCY	(SEQ ID NO：54)
			Peptide 5	DDDCCEYCCNPACTGCY	(SEQ ID NO：55)
Peptide 6	dDDDCCEYCCNPACTGCY	(SEQ ID NO：47)
			Peptide 7	GDDCCEYCCNPACTGCY	(SEQ ID NO：56)
Peptide 8	PDDCCEYCCNPACTGCY	(SEQ ID NO：57)
			Peptide 9	ADDCCEYCCNPACTGCY	(SEQ ID NO：58)
Peptide 10	dADDCCEYCCNPACTGCY	(SEQ ID NO：48)
			Peptide 11	NDDCCELCCNPACTGCY	(SEQ ID NO：59)
Peptide 12	dNDDCCELCCNPACTGCY	(SEQ ID NO：49)
			Peptide 13	ADDCCELCCNPACTGCY	(SEQ ID NO：60)
Peptide 14	dADDCCELCCNPACTGCY	(SEQ ID NO：50)
			Peptide 15	βADDCCELCCNPACTGCY	(SEQ ID NO：61)
Peptide 16	DDCCELCCNPACTGCY	(SEQ ID NO：62)
			Peptide 17	DDDCCELCCNPACTGCY	(SEQ ID NO：63)
Peptide 18	PDDCCELCCNPACTGCY	(SEQ ID NO：64)
			Peptide 19	GDDCCELCCNPACTGCY	(SEQ ID NO：65)
Peptide 20	DDDCCELCCNPACTGC	(SEQ ID NO：66)
			Peptide 21	DDDCCELCCNPACTGCdY	(SEQ ID NO：51)
Peptide 22	EEECCEYCCNPACTGCY	(SEQ ID NO：67)
			Peptide 23	dDDDCCELCCNPACTGC	(SEQ ID NO：52)

Example 1: accumulation of cGMP in T84 cells for GC-C Activity analysis

GC-C activity of cGMP accumulation in T84 cells was analyzed for the cGMP assay, 4.5 × 10⁵cells/mL of T84 cells were grown overnight in 24-well tissue culture plates. The following day, T84 cells were washed twice with 1mL of DMEM +20mM MES (pH 5) or DMEM +50mM sodium bicarbonate (NaBicarb, pH 8), where these buffers were serum free. After the second wash, the cells were incubated with 450 μ L of 1mM Isobutylmethylxanthine (IBMX) in pH 5 or pH 8 buffer for 10 min at 37 ℃ to inhibit any phosphodiesterase activity. The peptide was then diluted to 10x concentration in pH 5 or pH 8 buffer. 50 μ L of the peptide solution was diluted with T84 cells to a final volume of 500 μ L to give a 1X concentration of each peptide. Eleven-point curve analysis (in nM) was performed for each peptide at the final peptide concentration tested in each assay: 10000. 3000, 1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1.

There is no peptide control for determining endogenous levels of cGMP. The peptides were incubated at 37 ℃ for 30 minutes. After 30min, the supernatant was removed and the cells were lysed with 0.1M HCl on ice for 30 min.

After 30 minutes, the lysate was pipetted and placed into a 96-well HPLC plate and centrifuged at 10,000g for 10 minutes to remove any cellular debris. The supernatant was removed from the above centrifugation and placed into a new 96 well HPLC plate. The samples were diluted with an equal volume of 1M ammonium acetate (pH 7) to neutralize the samples for better chromatography. A2 x cGMP standard curve was prepared in 0.1M HCl and subsequently diluted with an equal volume of 1M ammonium acetate, with the final concentrations (in nM) as follows: 1024. 512, 256, 128, 64, 32, 16, 8, 4, 2, 1.

The cGMP concentration of each sample was determined using LC/MS parameters (table 1 below) and the calculated standard curve. EC (EC)₅₀Values were calculated from concentration-response curves generated with GraphPad Prism Software.

