US20040266989A1 - Methods and compositions for the treatment of gastrointestinal disorders - Google Patents

Methods and compositions for the treatment of gastrointestinal disorders Download PDF

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US20040266989A1
US20040266989A1 US10/766,735 US76673504A US2004266989A1 US 20040266989 A1 US20040266989 A1 US 20040266989A1 US 76673504 A US76673504 A US 76673504A US 2004266989 A1 US2004266989 A1 US 2004266989A1
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Mark Currie
Shalina Mahajan-Miklos
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Microbia Inc
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Microbia Inc
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=32854294&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20040266989(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to US10/766,735 priority Critical patent/US20040266989A1/en
Application filed by Microbia Inc filed Critical Microbia Inc
Priority to US10/796,719 priority patent/US7304036B2/en
Priority to US10/845,895 priority patent/US20060281682A1/en
Priority to US10/899,806 priority patent/US7371727B2/en
Assigned to MICROBIA, INC. reassignment MICROBIA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CURRIE, MARK G., MAHAJAN-MIKLOS, SHALINA
Publication of US20040266989A1 publication Critical patent/US20040266989A1/en
Priority to US11/054,071 priority patent/US7772188B2/en
Assigned to MICROBIA, INC. reassignment MICROBIA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MILNE, G. TODD, NORMAN, THEA
Priority to US11/930,696 priority patent/US7704947B2/en
Priority to US12/120,023 priority patent/US7910546B2/en
Priority to US12/754,138 priority patent/US8080526B2/en
Priority to US12/788,979 priority patent/US8110553B2/en
Abandoned legal-status Critical Current

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Definitions

  • This invention relates to methods and compositions for treating various disorders, including gastrointestinal disorders, obesity, congestive heart failure and benign prostatic hyperplasia.
  • IBS Irritable bowel syndrome
  • c-IBS constipation-predominant
  • d-IBS diarrhea-predominant
  • a-IBS alternating between the two
  • IBS is considered to be a “biopsychosocial” disorder resulting from a combination of three interacting mechanisms: altered bowel motility, an increased sensitivity of the intestine or colon to pain stimuli (visceral sensitivity) and psychosocial factors (Camilleri 2001, Gastroenterology 120:652-668).
  • NO inducible nitric oxide
  • iNOS synthase
  • the present invention features compositions and related methods for treating IBS and other gastrointestinal disorders and conditions (e.g., gastrointestinal motility disorders, functional gastrointestinal disorders, gastroesophageal reflux disease (GERD), Crohn's disease, ulcerative colitis, Inflammatory bowel disease, functional heartburn, dyspepsia (including functional dyspepsia or nonulcer dyspepsia), gastroparesis, chronic intestinal pseudo-obstruction (or colonic pseudo-obstruction), and disorders and conditions associated with constipation, e.g., constipation associated with use of opiate pain killers, post-surgical constipation, and constipation associated with neuropathic disorders as well as other conditions and disorders.
  • the compositions feature peptides that activate the guanylate cyclase C (GC—C) receptor.
  • GC—C guanylate cyclase C
  • the present invention also features compositions and related methods for treating obesity, congestive heart failure and benign prostatic hyperplasia (BPH).
  • BPH benign prostatic hyperplasia
  • the peptides are useful because they can increase gastrointestinal motility.
  • the peptides are useful, in part, because they can decrease inflammation.
  • the peptides are also useful because they can decrease gastrointestinal pain or visceral pain.
  • the invention features pharmaceutical compositions comprising certain peptides that are capable of activating the guanylate-cyclase C (GC—C) receptor. Also within the invention are pharmaceutical compositions comprising a peptide of the invention as well as combination compositions comprising a peptide of the invention and a second therapeutic agent, e.g., an agent for treating constipation (e.g., SPI-0211; Sucampo Pharmaceuticals, Inc.; Bethesda, Md.) or some other gastrointestinal disorder.
  • a second therapeutic agent include: acid reducing agents such as proton pump inhibitors and H2 receptor blockers, pro-motility agents such as 5HT receptor agonists (e.g.
  • Zelnorm® anti-inflammatory agents
  • antispasmodics antidepressants
  • centrally-acting analgesic agents such as opiod receptor agonists, opiod receptor antagonists, agents for the treatment of Inflammatory bowel disease, Crohn's disease and ulcerative colitis (e.g., Traficet-ENTM (ChemoCentryx, Inc.; San Carlos, Calif.) agents that treat gastrointestinal or visceral pain and cGMP phosphodiesterase inhibitors (motapizone, zaprinast, and suldinac sulfone).
  • the pharmaceutical compositions can include an analgesic agent selected from the group consisting of: Ca channel blockers (e.g., ziconotide), 5HT receptor antagonists (for example 5HT3, 5HT4 and 5HT1 receptor antagonists), opioid receptor agonists (e.g., loperamide, fedotozine, and fentanyl, naloxone, naltrexone, methyl nalozone, nalmefene, cypridime, beta funaltrexamine, naloxonazine, naltrindole, and nor-binaltorphimine, morphine, diphenyloxylate, enkephalin pentapeptide, and trimebutine), NK1 receptor antagonists (e.g., ezlopitant and SR-14033), CCK receptor agonists (e.g., loxiglumide), NK1 receptor antagonists, NK3 receptor antagonists (e.g., talnetant,
  • the invention includes methods for treating various gastrointestinal disorders by administering a peptide that acts as a partial or complete agonist of the GC—C receptor.
  • the peptide includes at least six cysteines that form three disulfide bonds. In certain embodiments the disulfide bonds are replaced by other covalent cross-links and in some cases the cysteines are substituted by other residues to provide for alternative covalent cross-links.
  • the peptides may also include at least one trypsin or chymotrypsin cleavage site and/or a carboxy-terminal analgesic peptide or small molecule, e.g., AspPhe or some other analgesic peptide.
  • the analgesic peptide or small molecule may be preceded by a chymotrypsin or trypsin cleavage site that allows release of the analgesic peptide or small molecule.
  • the peptides and methods of the invention are also useful for treating pain and inflammation associated with various disorders, including gastrointestinal disorders.
  • Certain peptides include a functional chymotrypsin or trypsin cleavage site located so as to allow inactivation of the peptide upon cleavage.
  • Certain peptides having a functional cleavage site undergo cleavage and gradual inactivation in the digestive tract, and this is desirable in some circumstances.
  • a functional chymotrypsin site is altered, increasing the stability of the peptide in vivo.
  • the invention includes methods for treating other disorders such as congestive heart failure and benign prostatic hyperplasia by administering a peptide or small molecule (parenterally or orally) that acts as an agonist of the GC—C receptor.
  • a peptide or small molecule parenterally or orally
  • Such agents can be used in combination with natriuretic peptides (e.g., atrial natriuretic peptide, brain natriuretic peptide or C-type natriuretic peptide), a diuretic, or an inhibitor of angiotensin converting enzyme.
  • the invention features methods and compositions for increasing intestinal motility.
  • Intestinal motility involves spontaneous coordinated dissentions and contractions of the stomach, intestines, colon and rectum to move food through the gastrointestinal tract during the digestive process.
  • the peptides include either one or two or more contiguous negatively charged amino acids (e.g., Asp or Glu) or one or two or more contiguous positively charged residues (e.g., Lys or Arg) or one or two or more contiguous positively or negatively charged amino acids at the carboxy terminus.
  • all of the flanking amino acids at the carboxy terminus are either positively or negatively charged.
  • the carboxy terminal charged amino acids are preceded by a Leu.
  • amino acid sequences can be added to the carboxy terminus of the peptide: Asp; Asp Lys; Lys Lys Lys Lys Lys Lys Lys Lys (SEQ ID NO:123); Asp Lys Lys Lys Lys Lys (SEQ ID NO:124); Leu Lys Lys; and Leu Asp. It is also possible to simply add Leu at the carboxy terminus.
  • the invention features a peptide comprising, consisting of, or consisting essentially of the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xa4 Xaa 5 is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing.
  • Xaa 8 , Xaa 9 , Xaa 12 , Xaa 13 , Xaa 14 , Xaa 17 , and Xaa 19 can be any amino acid.
  • Xaa 5 is Asn, Trp, Tyr, Asp, or Phe.
  • Xaa 5 can also be Thr or Ile.
  • Xaa 5 is Tyr, Asp or Trp.
  • Xaa 8 is Glu, Asp, Gln, Gly or Pro.
  • Xaa 8 is Glu; in some embodiments Xaa 9 is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe in some embodiments Xaa 9 is Leu, Ile, Val, Lys, Arg, Trp, Tyr or Phe.
  • an amino acid can be replace by a non-naturally occurring amino acid or a naturally or non-naturally occurring amino acid analog.
  • an aromatic amino acid can be replaced by 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr).
  • Phg and norTyr and other amino acids including Phe and Tyr can be substituted by, e.g., a halogen, —CH3, —OH, —CH 2 NH 3 , —C(O)H, —CH 2 CH 3 , —CN, —CH 2 CH 2 CH 3 , —SH, or another group.
  • Xaa 12 is Asn, Tyr, Asp or Ala. In other embodiments Xaa 12 is Asn. In some embodiments Xaa 13 is Ala, Pro or Gly, and in other embodiments it is Pro. In some embodiments Xaa 14 is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp, and in other embodiments it is Ala or Gly, and in still other embodiments it is Ala.
  • the invention also features methods for treating a gastrointestinal disorder (e.g., a gastrointestinal motility disorder, a functional gastrointestinal disorder, gastroesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronic intestinal pseudo-obstruction, colonic pseudo-obstruction), obesity, congestive heart failure or benign prostatic hyperplasia by administering a composition comprising an aforementioned peptide
  • a gastrointestinal disorder e.g., a gastrointestinal motility disorder, a functional gastrointestinal disorder, gastroesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronic intestinal pseudo-obstruction, colonic pseudo-obstruction
  • a gastrointestinal disorder e.g., a gastrointestinal motility disorder, a functional gastrointestinal disorder, gastroesophageal reflux disease, functional heartburn
  • Xaa 9 is Trp, Tyr or Phe or when Xaa 16 is Trp the peptide has a potentially functional chymotrypsin cleavage site that is located at a position where cleavage will inactivate GC—C receptor binding by the peptide.