Table 1: LC/MS parameters

Effect of pH on cGMP production

The ability of GC-C agonist peptides to stimulate cGMP synthesis in human T84 cells at pH 5 and pH 8 was compared. Figure 1 shows the effect on T84cGMP dose response for peptide 1 and peptide 17. Table 2 lists exemplary peptides of the invention tested, summarizing the EC for various peptides at pH 5 and pH 8₅₀The value is obtained. The EC of various peptides at pH 8 and pH 5 is also provided₅₀A ratio.

Table 2: cGMP response in T84 cells

EC at pH 8₅₀Not quantifiable, but greater than 1 mM.

Due to EC at pH 8₅₀Cannot be quantified, so EC cannot be calculated₅₀pH 8/EC₅₀Ratio of pH 5.

Example 2: is thin at T84Competitive radioligand binding on cells

A competitive radioligand binding assay was performed using whole human T84 cells from the American Type Culture Collection (ATCC; Manassas, Va.). T84 cells were cultured in Dulbecco's Modified Eagle Medium (Dulbecco's Modified Eagle Medium): ham's F-1250/50 medium (DMEM/F12) + 5% Fetal Bovine Serum (FBS) were grown as monolayers on T-150 plastic bottles to 60-70% confluence. Cells were harvested by gentle scraping with a cell scraper and harvested by centrifugation at 2000g for 10 min at 4 ℃. Cells were washed twice by gently resuspending in Phosphate Buffered Saline (PBS) and harvested by centrifugation as described above.

Prepared by dissolving 100. mu.g of NTFYCCELCCNPACAGCY (SEQ ID NO: 71) (EnterotoxinSTp; Bachem H-6248) in 0.5mL of water, sending it to Perkin-Elmer Life and Analytical Sciences (N.Billerica, MA) using the lactoperoxidase method described in Marchanoli, J.J., "An enzymatic method for the transduction of immunoglobulins and other proteins", biochem.J. (1969)113, 299-305¹²⁵I]-an STp radioligand. The Perkin-Elmer purified the labeled tracer by HPLC using a Waters C-18. mu. Bondapak column (25cm) previously equilibrated with 10mM ammonium acetate (pH 5.8). A gradient of 0 to 25% acetonitrile was applied to the column over 60min, followed by a further gradient of 20min of 25% acetonitrile. The process separates the two monoiodinated forms from each other and from the unlabeled precursor. The second monoiodinated peak (peak 2, which elutes after 64min and corresponds to the iodination of the fourth tyrosine) was used as a labelled tracer in the assay. The labeled tracer has a specific activity of 2200 Ci/mmol. The tracer was stored in small portions at-20 ℃ by the arrival of Perkin-Elmer.

The conjugation reaction was performed in 0.2mL of a solution containing 2.5 × 10⁵T84 cell (0.25mg protein), 200,000cpm¹²⁵I]STp (41fmol, 200pM), 0.1 to 3,000nM competitor and 0.5% Bovine Serum Albumin (BSA) were combined in duplicate. Binding assays at pH 5.0 were performed in DMEM/20mM 2- (N-morpholino) ethanesulfonic acid (MES). At pH 8.Binding assays for 0 were performed in DMEM/20mM N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES)/50mM sodium bicarbonate. Control reactions contained no competitor (total) or no cells.

First a buffer solution was prepared and subsequently protease-free BSA was added to 0.5%. The radioligand was added to a final concentration of 0.001. mu. Ci/. mu.L. Competitor peptide stock solutions were prepared by dissolving the peptide to 1mg/mL in 50mM sodium phosphate pH 6.0. The concentration was calculated from the molecular weight of the peptide provided by the assay report. Competitor dilutions were prepared in 50mM sodium phosphate (pH 6.0) containing 20-fold the final concentration of the peptide to be detected in the binding reaction (20X competitor).

The binding reactions were combined in the following order:

i. radioligand and BSA in buffer solution.

ii.10. mu.L of 20X competitor.