  • Xaa 9 is Lys or Arg or when Xaa 16 is Lys or Arg, the peptide has a potentially functional trypsin cleavage site that is located at a position where cleavage will inactivate GC—C receptor binding by the peptide.
  • the peptide has a chymotrypsin cleavage site that is located at a position where cleavage will liberate the portion of the peptide carboxy-terminal to Xaa 19 .
  • the peptide can have a chymotrypsin cleavage site that is located at a position where cleavage will liberate the portion of the peptide amino-terminal to Xaa 19 .
  • the same effect is seen when Xaa 19 is His.
  • the peptide When Xaa 19 is Lys or Arg, the peptide has a trypsin cleavage site that is located at a position where cleavage will liberate portion of the peptide carboxy-terminal to Xaa 19 . Thus, if the peptide includes an analgesic peptide carboxy-terminal to Xaa 19 , the peptide will be liberated in the digestive tract upon exposure to the appropriate protease.
  • analgesic peptides which can be included in the peptide are: AspPhe (as Xaa 20 Xaa 21 ), endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron, and substance P and other analgesic peptides described herein. These peptides can, for example, be used to replace Xaa 20 Xaa 21 .
  • the peptide has a chymotrypsin cleavage site that is located at a position where cleavage will liberate the portion of the peptide amino-terminal to Xaa 1 (or Xaa 2 or Xaa 3 ) along with Xaa 1 , Xaa 2 or Xaa 3 .
  • the peptide has a trypsin cleavage site that is located at a position where cleavage will liberate portion of the peptide amino-terminal to Xaa 1 along with Xaa 1 , Xaa 2 or Xaa 3 ).
  • the peptide can have a chymotrypsin cleavage site that is located at a position where cleavage will liberate the portion of the peptide amino-terminal to Xaa 1 .
  • the peptide includes an analgesic peptide amino-terminal to Xaa 1 , the peptide will be liberated in the digestive tract upon exposure to the, appropriate protease.
  • analgesic peptides which can be included in the peptide are: AspPhe, endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron, and substance p and other analgesic peptides described herein.
  • the peptides of the invention bear some sequence similarity to ST peptides. However, they include amino acid changes and/or additions that improve functionality. These changes can, for example, increase or decrease activity (e.g., increase or decrease the ability of the peptide to stimulate intestinal motility), alter the ability of the peptide to fold correctly, the stability of the peptide, the ability of the peptide to bind the GC—C receptor and/or decrease toxicity. In some cases the peptides may function more desirably than wild-type ST peptide. For example, they may limit undesirable side effects such as diarrhea and dehydration.
  • one or both members of one or more pairs of Cys residues which normally form a disulfide bond can be replaced by homocysteine, 3-mercaptoproline (Kolodziej et al. 1996 Int J Pept Protein Res 48:274); ⁇ , ⁇ dimethylcysteine (Hunt et al. 1993 Int J Pept Protein Res 42:249) or diaminopropionic acid (Smith et al. 1978 J Med Chem 21:117) to form alternative internal cross-links at the positions of the normal disulfide bonds.
  • one or more disulfide bonds can be replaced by alternative covalent cross-links, e.g., an amide bond, an ester linkage, an alkyl linkage, a thio ester linkage, a lactam bridge, a carbamoyl linkage, a urea linkage, a thiourea linkage, a phosphonate ester linkage, an alkyl linkage, and alkenyl linkage, an ether, a thioether linkage, or an amino linkage.
  • alternative covalent cross-links e.g., an amide bond, an ester linkage, an alkyl linkage, a thio ester linkage, a lactam bridge, a carbamoyl linkage, a urea linkage, a thiourea linkage, a phosphonate ester linkage, an alkyl linkage, and alkenyl linkage, an ether, a thioether linkage, or an amino
  • the peptide can include an amino terminal sequence that facilitates recombinant production of the peptide and is cleaved prior to administration of the peptide to a patient.
  • the peptide can also include other amino terminal or carboxyterminal amino acids.
  • the additional amino acids protect the peptide, stabilize the peptide or alter the activity of the peptide.
  • some or all of these additional amino acids are removed prior to administration of the peptide to a patient.
  • the peptide can include 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 80, 90, 100 or more amino acids at its amino terminus or carboxy terminus or both.
  • the number of flanking amino acids need not be the same. For example, there can be 10 additional amino acids at the amino terminus of the peptide and none at the carboxy terminus.
  • the peptide comprises the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is missing; Xaa 8 is Glu; Xaa 9 is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa 12 is Asn; Xaa 13 is Pro; Xaa 14 is Ala; Xaa 16 is Thr, Ala, Lys, Arg, Trp; Xaa 17 is Gly; Xaa 19 is Tyr or Leu; and
  • the invention also features a therapeutic or prophylactic method comprising administering a peptide comprising the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa 8 is Glu, Asp, Gln, Gly or Pro
  • the peptide comprises the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is missing; Xaa 8 is Glu; Xaa 9 is Leu, Ile, Lys, Arg, Trp, Tyr, or Phe; Xaa 12 is Asn; Xaa 13 is Pro; Xaa 14 is Ala; Xaa 16 is Thr, Ala, Lys, Arg, Trp or Xaa 16 is any amino acid or
  • the invention features, a purified polypeptide comprising the amino acid sequence (II):
  • Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn;
  • Xaa 8 is Glu or Asp
  • Xaa 9 is Leu, Ile, Val, Trp, Tyr or Phe;
  • Xaa 16 is Thr, Ala, Trp;
  • Xaa 19 is Trp, Tyr, Phe or Leu or is missing; and Xaa 20 Xaa 21 is AspPhe.
  • the invention features a purified polypeptide comprising the amino acid sequence (II): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Asn 12 Pro 13 Ala 14 Cys 15 Xaa 16 Gly 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:120) wherein, Xaa 9 is Leu, Ile or Val and Xaa 16 is Trp, Tyr or Phe; Xaa 9 is Trp, Tyr or Phe, and Xaa 16 is Thr or Ala; Xaa 19 is Trp, Tyr, Phe and Xaa 20 Xaa 21 is AspPhe; and Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn; the peptide comprises fewer than 50, 40,
  • the peptides can be co-administered with or linked, e.g., covalently linked to any of a variety of other peptides including analgesic peptides or analgesic compounds.
  • a therapeutic peptide of the invention can be linked to an analgesic agent selected from the group consisting of: Ca channel blockers (e.g., ziconotide), complete or partial 5HT receptor antagonists (for example 5HT3, 5HT4 and 5HT1 receptor antagonists), complete or partial 5HT receptor agonists including 5HT3, 5HT4 (for example tegaserod, mosapride and renzapride) and 5HT1 receptor agonists, CRF receptor agonists (NBI-34041), ⁇ -3 adrenoreceptor agonists, opioid receptor agonists (e.g., loperamide, fedotozine, and fentanyl, naloxone, naltrexone, methyl nalozone,
  • Amino acid, non-amino acid, peptide and non-peptide spacers can be interposed between a peptide that is a GC—C receptor agonsit and a peptide that has some other biological function, e.g., an analgesic peptide or a peptide used to treat obesity.
  • the linker can be one that is cleaved from the flanking peptides in vivo or one that remains linked to the flanking peptides in vivo.
  • glycine, beta-alanine, glycyl-glycine, glycyl-beta-alanine, gamma-aminobutyric acid, 6-aminocaproic acid, L-phenylalanine, L-tryptophan and glycil-L-valil-L-phenylalanine can be used as a spacer (Chaltin et al. 2003 Helvetica Chimica Acta 86:533-547; Caliceti et al. 1993 FARMCO 48:919-32) as can polyethylene glycols (Butterworth et al. 1987 J. Med. Chem 30:1295-302) and maleimide derivatives (King et al.
  • the peptides can include the amino acid sequence of a peptide that occurs naturally in a vertebrate (e.g., mammalian) species or in a bacterial species.
  • the peptides can be partially or completely non-naturally occurring peptides.
  • peptidomimetics corresponding to the peptides of the invention.
  • the patient is suffering from a gastrointestinal disorder; the patient is suffering from a disorder selected from the group consisting of: a gastrointestinal motility disorder, irritable bowel syndrome, chronic constipation, a functional gastrointestinal disorder, gastroesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronic intestinal pseudo-obstruction, Crohn's disease, ulcerative colitis, Irritable bowel syndrome, colonic pseudo-obstruction, obesity, congestive heart failure, or benign prostatic hyperplasia; the composition is administered orally; the peptide comprises 30 or fewer amino acids, the peptide comprises 20 or fewer amino acids, and the peptide comprises no more than 5 amino acids prior to Cys 6 ; the peptide comprises 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, or 30 or fewer amino acids.
  • the peptide comprises 20 or fewer amino acids. In other embodiments the peptide comprises no more than 20, 15, 10, or 5 peptides subsequent to Cys 18 .
  • Xaa 19 is a chymotrypsin or trypsin cleavage site and an analgesic peptide is present immediately following Xaa 19 .
  • the invention features a method for treating a patient suffering from constipation.
  • Clinically accepted criteria that define constipation range from the frequency of bowel movements, the consistency of feces and the ease of bowel movement.
  • One common definition of constipation is less than three bowel movements per week.
  • Other definitions include abnormally hard stools or defecation that requires excessive straining (Schiller 2001, Aliment Pharmacol Ther 15:749-763).
  • Constipation may be idiopathic (functional constipation or slow transit constipation) or secondary to other causes including neurologic, metabolic or endocrine disorders.
  • Constipation may also be the result of surgery (postoperative ileus) or due to the use of drugs such as analgesics (like opiods), antihypertensives, anticonvulsants, antidepressants, antispasmodics and antipsychotics.