T84 cells.

The binding reactions were mixed gently and incubated at 37 ℃ for 1 hour. The membrane bound was separated from free radioligand by applying the binding reaction to a 2.5cm Whatman GF/C glass fiber filter (pretreated with 1% polyvinylpyrrolidone in PBS) using vacuum filtration. The filters were rinsed twice with 5mL ice-cold PBS buffer and the retained radioligand was measured in a scintillation counter. The determination of specific binding was performed by subtracting the bound radioactivity from the reaction containing excess competitor (1 μ M) from the bound radioactivity of the various samples. Competitive radioligand-binding curves were generated using GraphPadPrism (GraphPad Software, San Diego, CA) and the data were analyzed using non-linear regression to calculate results that resulted in 50% radioligand binding (IC)₅₀) The concentration of the competitor of (a). Apparent dissociation equilibrium constants (K) of various competitors_i) By IC₅₀Values and using Cheng and Prusoff (1973) biochem. 3099 estimation of the predetermined radioligand dissociation constant obtained by the method of 3108To obtain the final product. The 200pM radioligand concentration used in the assay was very small compared to its dissociation constant, hence the calculated IC₅₀And K_iThe values (table 3) are virtually identical.

Table 3: binding affinity comparison

Example 3: gastrointestinal transport in mice

The purpose of this assay was to examine the effect of guanylate cyclase C peptide on gastrointestinal transport in vivo in mice. Orally administered doses of guanylate cyclase C agonists have been shown to increase% carbon meal stroke in mice. The assay may be indicative of the activity of the described guanylate cyclase C peptides to increase upper GI transport in the small intestine of a patient.

For the experiments, female CD-1 mice weighing 25-30g (n ═ 10 per group) were fasted overnight and given unlimited amounts of water. Activated charcoal (20 g; 100 mesh; Sigmacat #242276) was suspended in 200mL of gum arabic (100mg/mL) and stirred for at least one hour. The detection peptide was prepared in 20mM Tris (pH 6.9) mediator.

Test peptides and primers were administered by oral gavage at a dose of 200 μ L. Seven minutes after administration of the test peptide, 200 μ L of charcoal/gum arabic suspension was administered by oral gavage. After 15 minutes, by overdosing with CO₂The stroke (%) was determined as (charcoal stroke/total length of small intestine) × 100, the data was entered into the GraphPad Prism software program and analyzed by ANOVA using Bonferroni multiple comparison test after the event₅₀。

Dose-dependent effects of acute doses of exemplary peptides on GI trafficking were determined in female CD mice. The travel of the carbon front after seven minutes, expressed as a percentage of the total length of the small intestine, was used to calculate ED₅₀Values (table 4).

Table 4: potency of exemplary peptides in the mouse gastrointestinal transit assay.

Peptides	ED₅₀μg/kg
		Peptide 1	0.43
Peptide 2	2.06
		Peptide 4	6.3
Peptide 5	7.54
		Peptide 10	13.7
Peptide 14	3.04
		Peptide 17	2.9

Example 4: levels and scores of cGMP in ligated intestinal loops (ligated intestinal loops) in rodents Influence of secretion

The effect of the GC-C agonist peptide on secretion was studied by injecting the GC-C agonist peptide described herein directly into an isolated loop (isolated loop) of a wild-type rat. This was done by surgical ligation of three loops in the small intestine of each rat. The method of ligation loop formation is similar to that described in London et al, (1997) Am J Physiol, p.G93-105. The loop is approximately centered and 1-3cm in length. The loop was injected with 200. mu.L peptide/GC-C agonist (0.1-5. mu.g) or a mediator (20mM Tris, pH 7.5 or Krebs Ringer, 10mM Glucose, HEPES buffer (KRGH)). The loop was excised after a recovery time of up to 90 minutes. The weight of each ring was recorded before and after the liquid contained in each ring was removed. The length of each loop is also recorded. The weight to length ratio (W/L) of each loop was calculated to determine the effect of the GC-C agonist peptides described herein on secretion. The loop liquid volume is also determined. Data showing increased fluid and sodium bicarbonate secretion from the ligated duodenal loop of rats after 2.5 μ g peptide injection per loop are provided in table 5.