  • the method comprising administering a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa 8 is Glu, Asp, Gln, Gly or Pro; Xa
  • the peptide comprises the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is missing; Xaa 8 is Glu; Xaa 9 is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa 12 is Asn; Xaa 13 is Pro; Xaa 14 is Ala; Xaa 16 is Thr, Ala, Lys, Arg, Trp; Xaa 17 is Gly; Xaa 19 is Tyr or
  • the constipation is associated with use of a therapeutic agent; the constipation is associated with a neuropathic disorder; the constipation is post-surgical constipation (postoperative ileus); and the constipation associated with a gastrointestinal disorder; the constipation is idiopathic (functional constipation or slow transit constipation); the constipation is associated with neuropathic, metabolic or endocrine disorder (e.g., diabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcaemia, Multiple Sclerosis, Parkinson's disease, spinal cord lesions, neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprung's disease or cystic fibrosis).
  • neuropathic, metabolic or endocrine disorder e.g., diabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcaemia, Multiple Sclerosis, Parkinson's disease, spinal cord lesions, neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprung's disease or
  • Constipation may also be the result of surgery (postoperative ileus) or due the use of drugs such as analgesics (e.g., opiods), antihypertensives, anticonvulsants, antidepressants, antispasmodics and antipsychotics.
  • the invention features a method for treating a patient suffering a gastrointestinal disorder, the method comprising administering to the patient a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn, Trp, Tyr, Asp, Ile, Thr, or
  • the peptide comprises the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xa 5 is missing; Xaa 8 is Glu; Xaa 9 is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa 12 is Asn; Xaa 13 is Pro; Xaa 14 is Ala; Xaa 16 is Thr, Ala, Lys, Arg, Trp; Xaa 17 is Gly; Xaa 19 is Tyr or
  • the patient is suffering from a gastrointestinal disorder; the patient is suffering from a disorder selected from the group consisting of: a gastrointestinal motility disorder, irritable bowel syndrome, chronic constipation, a functional gastrointestinal disorder, gastroesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronic intestinal pseudo-obstruction, Crohn's disease, ulcerative colitis, Inflammatory bowel disease, colonic pseudo-obstruction, obesity, congestive heart failure, or benign prostatic hyperplasia.
  • a gastrointestinal motility disorder irritable bowel syndrome
  • chronic constipation a functional gastrointestinal disorder
  • gastroesophageal reflux disease functional heartburn
  • dyspepsia functional dyspepsia
  • nonulcer dyspepsia gastroparesis
  • chronic intestinal pseudo-obstruction Crohn's disease
  • ulcerative colitis ulcerative colitis
  • Xaa 9 is Leu, Ile or Val and Xaa 16 is Trp, Tyr or Phe; Xaa 9 is Trp, Tyr or Phe and Xaa 16 is Thr or Ala; Xaa 19 is Trp, Tyr, Phe; Xaa 19 is Lys or Arg;Xaa 20 Xaa 21 is AspPhe; Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn.
  • the invention features a method for increasing gastrointestinal motility in a patient, the method comprising: administering to the patient a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn, Trp, Tyr, Asp, Ile, Thr, or
  • the peptide comprises the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xa 5 is missing; Xaa 8 is Glu; Xaa 9 is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa 12 is Asn; Xaa 13 is Pro; Xaa 14 is Ala; Xaa 16 is Thr, Ala, Lys, Arg, Trp; Xaa 17 is Gly; Xaa 19 is Tyr or Leu;
  • the invention features a method for increasing the activity of an intestinal guanylate cyclase (GC—C) receptor in a patient, the method comprising: administering to the patient a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn
  • the peptide comprises the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xa 5 is missing; Xaa 8 is Glu; Xaa 9 is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa 12 is Asn; Xaa 13 is Pro; Xaa 14 is Ala; Xaa 16 is Thr, Ala, Lys, Arg, Trp; Xaa 17 is Gly; Xaa 19 is Tyr or Leu;
  • the invention features an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence: (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is Asn Ser Ser Asn Tyr or is missing or Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa 8 is Glu, Asp, Gl
  • the peptide comprises the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is missing; Xaa 8 is Glu; Xaa 9 is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa 12 is Asn; Xaa 13 is Pro; Xaa 14 is Ala; Xaa 16 is Thr, Ala, Lys, Arg, Trp; Xaa 17 is Gly; Xaa 19 is Tyr or Leu;
  • the invention features a method for treating constipation, the method comprising administering an agonist of the intestinal guanylate cyclase (GC—C) receptor.
  • the agonist is a peptide
  • the peptide includes four Cys that form two disulfide bonds
  • the peptide includes six Cys that form three disulfide bonds.
  • the invention features a method for treating a gastrointestinal disorder, a gastrointestinal motility disorder, irritable bowel syndrome, chronic constipation, a functional gastrointestinal disorder, gastroesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronic intestinal pseudo-obstruction, colonic pseudo-obstruction, Crohn's disease, ulcerative colitis, Inflammatory bowel disease, obesity, congestive heart failure, or benign prostatic hyperplasia, the method comprising administering an agonist of the intestinal guanylate cyclase (GC—C) receptor either orally, by rectal suppository, or parenterally.
  • the agonist is a peptide
  • the peptide includes four Cys that form two disulfide bonds
  • the peptide includes six Cys that form three disulfide bonds.
  • the invention features a method for treating a gastrointestinal disorder selected from the group consisting of: a gastrointestinal motility disorder, irritable bowel syndrome, chronic constipation, a functional gastrointestinal disorder, gastroesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronic intestinal pseudo-obstruction, colonic pseudo-obstruction, Crohn's disease, ulcerative colitis, Inflammatory bowel disease, the method comprising administering an agonist of the intestinal guanylate cyclase (GC—C) receptor.
  • the composition is administered orally; the peptide comprises 30 or fewer amino acids, the peptide comprises 20 or fewer amino acids, and the peptide comprises no more than 5 amino acids prior to Cys 5 .
  • the agonist is a peptide
  • the peptide includes four Cys that form two disulfide bonds
  • the peptide includes six Cys that form three disulfide bonds.
  • the invention features a method for treating obesity, the method comprising administering an agonist of the intestinal guanylate cyclase (GC—C) receptor.
  • the agonist is a peptide
  • the peptide includes four Cys that form two disulfide bonds
  • the peptide includes six Cys that form three disulfide bonds.
  • the invention features a method for treating obesity, the method comprising administering a polypeptide comprising the amino acid sequence: (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa 8 is Glu, Asn, Trp, Tyr, Asp, Ile,
  • the peptide comprises the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is missing; Xaa 8 is Glu; Xaa 9 is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa 12 is Asn; Xaa 13 is Pro; Xaa 14 is Ala; Xaa 16 is Thr, Ala, Lys, Arg, Trp; Xaa 17 is Gly; Xaa 19 is Tyr or Leu; and Xaa 20
  • the invention features a pharmaceutical composition comprising a polypeptide described herein.
  • the invention features a method for treating congestive heart failure, the method comprising: administering to the patient a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID NO:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe
  • the peptide can be administered in combination with another agent for treatment of congestive heart failure, for example, a natriuretic peptide such as atrial natriuretic peptide, brain natriuretic peptide or C-type natriuretic peptide), a diuretic, or an inhibitor of angiotensin converting enzyme.
  • a natriuretic peptide such as atrial natriuretic peptide, brain natriuretic peptide or C-type natriuretic peptide
  • a diuretic such as an agent for treatment of congestive heart failure
  • angiotensin converting enzyme for example, a natriuretic peptide such as atrial natriuretic peptide, brain natriuretic peptide or C-type natriuretic peptide
  • an inhibitor of angiotensin converting enzyme for example, a natriuretic peptide such as atrial
  • the peptide comprises the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID No: 119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is missing; Xaa 8 is Glu; Xaa 9 is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa 12 is Asn; Xaa 13 is Pro; Xaa 14 is Ala; Xaa 16 is Thr, Ala, Lys, Arg, Trp; Xaa 17 is Gly; Xaa 19 is Tyr or Leu;
  • the invention features a method for treating benign prostatic hyperplasia, the method comprising: administering to the patient a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID No:119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa 1 Xaa 2 Xaa 3 Xaa 4 is missing and Xaa 5 is Asn, Trp, Tyr, Asp, Ile, Thr, or
  • the peptide can be administered in combination with another agent for treatment of BPH, for example, a 5-alpha reductase inhibitor (e.g., finasteride) or an alpha adrenergic inhibitor (e.g., doxazosine).
  • a 5-alpha reductase inhibitor e.g., finasteride
  • an alpha adrenergic inhibitor e.g., doxazosine
  • the peptide comprises the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID No: 119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is missing; Xaa 8 is Glu; Xaa 9 is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa 12 is Asn; Xaa 13 is Pro; Xaa 14 is Ala; Xaa 16 is Thr, Ala, Lys, Arg, Trp; Xaa 17 is Gly; Xaa 19 is Tyr or Leu;
  • the invention features a method for treating or reducing pain, including visceral pain, pain associated with a gastrointestinal disorder or pain associated with some other disorder, the method comprising: administering to a patient a composition comprising a is purified polypeptide comprising the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 , (SEQ ID No: 119) e.g., a purified polypeptide comprising an amino acid sequence disclosed herein.
  • a composition comprising a is purified polypeptide comprising the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 X
  • the invention features a method for treating inflammation, including inflammation of the gastrointestinal tract, e.g., inflammation associated with a gastrointestinal disorder or infection or some other disorder, the method comprising: administering to a patient a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 , (SEQ ID No: 119) e.g., a purified polypeptide comprising an amino acid sequence disclosed herein.
  • a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7
  • the peptide includes a peptide comprising or consisting of the amino acid sequence Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys Cys Glu Xaa 9 Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Xaa 20 Xaa 21 (II) (SEQ ID NO:66) wherein Xaa 9 is any amino acid, wherein Xaa 9 is any amino acid other than Leu, wherein Xaa 9 is selected from Phe, Trp and Tyr; wherein Xaa 9 is selected from any other natural or non-natural aromatic amino acid, wherein Xaa 9 is Tyr; wherein Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is Asn Ser Ser Asn Tyr (SEQ ID NO:121); wherein Xaa 1 , Xaa 2 , Xaa 3 , Xaa 4 , and
  • a peptide comprising the sequence (I): Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys 6 Cys 7 Xaa 8 Xaa 9 Cys 10 Cys 11 Xaa 12 Xaa 13 Xaa 14 Cys 15 Xaa 16 Xaa 17 Cys 18 Xaa 19 Xaa 20 Xaa 21 (SEQ ID No: 119) wherein: Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 is missing and/or the sequence Xaa 19 Xaa 20 Xaa 21 is missing peptide can still contain additional carboxyterminal or amino terminal amino acids or both
  • peptides comprising, consisting of or consisting essentially of the amino acid sequence Xaa 1 Xaa 2 Xaa 3 Xaa 4 Xaa 5 Cys Cys Glu Xaa 9 Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Xaa 20 Xaa 21 (II) (SEQ ID NO:66) are the following peptides: Gln Ser Ser Asn Tyr Cys Cys Glu (SEQ ID NO:67) Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asn Thr Ser Asn Tyr Cys Cys Glu (SEQ ID NO:68) Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asn Leu Ser Asn Tyr Cys Cys Glu (SEQ ID NO:69) Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asn Ile Ser Asn Tyr Cys Cys Glu (SEQ ID NO:66) are the following peptid
  • the invention features a method for treating congestive heart failure, the method comprising administering a complete or partial agonist of the intestinal guanylate cyclase (GC—C) receptor.
  • the agonist can be administered in combination with another agent for treatment of congestive heart failure, for example, a natriuretic peptide such as atrial natriuretic peptide, brain natriuretic peptide or C-type natriuretic peptide), a diuretic, or an inhibitor of angiotensin converting enzyme.
  • the invention features a method for treating BPH, the method comprising administering a complete or partial agonist of the intestinal guanylate cyclase (GC—C) receptor.
  • the agonist can be administered in combination with another agent for treatment of BPH, for example, a 5-alpha reductase inhibitor (e.g., finasteride) or an alpha adrenergic inhibitor (e.g., doxazosine).
  • the invention features a method for treating obesity, the method comprising administering a complete or partial agonist of the intestinal guanylate cyclase (GC—C) receptor.
  • GC—C intestinal guanylate cyclase
  • the agonist can be administered in combination with another agent for treatment of obesity, for example, gut hormone fragment peptide YY 3-36 (PYY 3-36 )( N. Engl. J. Med.
  • a peptide useful for treating obesity can be administered as a co-therapy with a peptide of the invention either as a distinct molecule or as part of a fusion protein with a peptide of the invention.
  • PYY 3-36 can be fused to the carboxy or amino terminus of a peptide of the invention.
  • Such a fusion protein can include a chymostrypsin or trypsin cleavage site that can permit cleavage to separate the two peptides.
  • the peptides and agonist of the intestinal guanylate cyclase (GC—C) receptor can be used to treat constipation or decreased intestinal motility, slow digestion or slow stomach emptying.
  • the peptides can be used to relieve one or more symptoms of IBS (bloating, pain, constipation), GERD (acid reflux into the esophagus), functional dyspepsia, or gastroparesis (nausea, vomiting, bloating, delayed gastric emptying) and other disorders described herein.
  • FIG. 1 a depicts the results of LCMS analysis of recombinant MM-416776 peptide and MD-915 peptide.
  • FIGS. 1 b and c depict the results of LCMS analysis of synthetic MD-1100 peptide and the blank.
  • FIG. 2 depicts the results of the intestinal GC—C receptor activity assay of synthetic MM-416776 peptide, MD-915 peptide and two different MD-1100 peptides.
  • FIG. 3 a depicts the effect of recombinant MM-416776 peptide and Zelnorm® in a murine gastrointestinal transit model.
  • FIG. 3 b depicts the effect of synthetic MD-1100 peptide and Zelnorm® in an acute murine gastrointestinal transit model.
  • FIG. 3 b depicts the effect of synthetic MD-1100 peptide and Zelnorm® in an chronic murine gastrointestinal transit model.
  • FIGS. 4 a and 4 b depict the effect of peptides MD-915, MD-1100, and MM-416776 in an acute murine gastrointestinal transit model.
  • FIG. 4 c depicts the effect of MD-1100 peptide in a chronic murine gastrointestinal transit model.
  • FIG. 5 a depicts the effect of MM-416776 peptide and Zelnorm® in a suckling mouse intestinal secretion model.
  • FIG. 5 b depicts the effects of MD-1100 and Zelnorm® in a mouse intestinal secretion model.
  • FIGS. 6 a and 6 b depict the effects of MM 416776, MD-1100 and MD-915 peptides in a mouse intestinal secretion model.
  • FIG. 7 shows the results of experiment in which MD-1100 activity was analyzed in the TNBS colonic distention model.
  • FIGS. 8 a and 8 b show the effects of differing doses of MD-915 and MD-1100 in the PBQ writhing assay.
  • FIG. 9 shows the results of Kd determination analysis using MD-1100 in a competitive radioligand binding assay.
  • FIGS. 10 a and 10 b show bioavailability data for IV and orally administered MD-1100 as detected by an ELISA assay and LCMS.
  • the peptides of the invention bind to the intestinal guanylate cyclase (GC—C) receptor, a key regulator of fluid and electrolyte balance in the intestine.
  • GC—C intestinal guanylate cyclase
  • this receptor which is located on the apical membrane of the intestinal epithelial surface, causes an increase in intestinal epithelial cyclic GMP (cGMP).
  • cGMP intestinal epithelial cyclic GMP
  • This increase in cGMP is believed to cause a decrease in water and sodium absorption and an increase in chloride and potassium ion secretion, leading to changes in intestinal fluid and electrolyte transport and increased intestinal motility.
  • the intestinal GC—C receptor possesses an extracellular ligand binding region, a transmembrane region, an intracellular protein kinase-like region and a cyclase catalytic domain. Proposed functions for the GC—C receptor are fluid and electrolyte homeostasis, the regulation of epithelial cell proliferation and the induction of apoptosis (Shalubhai 2002 Curr Opin Drug Dis Devel 5:261-268).
  • GC—C In addition to being expressed in the intestine by gastrointestinal epithelial cells, GC—C is expressed in extra-intestinal tissues including kidney, lung, pancreas, pituitary, adrenal, developing liver (reviewed in Vaandrager 2002, Mol Cell Biochem 230:73-83) and male and female reproductive tissues (reviewed in Vaandrager 2002 Mol Cell Biochem 230:73-83)) This suggests that the GC—C receptor agonists can be used in the treatment of disorders outside the GI tract, for example, congestive heart failure and benign prostatic hyperplasia.
  • Ghrelin a peptide hormone secreted by the stomach, is a key regulator of appetite in humans. Ghrelin expression levels are regulated by fasting and by gastric emptying (Kim et al., 2003, Neuroreprt 14:1317-20; Gualillo et al., 2003, FEBS Letts 552: 105-9). Thus, by increasing gastrointestinal motility, GC—C receptor agonists may also be used to regulate obesity.
  • the GC—C receptor is activated by guanylin (Gn) (U.S. Pat. No. 5,96,097), uroguanylin (Ugn) (U.S. Pat. No. 5,140,102) and lymphoguanylin (Forte et al., 1999, Endocrinology 140:1800-1806).
  • Gn guanylin
  • Ugn U.S. Pat. No. 5,140,102
  • lymphoguanylin guanylin
  • these agents are 10-100 fold less potent than a class of bacterially derived peptides, termed ST (reviewed in Gianella 1995 J Lab Clin Med 125:173-181).
  • ST peptides are considered super agonists of GC—C and are very resistant to proteolytic degradation.
  • ST peptide is capable of stimulating the enteric nervous system (Rolfe et al., 1994, J Physiolo 475: 531-537; Rolfe et al., 1999, Gut 44: 615-619; Nzegwu et al., 1996, Exp Physiol 81: 313-315).
  • cGMP has been reported to have anitnociceptive effects in multiple animal models of pain (Lazaro Ibanez et al., 2001, Eur J Pharmacol 426: 39-44; Soares et al., 2001, British J Pharmacol 134: 127-131; Jain et al., 2001, Brain Res 909:170-178; Amarante et al., 2002, Eur J Pharmacol 454:19-23).
  • GC—C agonists may have both an analgesic as well an anti-inflammatory effect.
  • ST peptides are derived from a preproprotein that generally has at least 70 amino acids.
  • the pre and pro regions are cleaved as part of the secretion process, and the resulting mature protein, which generally includes fewer than 20 amino acids, is biologically active.
  • E. coli ST Ib (Moseley et al. (1983) Infect. Immun. 39:1167) having the mature amino acid sequence Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:1); E. coli ST Ia (So and McCarthy (1980) Proc. Natl. Acad. Sci. USA 77:4011) having the mature amino acid sequence Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys Tyr (SEQ ID NO:2); E.
  • enterocolitica ST peptides Y-ST(Y-STa), Y-STb, and Y-STc (reviewed in Huang et al. (1997) Microb. Pathog. 22:89) having the following pro-form amino acid sequences: Gln Ala Cys Asp Pro Pro Ser Pro Pro Ala Glu Val Ser Ser Asp Trp Asp Cys Cys Asp Val Cys Cys Asn Pro Ala Cys Ala Gly Cys (SEQ ID NO:5) (as well as a Ser-7 to Leu-7 variant of Y-STa (SEQ ID NO:122), (Takao et al. (1985) Eur. J. Biochem.