Table 5: secretion of liquid and sodium bicarbonate in rat intestinal Ring

OTHER EMBODIMENTS

All publications and patents cited in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If the meaning of a term in any patent or publication incorporated by reference conflicts with the meaning of the term used in the present disclosure, the meaning of the term in the present disclosure shall govern. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, 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 invention as defined in the following claims.

Sequence listing

<110>IRONWOOD PHARMACEUTICALS, INC.

Currie, Mark G.

Zimmer, Daniel P.

Fretzen, Angelika

Kessler, Marco

Cali, Brian M.

<120> treatment for gastrointestinal disorders

<130>223355-076PCT1-305639

<140>PCT/US2010/xxxxxx

<141>2010-12-02

<150>US 61/267,316

<151>2009-12-07

<160>71

<170> PatentIn 3.5 edition

<210>1

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> Xaa1 represents Asn, D-Asn, Gln, D-Gln, Pro, D-Pro, Ala, beta-Ala,

D-Ala, Val, D-Val, Gly, Thr, D-Thr, Asp, D-Asp, beta-carboxylation

Asp, Glu, D-Glu, gamma-carboxylated Glu, alpha-amino suberic acid

(Asu), alpha-aminoadipic acid (Aad) or alpha-aminopimelic acid

<220>

<221> variants

<222>(1)..(1)

<223> Xaa1 can be substituted on the amino group with methyl, oxalic, malonic, succinic, glutaric, succinic,

Adipic, pimelic or suberic acid modification

<220>

<221> variants

<222>(1)..(1)

<223> if Xaa2 and Xaa3 are both absent, then Xaa1 must be β -carboxylated Asp or

Gamma-carboxylated Glu, or Xaa1 must be Asp, D-Asp, Glu, D-Glu, Asu,

Aad or Apm, and must be substituted on its amino group

<220>

<221> variants

<222>(1)..(1)

<223> to < take on >. oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid,

Pimelic acid or suberic acid modification.

<220>

<221>MISC_FEATURE

<222>(2)..(3)

<223> Xaa2 and Xaa3 are independently Asp, β -carboxylated Asp, Glu, γ -carboxylated Glu,

Asu, Aad, Apm or null

<220>

<221>MISC_FEATURE

<222>(4)..(4)

<223> Xaa4 is Cys or D-Cys

<220>

<221>MISC_FEATURE

<222>(6)..(6)

<223> Xaa6 is Asp or Glu

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223> Xaa7 is Tyr, Leu, Phe or Ile

<220>

<221>MISC_FEATURE

<222>(8)..(8)

<223> Xaa8 is Cys or D-Cys

<220>

<221>MISC_FEATURE

<222>(14)..(14)

<223> Xaa14 is Thr, Ala or Phe

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> Xaa16 is Cys or D-Cys

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> Xaa17 is Tyr, D-Tyr or absent

<400>1

Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Asn Pro Ala Cys Xaa Gly Xaa

1 5 10 15

Xaa

<210>2

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<222>(1)..(1)

<223> Xaa1 is Asn, D-Asn, Gln, D-Gln, Pro, D-Pro, Ala, beta-Ala, D-Ala,

val, D-Val, Gly, Thr, D-Thr, Asp, D-Asp, Glu or D-Glu

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223> Xaa2 is Asp or Glu

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223> Xaa3 is Asp or Glu or absent

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223> Xaa7 is Tyr or Leu

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> Xaa17 is Tyr or absent

<400>2

Xaa Xaa Xaa Cys Cys Glu Xaa Cys Cys Asn Pro Ala Cys Thr Gly Cys

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Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