  • kristensenii ST peptide having the mature amino acid sequence Ser Asp Trp Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Ala Gly Cys (SEQ ID NO:8); V. cholerae non-01 ST peptide (Takao et al. (1985) FEBS lett. 193:250) having the mature amino acid sequence Ile Asp Cys Cys Glu Ile Cys Cys Asn Pro Ala Cys Phe Gly Cys Leu Asn (SEQ ID NO:9); and V. mimicus ST peptide (Arita (1991) et al. FEMS Microbiol. Lett.
  • the immature (including pre and pro regions) form of E. coli ST-1A (ST-P) protein has the sequence: mkklmlaifisvlsfpsfsqstesldsskekitletkkcdvvknnsekksenmnntfyccelccnpacagcy (SEQ ID NO:20; see GenBank® Accession No. P01559 (gi:123711).
  • the pre sequence extends from aa 1-19.
  • the pro sequence extends from aa 20-54.
  • the mature protein extends from 55-72.
  • coli ST-1B (ST-H) protein has the sequence: mkksilfiflsvlsfspfaqdakpvesskekitleskkcniakksnksgpesmnssnyccelccnpactgcy (SEQ ID NO: 21; see GenBank® Accession No. P07965 (gi:3915589).
  • enterocolitica ST protein has the sequence: mkkivfvlvlmlssfgafgqetvsgqfsdalstpitaevykqacdpplppaevssdwdccdvccnpacagc (SEQ ID NO:22) see GenBank® Accession No. S25659 (gi:282047).
  • the peptides of the invention like the bacterial ST peptides, have six Cys residues. These six Cys residues form three disulfide bonds in the mature and active form of the peptide. If the six Cys residues are identified, from the amino to carboxy terminus of the peptide, as A, B, C, D, E, and F, then the disulfide bonds form as follows: A-D, B-E, and C—F. The formation of these bonds is thought to be important for GC—C receptor binding.
  • Certain of the peptides of the invention include a potentially functional chymotrypsin cleavage site, e.g., a Trp, Tyr or Phe located between either Cys B and Cys D or between Cys E and Cys F. Cleavage at either chymotrypsin cleavage site reduces or eliminates the ability of the peptide to bind to the GC—C receptor.
  • a potentially functional chymotrypsin cleavage site e.g., a Trp, Tyr or Phe located between either Cys B and Cys D or between Cys E and Cys F.
  • chymotrypsinogen is produced in the pancreas.
  • this inactive enzyme reaches the small intestine it is converted to active chymotrypsin by the excision of two di-peptides.
  • Active chymotrypsin can potentially cleave peptides at the peptide bond on the carboxy-terminal side of Trp, Tyr or Phe.
  • the presence of active chymotrypsin in the intestinal tract can potentially lead to cleavage of certain of the peptides of the invention having an appropriately positioned functional chymotrypsin cleavage site. It is expected that chymotrypsin cleavage will moderate the action of a peptide of the invention having an appropriately positioned chymotrypsin cleavage site as the peptide passes through the intestinal tract.
  • Trypsinogen like chymotrypsin, is a serine protease that is produced in the pancreas and is present in the digestive tract.
  • the active form, trypsin will cleave peptides having a Lys or Arg.
  • the presence of active trypsin in the intestinal tract can lead to cleavage of certain of the peptides of the invention having an appropriately positioned functional trypsin cleavage site. It is expected that chymotrypsin cleavage will moderate the action of a peptide of the invention having an appropriately positioned trypsin cleavage site as the peptide passes through the intestinal tract.
  • Certain of the peptides of the invention include analgesic or antinociceptive tags such as the carboxy-terminal sequence AspPhe immediately following a Trp, Tyr or Phe that creates a functional chymotrypsin cleavage site or following Lys or Arg that creates a functional trypsin cleavage site.
  • Chymotrypsin in the intestinal tract can potentially cleave such peptides immediately carboxy terminal to the Trp, Phe or Tyr residue, releasing the dipeptide, AspPhe. This dipeptide has been shown to have analgesic activity in animal models (Abdikkahi et al.
  • analgesic peptides can be present at the carboxy terminus of the peptide (following a functional cleavage site) including: endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron, and substance P.
  • a number of the useful peptides are based on the core sequence: Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:29).
  • the core sequence is followed by Asp Phe.
  • the carboxy terminal Tyr in the core sequence can allow the Asp Phe dipeptide to be released by chymotrypsin in the digestive tract.
  • the core sequence can be optionally be preceded by Asn Ser Ser Asn Tyr or Asn.
  • useful variants based on the core sequence include: Asn Ser Ser Asn Tyr Cys Cys Glu (SEQ ID NO:26; Leu Cys Cys Asn Pro Ala Cys Thr MM-416776) Gly Cys Tyr Asn Ser Ser Asn Tyr Cys Cys Glu (SEQ ID NO:27) Leu Cys Cys Asn Pro Ala Cys Trp Gly Cys Tyr Asn Ser Ser Asn Tyr Cys Cys Glu (SEQ ID NO:28; Tyr Cys Cys Asn Pro Ala Cys Thr MD-915) Gly Cys Tyr Cys Cys Glu Leu Cys Cys Asn Pro (SEQ ID NO:29; Ala Cys Thr Gly Cys Tyr MM416774) Cys Cys Glu Leu Cys Cys Asn Pro (SEQ ID NO:30) Ala Cys Trp Gly Cys Tyr Cys Cys Glu Tyr Cys Cys Asn Pro (SEQ ID NO:31; Ala Cys Thr Gly Cys
  • the peptides of the invention are produced as a prepro protein that includes the amino terminal leader sequence: mkksilfiflsvlsfspfaqdakpvesskekitleskkcniakksnksgpesmn (SEQ ID NO:23).
  • the peptide is produced by a bacterial cell, e.g., E. coli
  • the forgoing leader sequence will be cleaved and the mature peptide will be efficiently secreted from the bacterial cell.
  • U.S. Pat. No. 5,395,490 describes vectors, expression systems and methods for the efficient production of ST peptides in bacterial cells and methods for achieving efficient secretion of mature ST peptides.
  • the vectors, expression systems and methods described in U.S. Pat. No. 5,395,490 can be used to produce the ST peptides and variant ST peptides of the present invention
  • the invention includes variant peptides which can include one, two, three, four, five, six, seven, eight, nine, or ten (in some embodiments fewer than 5 or fewer than 3 or 2 or fewer) amino acid substitutions compared to SEQ ID NOs:25 to 59 and 66 to 110.
  • the substitution(s) can be conservative or non-conservative.
  • the naturally-occurring amino acids can be substituted by D-isomers of any amino acid, non-natural amino acids, and other groups.
  • a conservative amino acid substitution results in the alteration of an amino acid for a similar acting amino acid, or amino acid of like charge, polarity, or hydrophobicity. At some positions, even conservative amino acid substitutions can reduce the activity of the peptide.
  • Useful peptides can be produced either in bacteria including, without limitation, E. coli, or in other existing systems for peptide or protein production (e.g., Bacillus subtilis, baculovirus expression systems using Drosophila Sf9 cells, yeast or filamentous fungal expression systems, mammalian cell expression systems), or they can be chemically synthesized.
  • bacteria including, without limitation, E. coli, or in other existing systems for peptide or protein production (e.g., Bacillus subtilis, baculovirus expression systems using Drosophila Sf9 cells, yeast or filamentous fungal expression systems, mammalian cell expression systems), or they can be chemically synthesized.
  • the nucleic acid molecule encoding the peptide will preferably also encode a leader sequence that permits the secretion of the mature peptide from the cell.
  • the sequence encoding the peptide can include the pre sequence and the pro sequence of, for example, a naturally-occurring bacterial ST peptide.
  • the secreted, mature peptide can be purified from the culture medium.
  • the sequence encoding a peptide of the invention is preferably inserted into a vector capable of delivering and maintaining the nucleic acid molecule in a bacterial cell.
  • the DNA molecule may be inserted into an autonomously replicating vector (suitable vectors include, for example, pGEM3Z and pcDNA3, and derivatives thereof).
  • the vector nucleic acid may be a bacterial or bacteriophage DNA such as bacteriophage lambda or M13 and derivatives thereof. Construction of a vector containing a nucleic acid described herein can be followed by transformation of a host cell such as a bacterium. Suitable bacterial hosts include but are not limited to, E. coli, B.
  • the genetic construct also includes, in addition to the encoding nucleic acid molecule, elements that allow expression, such as a promoter and regulatory sequences.
  • the expression vectors may contain transcriptional control sequences that control transcriptional initiation, such as promoter, enhancer, operator, and repressor sequences. A variety of transcriptional control sequences are well known to those in the art.
  • the expression vector can also include a translation regulatory sequence (e.g., an untranslated 5′ sequence, an untranslated 3′ sequence, or an internal ribosome entry site).
  • the vector can be capable of autonomous replication or it can integrate into host DNA to ensure stability during peptide production.
  • the protein coding sequence that includes a peptide of the invention can also be fused to a nucleic acid encoding a polypeptide affinity tag, e.g., glutathione S-transferase (GST), maltose E binding protein, protein A, FLAG tag, hexa-histidine, myc tag or the influenza HA tag, in order to facilitate purification.
  • GST glutathione S-transferase
  • the affinity tag or reporter fusion joins the reading frame of the peptide of interest to the reading frame of the gene encoding the affinity tag such that a translational fusion is generated. Expression of the fusion gene results in translation of a single polypeptide that includes both the peptide of interest and the affinity tag.
  • DNA sequence encoding a protease recognition site will be fused between the reading frames for the affinity tag and the peptide of interest.
  • Mature peptides and variants thereof can be synthesized by the solid-phase method using an automated peptide synthesizer.
  • the peptide can be synthesized on Cyc(4-CH 2 Bxl)-OCH 2 -4-(oxymethyl)-phenylacetamidomethyl resin using a double coupling program.
  • Protecting groups must be used appropriately to create the correct disulfide bond pattern.