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<221>MISC_FEATURE

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Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

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<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

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Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

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<210>6

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<212>PRT

<213> Artificial sequence

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<220>

<221>MISC_FEATURE

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Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

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<213> Artificial sequence

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<220>

<221>MISC_FEATURE

<222>(16)..(16)

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<400>7

Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

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<213> Artificial sequence

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<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

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<400>8

Asn Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

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<213> Artificial sequence

<220>

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<220>

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<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>9

Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

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<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

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<400>10

Asn Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>11

<211>16

<212>PRT

<213> Artificial sequence

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<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

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<400>11

Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>12

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<213> Artificial sequence

<220>

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<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

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<400>12

Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

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<213> Artificial sequence

<220>

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<223>bAla

<220>

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<400>13

Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

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<220>

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<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>14

Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>15

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<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>15

Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>16

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>16

Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>17

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MOD_RES

<222>(1)..(1)

<223>bAla

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>17

Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>18

<211>17

<212>PRT

<213> Artificial sequence

<220>

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<220>

<221>MOD_RES

<222>(1)..(1)

<223>bAla

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>18

Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys ThrGly Cys

1 5 10 15

Tyr

<210>19

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>19

Pro Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>20

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>20

Pro Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>21

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>21

Pro Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>22

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>22

Pro Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>23

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>23

Thr Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>24

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>24

Thr Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>25

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>25

Thr Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>26

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>26

Thr Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>27

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>27

Gly Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>28

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>28

Gly Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>29

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>29

Gly Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>30

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>30

Gly Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>31

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>31

Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>32

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>32

Asp Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>33

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>33

Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>34

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>34

Asp Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>35

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(15)..(15)

<223> L-amino acid residue or D-amino acid residue

<400>35

Glu Glu Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>36

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(15)..(15)

<223> L-amino acid residue or D-amino acid residue

<400>36

Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>37

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>37

Glu Glu Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

1 5 10 15

<210>38

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>38

Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

1 5 10 15

<210>39

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>39

Glu Glu Glu Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>40

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>40

Glu Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>41

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>41

Glu Glu Glu Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>42

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> L-amino acid residue or D-amino acid residue

<400>42

Glu Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>43

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(15)..(15)

<223> L-amino acid residue or D-amino acid residue

<400>43

Glu Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>44

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(15)..(15)

<223> L-amino acid residue or D-amino acid residue

<400>44

Glu Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>45

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>45

Glu Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

1 5 10 15

<210>46

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> L-amino acid residue or D-amino acid residue

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> L-amino acid residue or D-amino acid residue

<400>46

Glu Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

1 5 10 15

<210>47

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> D amino acid

<400>47

Asp Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>48

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> D amino acid

<400>48

Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>49

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> D amino acid

<400>49

Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>50

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> D amino acid

<400>50

Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>51

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> D amino acid

<400>51

Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 1015

Tyr

<210>52

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> D amino acid

<400>52

Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>53

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>53

Asn Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>54

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>54

Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

1 5 10 15

<210>55

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>55

Asp Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>56

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>56

Gly Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>57

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>57

Pro Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>58

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>58

Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>59

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>59

Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>60

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>60

Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>61

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MOD_RES

<222>(1)..(1)

<223>bAla

<400>61

Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>62

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>62

Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

1 5 10 15

<210>63

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>63

Asp AspAsp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>64

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>64

Pro Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>65

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>65

Gly Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>66

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>66

Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

<210>67

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400>67

Glu Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Tyr

<210>68

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> Xaa1 represents Asn, D-Asn, Gln, D-Gln, Pro, D-Pro, Ala, beta-Ala, D-Ala,

Val, D-Val, Gly, Thr, D-Thr, Asp, D-Asp, Glu or D-Glu

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223> Xaa3 is Asp or absent

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223> Xaa7 is Tyr or Leu

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> Xaa17 is Tyr or absent

<400>68

Xaa Asp Xaa Cys Cys Glu Xaa Cys Cys Asn Pro Ala Cys Thr Gly Cys

1 5 10 15

Xaa

<210>69

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> (peptide 1)