  • protecting groups can be used: t-butyloxycarbonyl (alpha-amino groups); acetamidomethyl (thiol groups of Cys residues B and E); 4-methylbenyl (thiol groups of Cys residues C and F); benzyl (y-carboxyl of glutamic acid and the hydroxyl group of threonine, if present); and bromobenzyl (phenolic group of tyrosine, if present).
  • Coupling is effected with symmetrical anhydride of t-butoxylcarbonylamino acids or hydroxybenzotriazole ester (for asparagine or glutamine residues), and the peptide is deprotected and cleaved from the solid support in hydrogen fluoride, dimethyl sulfide, anisole, and p-thiocresol using 8/1/1/0.5 ratio (v/v/v/w) at 0° C. for 60 min.
  • the disulfide bond between Cys residues C and F is formed by first dissolving the peptide in 50% acetic acid in water. Saturated iodine solution in glacial acetic acid is added (1 ml iodine solution per 100 ml solution). After incubation at room temperature for 2 days in an enclosed glass container, the solution is diluted five-fold with deionized water and extracted with ethyl ether four times for removal of unreacted iodine. After removal of the residual amount of ethyl ether by rotary evaporation the solution of crude product is lyophilized and purified by successive reverse-phase chromatography.
  • peptides and other agents to bind to the intestinal GC—C receptor can be tested as follows.
  • Cells of the T84 human colon carcinoma cell line (American Type Culture Collection (Bethesda, Md.) are grown to confluence in 24-well culture plates with a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium (DMEM), supplemented with 5% fetal calf serum. Cells used in the assay are typically between passages 54-60. Briefly, T84 cell monolayers in 24-well plates are washed twice with 1 ml of binding buffer (DMEM containing 0.05% bovine serum albumin and 25 mM HEPES, pH 7.2), then incubated for 30 min at 37° C.
  • binding buffer DMEM containing 0.05% bovine serum albumin and 25 mM HEPES, pH 7.2
  • E. coli ST peptide in the presence of mature radioactively labeled E. coli ST peptide and the test material at various concentrations.
  • the cells are then washed four times with 1 ml of DMEM and solubilized with 0.5 ml/well 1N NaOH.
  • the level of radioactivity in the solubilized material is then determined using standard methods.
  • MD-915 A variant ST peptide, referred to as MD-915, was reproduced recombinantly and tested in an animal model.
  • MD-915 has the sequence: Asn Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:28).
  • a peptide having the sequence of the wild-type ST peptide was also created (MM-416776).
  • MD-915 and MM-416776 peptides were produced as preproproteins using vectors produced as follows.
  • a sequence encoding a heat-stable enterotoxin pre-pro sequence was amplified from pGK51/pGSK51 (ATCC 67728) using oligonucleotide MO3514 (5′CACACCATATGAAGAAATCAATATTATTTATTTTTCTTTCTG 3′ (SEG ID NO:60)) and oligonucelotide M03515 (5′CACACCTCGAGTTAGGTCTCCATGCTTTCAGGACCACTTTTATTAC 3′ (SEQ ID NO: 61)).
  • the amplification product fragment was digested with NdeI/XhoI and ligated to the T7 expression vector, pET26b(+) (Novagen) digested with NdeI/XhoI thereby creating plasmid MB3976.
  • the region encoding the pre-pro protein was sequenced and found to encode the amino acid sequence: mkksilfiflsvlsfspfaqdakpagsskekitleskkcnivkk s n k s g pesm (SEQ ID NO: 24) which differs from the amino acid sequence of heat-stable enterotoxin a2 precursor (sta2; mkksilfiflsvlsfspfaqdakpagsskekitleskkcnivkknnesspesm (SEQ ID NO:25); GenBank® Accession No. Q47185, GI: 3913876) at three positions (indicated by underlining and bold text) near the C-terminus.
  • MB3976 complementary oligos encoding each ST peptide variant or wild-type ST peptide were annealed and cloned into the MB3976 expression vector.
  • MB3984 encoding MM-416776 peptide full length wild-type ST peptide as a prepro protein
  • MB 3976 was digested with BsaI/XhoI and ligated to annealed oligos MO3621 (5′GCATGAATAGTAGCAATTACTGCTGTGAATTGTGTTGTAATCCTGCTTGTACCGGGT GCTATTAATAAC 3′ (SEQ ID NO:62)
  • MO3622 5′TCGAGTTATTAATAGCACCCGGTACAAGCAGGATTACAACACAATTCACAGCAGTA ATTGCTACTATTC 3′ (SEQ ID NO:63)).
  • MB3985 (encoding MD-915 as a prepro protein) containing the following amino acid sequence, NSSNYCCEYCCNPACTGCY (SEQ ID NO:28) fused downstream of the pre-pro sequence
  • MB 3976 was digested with BsaI/XhoI and ligated to annealed oligos M03529 (5′GCATGAATAGTAGCAATTACTGCTGTGAATATTGTTGTAATCCTGCTTGTACCGGGT GCTATTAATAAC 3′ (SEQ ID NO:64)) and MO3530 (5′TCGAGTTATTAATAGCACCCGGTACAAGCAGGATTACAACAATATTCACAGCAGTA ATTGCTACTATTC 3′ (SEQ ID NO:65)).
  • the MD-915 peptide and the MM-416776 peptide were produced as follows.
  • the expression vectors were transformed into E. coli bacterial host BL21 ⁇ DE3 (Invitrogen). A single colony was innoculated and grown shaking overnight at 30° C. in L broth+25 mg/l kanamycin.
  • the overnight culture was added to 3.2 L of batch medium (Glucose 25 g/l, Caseamino Acids 5 g/l, Yeast Extract 5 g/l, KH 2 PO 4 13.3 g/l, (NH 4 ) 2 HPO 4 4 g/l, MgSO 4 -7H 2 0 1.2 g/l, Citric Acid 1.7 g/l, EDTA 8.4 mg/l, CoCl 2 -6H 2 O 2.5 mg/l, MnCl 2 -4H 2 O 15 mg/l, CuCl 2 -4H 2 0 1.5 mg/l, H 3 BO 3 3 mg/l, Na 2 MoO 4 -2H 2 0 2.5 mg/l, Zn Acetate-2H 2 0 13 mg/l, Ferric Citrate 100 mg/l, Kanamycin 25 mg/l, Antifoam DF 2 0 4 1 ml/l) and fermented using the following process parameters: pH 6.7—control with base only (28% NH 4 OH), 30° C
  • MD-915 peptide and MM-416776 peptide fractions were analyzed by standard LCMS and HPLC. LCMS analysis revealed that MD-915 is more homogeneous than MM-416776 (see FIG. 1 a; note that MD-915 peptide exhibits fewer peaks (Panel B) than MM-416776 (Panel A)).
  • Peptides were chemically synthesized by a commercial peptide synthesis company. Varying yields of peptides were obtained depending on the efficiency of chemical synthesis. Thus, the four peptides, in decreasing order of yield were: Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:31; MD-1100), 10-20% yield; Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:29; MM416774); Asn Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:28; MD-915); Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:26 MM-416776), ⁇ 5% yield.
  • the specific amino acid changes introduced into the peptides can create improved manufacturing properties
  • FIG. 1 b shows the total ion chromatograph profile of synthetically manufactured MD-1100.
  • FIG. 1 c shows the total ion chromatograph profile of the control blank sample. There is one major peak present in the MD-1100 sample that is not also present in the control sample. Quantitative analysis suggests the MD-1100 is >98% pure.
  • the ability of MD-915, MM-416776, and MD-1100 to activate the intestinal GC—C receptor was assessed in an assay employing the T84 human colon carcinoma cell line (American Type Culture Collection (Bethesda, Md.). For the assays cells were grown to confluency in 24-well culture plates with a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium (DMEM), supplemented with 5% fetal calf serum and were used at between passages 54 and 60.
  • DMEM Dulbecco's modified Eagle's medium
  • the dried samples were resuspended in 0.5 ml of phosphate buffer provided in the Cayman Chemical Cyclic GMP EIA kit (Cayman Chemical, Ann Arbor, Mich.). Cyclic GMP was measured by EIA according to procedures outlined in the Cayman Chemical Cyclic GMP EIA kit.
  • FIG. 2 shows the activity of chemically synthesized peptide variants in this GC—C receptor activity assay.
  • MM-416776 and two different MD-1100 peptides (MD-1100(a) and MD-1100(b), synthesized by two different methods) had activity comparable to MM-416776.
  • MD-915 and MM-416776 peptide were chemically synthesized in a manner identical to that of MD-1100(b).
  • GIT murine gastrointestinal transit
  • mice were fasted with free access to water for 12 to 16 hours before the treatment with peptide or control buffer.
  • the peptides were orally administered at 1 ⁇ g/kg-1 mg/kg of peptide in buffer (20 mM Tris pH 7.5) 7 minutes before being given an oral dose of 5% Activated Carbon (Aldrich 242276-250G).
  • Control mice were administered buffer only before being given a dose of Activated Carbon.
  • the mice were sacrificed and their intestines from the stomach to the cecum were dissected. The total length of the intestine as well as the distance traveled from the stomach to the charcoal front was measured for each animal and the results are expressed as the percent of the total length of the intestine traveled by the charcoal front.
  • mice All results are reported as the average of 10 mice ⁇ standard deviation. A comparison of the distance traveled by the charcoal between the mice treated with peptide versus the mice treated with vehicle alone was performed using a Student's t test and a statistically significant difference was considered for P ⁇ 0.05. P-values are calculated using a two-sided T-Test assuming unequal variances.
  • FIG. 3 a, b wild-type ST peptide (MM-416776, (Sigma-Aldrich, St Louis, Mo.; 0.1 mg/kg), synthetically manufactured MD-1100 and Zelnorm® (0.1 mg/kg), a drug approved for IBS that is an agonist for the serotonin receptor 5HT4, increase gastrointestinal transit rate in this model.
  • FIG. 4 a shows the result of a study demonstrating that intestinal transit rate increases with an increasing dosage of either recombinantly synthesized MM-416776 or MD-915.
  • FIG. 4 b shows the results of a study demonstrating both chemically synthesized MM-416776 or MD-1100 peptide increase intestinal transit rates more than either Tris buffer alone or an equivalent dose of Zelnorm®.
  • FIG. 4 c shows the results of a study demonstrating both chemically synthesized MD-1100 or Zelnorm® are effective in a mouse gastrointestinal motility assay upon chronic dosing (daily for 5 days). The results are shown side by side with acute dosing (1 day).
  • MM-416776 peptide and MD-915 were tested for their ability to increase intestinal secretion using a suckling mouse model of intestinal secretion.
  • a test compound is administered to suckling mice that are between 7 and 9 days old. After the mice are sacrificed, the gastrointestinal tract from the stomach to the cecum is dissected (“guts”). The remains (“carcass”) as well as the guts are weighed and the ratio of guts to carcass weight is calculated. If the ratio is above 0.09, one can conclude that the test compound increases intestinal secretion.
  • FIG. 5 a shows a dose response curve for wild-type ST peptide (MM-416776) in this model.
  • FIG. 5 b shows dose response curve for the MD-1100 peptide in this model.
  • FIG. 6 a shows a dose response curve for the recombinant MM-416776 peptide described above and the recombinant MD-915 peptide described above. As can be seen from FIG. 6 a, both peptides increase intestinal secretion in this model.
  • FIG. 6 b shows a dose response curve for chemically synthesized MD-915, MD-1100 and MM-416776 as well as wild-type ST peptide (purchased from Sigma-Aldrich, St Louis, Mo.).
  • TNBS trinitrobenzenesulphonic acid
  • FIG. 7 shows the results of experiment in which MD-1100 activity was analyzed in the TNBS colorectal model. Significant decreases in abdominal response are observed at 0.3 ⁇ g/kg and 3 ⁇ g/kg MD-1100. These results demonstrate that MD-1100 reduces pain associated with colorectal distension in this animal model.
  • Male Wistar Rats (200-250 g) are surgically implanted with nichrome wire electrodes as in the TNBS model. Ten days post surgical implantation, partial restraint stress (PRS), is performed as described by Williams et al. for two hours (Williams et al. 1988 Gastroenterology 64:611). Briefly, under light anesthesia with ethyl-ether, the foreshoulders, upper forelimbs and thoracic trunk are wrapped in a confining harness of paper tape to restrict, but not prevent body movements. Control sham-stress animals are anaesthetized but not wrapped. Thirty minutes before the end of the PRS session, the animals are administered test-compound or vehicle.
  • PRS partial restraint stress
  • the CRD distension procedure is performed as described above for the TNBS model with barostat at pressures of 15, 30, 45 and 60 mm Hg.
  • Statistical analysis on the number of bursts is determined and analyzed as in the TNBS model above.
  • the PBQ-induced writhing model can be used to assess pain control activity of the peptides and GC—C receptor agonists of the invention. This model is described by Siegmund et al. (1957 Proc. Soc. Exp. Bio. Med. 95:729-731). Briefly, one hour after oral dosing with a test compound, e.g., a peptide, morphine or vehicle, 0.02% phenylbenzoquinone (PBQ) solution (12.5 mL/kg) is injected by intraperitoneal route into the mouse.
  • PBQ phenylbenzoquinone
  • the number of stretches and writhings are recorded from the 5 th to the 10 th minute after PBQ injection, and can also be counted between the 35 th and 40 th minute and between the 60 th and 65 th minute to provide a kinetic assessment.
  • the results are expressed as the number of stretches and writhings (mean ⁇ SEM) and the percentage of variation of the nociceptive threshold calculated from the mean value of the vehicle-treated group.
  • the statistical significance of any differences between the treated groups and the control group is determined by a Dunnett's test using the residual variance after a one-way analysis of variance (P ⁇ 0.05) using SigmaStat Software.
  • FIGS. 8 a and 8 b show the effect of different doses of MD-915 and MD-1100 in the PBQ writhing assay.
  • Indomethacin an NSAID (nonsteroidal anti-inflammatory drug) with known pain control activity, was used as the positive control in the assay.
  • Significant reductions in writhings were observed for MD-915 (1 mg/kg dose) and MD-1100 (2.5 mg/kg dose) compared to the vehicle control. Loss of efficacy at the highest dose tested has also been observed for multiple other compounds (such as 5HT-3 antagonists) tested in similar assays.
  • the results of this study suggest that both MD-915 and MD-1100 have antinociceptive effects in this visceral pain model comparable to the intermediate doses of indomethacin.
  • Intestinal mucosa was extruded from the intestine by gentle pressure between a piece of parafilm and a P-1000 pipette tip. Intestinal epithelial cells were placed in 2 ml PBS and pipetted up and down with a 5 ml pipette to make a suspension of cells. Protein concentration in the suspension was measured using the Bradford method ( Anal. Biochem. 72: 248-254 (1976)).
  • a competition binding assay was performed based on the method of Giannella et al. ( Am. J. Physiol. 245: G492-G498) between [ 125 I] labeled MM-416776 and MD-1100.
  • the assay mixture contained: 0.5 ml of DME with 20 mM HEPES-KOH pH 7.0, 0.9 mg of the cell suspension listed above, 21.4 fmol[ 125 I]-MM-416776 (42.8 pM), and different concentrations of competitor MD-1100 (0.01 to 1000 nM). The mixture was incubated at room temperature for 1 hour, and the reaction stopped by applying the mixture to GF/B glass-fiber filters (Whatman).
  • FIG. 9 shows that the Kd for MD-1100 in this assay is 4.5 nm.
  • %B/Bo is the percentage of the ratio of radioactivity trapped in each sample (B) compared to the radioactivity retained in a control sample with no cold competitor (Bo).
  • Giannella et al. Am. J Physiol. 245: G492-G4978 observed that the Kd for wild-type ST peptide in this same assay was ⁇ 13 nm.
  • FIG. 10 a show absorption data for intravenously and orally administered MD-1100 as detected by the ELISA assay. MD-1100 appears to be minimally systemically absorbed and is ⁇ 2.2% bioavailable.
  • the sample was eluted from the analytical column with a reverse phase gradient (Mobile Phase A: 10 mM ammonium hydroxide in dH 2 O, Mobile Phase B: 10 mM ammonium hydroxide in 80% acetonitrile and 20% methanol; 20% B for the first 3 minutes then ramping to 95% B over 4 min. and holding for 2 min., all at a flow rate of 0.4 mL/min.). At 9.1 minutes, the gradient returns to the initial conditions of 20%B for 1 min.
  • Mobile Phase A 10 mM ammonium hydroxide in dH 2 O
  • Mobile Phase B 10 mM ammonium hydroxide in 80% acetonitrile and 20% methanol
  • MRM triple-quadrapole mass spectrometry
  • FIG. 10 b shows absorption data for IV and orally administered MD-1100 as detected by LCMS.
  • MD-1100 appears similarly minimally systemically absorbed and is ⁇ 0.11% bioavailable.
  • the peptides and agonists of the invention are preferably administered orally, e.g., as a tablet, gel, paste, slurry, liquid, powder or in some other form.
  • Orally administered compositions can include binders, flavoring agents, and humectants.
  • the peptides and agonists can be co-administered with other agents used to treat gastrointestinal disorders including but not limited to acid suppressing agents such as Histamine-2 receptor agonists (H2As) and proton pump inhibitors (PPIs).
  • H2As Histamine-2 receptor agonists
  • PPIs proton pump inhibitors
  • the peptides and agonists can also be administered by rectal suppository.
  • peptides and agonists are preferably administered parenterally or orally.
  • the peptides described herein can be used alone or in combination with other agents.
  • the peptides can be administered together with an analgesic peptide or compound.
  • the analgesic peptide or compound can be covalently attached to a peptide described herein or it can be a separate agent that is administered together with or sequentially with a peptide described herein in a combination therapy.
  • Combination therapy can be achieved by administering two or more agents, e.g., a peptide described herein and an analgesic peptide or compound, each of which is formulated and administered separately, or by administering two or more agents in a single formulation.
  • agents e.g., a peptide described herein and an analgesic peptide or compound, each of which is formulated and administered separately, or by administering two or more agents in a single formulation.
  • two agents can be formulated together and administered in conjunction with a separate formulation containing a third agent. While the two or more agents in the combination therapy can be administered simultaneously, they need not be.
  • administration of a first agent (or combination of agents) can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks.
  • the two or more agents can be administered within minutes of each other or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of each other. In some cases even longer intervals are possible. While in many cases it is desirable that the two or more agents used in a combination therapy be present in within the patient's body at the same time, this need not be so.
  • Combination therapy can also include two or more administrations of one or more of the agents used in the combination. For example, if agent X and agent Y are used in a combination, one could administer them sequentially in any combination one or more times, e.g., in the order X—Y—X, X—X—Y, Y—X—Y, Y—Y—X, X—X—Y—Y, etc.
  • the agents alone or in combination, can be combined with any pharmaceutically acceptable carrier or medium. Thus, they can be combined with materials that do not produce an adverse, allergic or otherwise unwanted reaction when administered to a patient.
  • the carriers or mediums used can include solvents, dispersants, coatings, absorption promoting agents, controlled release agents, etc.
  • the agents either in their free form or as a salt can be combined with a polymer such as polylactic-glycoloic acid (PLGA), poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) (WO 01/12233), polyglycolic acid (U.S. Pat. No. 3,773,919), polylactic acid (U.S. Pat. No. 4,767,628); poly( ⁇ -caprolactone) to create a sustained release formulation.
  • PLGA polylactic-glycoloic acid
  • P(I)LGT poly-(I)-lactic-glycolic-tartaric acid
  • P(I)LGT) WO 01/12233
  • polyglycolic acid U.S. Pat. No. 3,773,919
  • polylactic acid U.S. Pat. No. 4,767,628
  • poly( ⁇ -caprolactone) poly( ⁇ -caprolactone)
  • Such formulations can be used to implants that release a peptide or another agent over a period of a few days, a few weeks or several months depending on the polymer, the particle size of the polymer, and the size of the implant (see, e.g., U.S. Pat. No. 6,620,422).