<400>69

Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

1 5 10

<210>70

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> (peptide 2)

<400>70

Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr

1 5 10

<210>71

<211>18

<212>PRT

<213> Escherichia coli (Escherichia coli)

<400>71

Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly

1 5 10 15

Cys Tyr

Claims

1. A peptide or a pharmaceutically acceptable salt thereof, wherein the peptide consists of the sequence:

D-Asp Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:47)；

D-Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:48)；

D-Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:49)；

D-Ala Asp Asp Cys Cys Glu Leu CysCys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:50)；

Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys D-Tyr(SEQID NO:51)；

Asn Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:53)；

Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQ IDNO:54)；

Asp Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:55)；

Gly Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:56)；

Pro Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:57)；

Ala Asp Asp Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:58)；

Asn Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:59)；

Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:60)；

β-Ala Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:61)；

Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQ IDNO:62)；

Asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:63)；

Pro Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:64)；

Gly Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:65)；

asp Asp Asp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys (SEQ ID NO: 66); or

Glu Glu Glu Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQID NO:67)。

2. The peptide or pharmaceutically acceptable salt thereof according to claim 1, wherein the peptide or pharmaceutically acceptable salt thereof is isolated or purified.

3. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, a peptide according to claim 1 and further selected from (i) selected from Mg²⁺、Ca²⁺、Zn²⁺、Mn²⁺、K⁺、Na⁺Or Al³⁺Or (ii) one or more reagents of a natural amino acid, wherein the Mg²⁺、Ca²⁺、Zn²⁺、Mn²⁺、K⁺、Na⁺Or Al³⁺Providing 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;

wherein the natural amino acid is histidine, phenylalanine, alanine, glutamic acid, aspartic acid, glutamine, leucine, methionine, asparagine, tyrosine, threonine, isoleucine, tryptophan or valine.

4. The pharmaceutical composition of claim 3, further comprising a pharmaceutically acceptable antioxidant, binder, additive or filler, said pharmaceutically acceptable binder or additive being selected from polyvinyl alcohol, polyvinylpyrrolidone (povidone), starch, maltodextrin or cellulose ether.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable binder or additive is a cellulose ether selected from the group consisting of: methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.

6. The pharmaceutical composition according to claim 4, wherein the pharmaceutically acceptable filler is cellulose, isomalt, mannitol, lactose or dibasic calcium phosphate.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable filler is cellulose selected from the group consisting of micro-fine cellulose and microcrystalline cellulose.

8. The pharmaceutical composition of claim 3, further comprising an additional therapeutic agent selected from one or more of an analgesic, an antidepressant, a prokinetic agent, an antiemetic, an antibiotic, a proton pump inhibitor, an acid blocker, a PDE5 inhibitor, an acid pump antagonist, a GABA-B agonist, a bile acid sequestrant, or a mucosal protectant.

9. Use of a pharmaceutical composition according to any one of claims 3-7 in the manufacture of a medicament for the treatment of a gastrointestinal disorder.

10. The use of claim 9, wherein the gastrointestinal disorder is gastroparesis, post-operative gastrointestinal obstruction, functional esophageal disorder, functional gastroduodenal disorder, gastroesophageal reflux disease, celiac disease, mucositis, or a duodenal or gastric ulcer.

11. The use of claim 10, wherein the gastrointestinal disorder is gastroparesis selected from idiopathic, diabetic, or post-operative gastroparesis.

12. The use of claim 10, wherein the gastrointestinal disorder is a functional esophageal disorder selected from functional heartburn, presumed esophageal-derived functional chest pain, functional dysphagia, or hysteria.

13. The use according to claim 10, wherein the gastrointestinal disorder is a functional gastroduodenal disorder selected from a functional dyspepsia, a belching disorder, a nausea or vomiting disorder, or a rumination syndrome.