  • Other sustained release formulations are described in EP 0 467 389 A2, WO 93/241150, U.S. Pat. No. 5,612,052; WO 97/40085, WO 94/155587, U.S. Pat. No. 5,672,659, U.S. Pat. No. 5,893,985, U.S. Pat. No. 5,134,122, U.S. Pat. No. 5,192,741, U.S. Pat.
  • sustained release formulations microparticles of peptide are combined with microparticles of polymer.
  • One or more sustained release implants can be placed in the large intestine, the small intestine or both.
  • the agents can be administered, e.g., by intravenous injection, intramuscular injection, subcutaneous injection, or by other routes.
  • the agents can be administered orally, e.g., as a tablet, gel, paste, slurry, liquid, powder or in some other form.
  • Orally administered compositions can include binders, flavoring agents, and humectants.
  • the agents can be included in dentifrices or oral washes.
  • oral formulations can include abrasives and foaming agents.
  • the agents can also be administered transdermally or in the form a suppository.
  • the agents can be a free acid or base, or a pharmacologically acceptable salt thereof. Solids can be dissolved or dispersed immediately prior to administration or earlier. In some circumstances the preparations include a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injection can include sterile aqueous or organic solutions or dispersions which include, e.g., water, an alcohol, an organic solvent, an oil or other solvent or dispersant (e.g., glycerol, propylene glycol, polyethylene glycol, and vegetable oils). Pharmaceutical agents can be sterilized by filter sterilization or by other suitable means.
  • Suitable pharmaceutical compositions in accordance with the invention will generally include an amount of the active compound(s) with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give a range of final concentrations, depending on the intended use.
  • an acceptable pharmaceutical diluent or excipient such as a sterile aqueous solution.
  • the techniques of preparation are generally well known in the art, as exemplified by Remington's Pharmaceutical Sciences (18th Edition, Mack Publishing Company, 1995).
  • the agents described herein and combination therapy agents can be packaged as a kit that includes single or multiple doses of two or more agents, each packaged or formulated individually, or single or multiple doses of two or more agents packaged or formulated in combination.
  • one or more agents can be present in first container, and the kit can optionally include one or more agents in a second container.
  • the container or containers are placed within a package, and the package can optionally include administration or dosage instructions.
  • a kit can include additional components such as syringes or other means for administering the agents as well as diluents or other means for formulation.
  • the peptides described herein can be used in combination therapy with an analgesic agent, e.g., an analgesic compound or an analgesic peptide.
  • analgesic agent e.g., an analgesic compound or an analgesic peptide.
  • the analgesic agent can optionally be covalently attached to a peptide described herein.
  • analgesic agents are: Ca channel blockers, 5HT receptor antagonists (for example 5HT3, 5HT4 and 5HT1 receptor antagonists), opioid receptor agonists (loperamide, fedotozine, and fentanyl), NK1 receptor antagonists, CCK receptor agonists (e.g., loxiglumide), NK1 receptor antagonists, NK3 receptor antagonists, norepinephrine-serotonin reuptake inhibitors (NSRI), vanilloid and cannabanoid receptor agonists, and sialorphin.
  • 5HT receptor antagonists for example 5HT3, 5HT4 and 5HT1 receptor antagonists
  • opioid receptor agonists loperamide, fedotozine, and fentanyl
  • NK1 receptor antagonists e.g., CCK receptor agonists (e.g., loxiglumide)
  • NK1 receptor antagonists e.g., loxiglumide
  • NK1 receptor antagonists e.g.,
  • sialorphin-related peptides including those comprising the amino acid sequence QHNPR (SEQ ID NO:111), including: VQHNPR (SEQ ID NO:112); VRQHNPR (SEQ ID NO:113); VRGQHNPR (SEQ ID NO:114); VRGPQHNPR (SEQ ID NO:115); VRGPRQHNPR (SEQ ID NO:116); VRGPRRQHNPR (SEQ ID NO:117); and RQHNPR (SEQ ID NO:118).
  • Sialorphin-related peptides bind to neprilysin and inhibit neprilysin-mediated breakdown of substance P and Met-enkephalin.
  • compounds or peptides that are inhibitors of neprilysin are useful analgesic agents which can be administered with the peptides of the invention in a co-therapy or linked to the peptides of the invention, e.g., by a covalent bond.
  • analgesic agents which can be administered with the peptides of the invention in a co-therapy or linked to the peptides of the invention, e.g., by a covalent bond.
  • Sialophin and related peptides are described in U.S. Pat. No. 6,589,750; U.S. 20030078200 A1; and WO 02/051435 A2.
  • Opioid receptor antagonists and agonists can be administered with the peptides of the invention in co-therapy or linked to the peptide of the invention, e.g., by a covalent bond.
  • opioid receptor antagonists such as naloxone, naltrexone, methyl nalozone, nalmefene, cypridime, beta funaltrexamine, naloxonazine, naltrindole, and nor-binaltorphimine are thought to be useful in the treatment of IBS. It can be useful to formulate opioid antagonists of this type is a delayed and sustained release formulation such that initial release of the antagonist is in the mid to distal small intestine and/or ascending colon.
  • Enkephalin pentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine) is an agonist of the mu and delta opioid receptors and is thought to be useful for increasing intestinal motility ( Eur. J Pharm. 219:445, 1992), and this peptide can be used in conjunction with the peptides of the invention. Also useful is trimebutine which is thought to bind to mu/delta/kappa opioid receptors and activate release of motilin and modulate the release of gastrin, vasoactive intestinal peptide, gastrin and glucagons.
  • Kappa opioid receptor agonists such as fedotozine, ketocyclazocine, and compounds described in WO 03/097051 A2 can be used with or linked to the peptides of the invention.
  • mu opioid receptor agonists such as morphine, diphenyloxylate, frakefamide (H-Tyr-D-Ala-Phe(F)-Phe-NH 2 ; WO 01/019849 A1) and loperamide can be used.
  • Tyr-Arg is a dipeptide that acts by stimulating the release of met-enkephalins to elicit an analgesic effect ( J. Biol. Chem 262:8165, 1987).
  • Kyotorphin can be used with or linked to the peptides of the invention.
  • CCK receptor agonists such as caerulein from amphibians and other species are useful analgesic agents that can be used with or linked to the peptides of the invention.
  • Conotoxin peptides represent a large class of analgesic peptides that act at voltage gated Ca channels, NMDA receptors or nicotinic receptors. These peptides can be used with or linked to the peptides of the invention.
  • Peptide analogs of thymulin can have analgesic activity and can be used with or linked to the peptides of the invention.
  • CCK (CCKa or CCKb) receptor antagonists including loxiglumide and dexloxiglumide (the R-isomer of loxiglumide) (WO 88/05774) can have analgesic activity and can be used with or linked to the peptides of the invention.
  • 5-HT4 agonists such as tegaserod/zelnorm and hydrogen acetate.
  • 5-HT4 agonists such as tegaserod/zelnorm and hydrogenacetate.
  • Such agonists are described in: EP1321142 A1, WO 03/053432A1, EP 505322 A1, EP 505322 B1, U.S. Pat. No. 5,510,353, EP 507672 A1, EP 507672 B1, and U.S. P 5,273,983.
  • Calcium channel blockers such as ziconotide and related compounds described in, for example, EP625162B1, U.S. Pat. No. 5,364,842, U.S. Pat. No. 5,587,454, U.S. Pat 5,824,645, U.S. Pat. No. 5,859,186, U.S. Pat. No. 5,994,305, U.S. Pat. No. 6,087,091, U.S. Pat. No. 6,136,786, WO 93/13128 A1, EP 1336409 A1, EP 835126 A1, EP 835126 B1, U.S. Pat. No. 5,795,864, U.S. Pat. No. 5,891,849, U.S. Pat. No. 6,054,429, WO 97/01351 A1, can be used with or linked to the peptides of the invention.
  • NK-1, NK-2, and NK-3 receptors can be can be used with or linked to the peptides of the invention.
  • NK1 receptor antagonists such as: aprepitant (Merck & Co Inc), vofopitant, ezlopitant (Pfizer, Inc.), R-673 (Hoffmann-La Roche Ltd), SR-14033 and related compounds described in, for example, EP 873753 A1, US 20010006972 A1, US 20030109417 A1, WO 01/52844 A1, can be used with or linked to the peptides of the invention.
  • NK-2 receptor antagonists such as nepadutant (Menarini Ricerche SpA), saredutant (Sanofi-Synthelabo), SR-144190 (Sanofi-Synthelabo) and UK-290795 (Pfizer Inc) can be used with or linked to the peptides of the invention.
  • NK3 receptor antagonists such as osanetant (Sanofi-Synthelabo), talnetant and related compounds described in, for example, WO 02/094187 A2, EP 876347 A1, WO 97/21680 A1, U.S. Pat. No. 6,277,862, WO 98/11090, WO 95/28418, WO 97/19927, and Boden et al. ( J Med Chem. 39:1664-75, 1996) can be used with or linked to the peptides of the invention.
  • Norepinephrine-serotonin reuptake inhibitors such as milnacipran and related compounds described in WO 03/077897 A1 can be used with or linked to the peptides of the invention.
  • Vanilloid receptor antagonists such as arvanil and related compounds described in WO 01/64212 A1 can be used with or linked to the peptides of the invention.
  • the resulting peptide may also include at least one trypsin or chymotrypsin cleavage site.
  • the analgesic peptide may be preceded by (if it is at the carboxy terminus) or followed by (if it is at the amino terminus) a chymotrypsin or trypsin cleavage site that allows release of the analgesic peptide.
  • analgesic peptides include: AspPhe, endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron, zicnotide, and substance P.
  • the peptides of the invention can be used for the treatment or prevention of cancer, pre-cancerous growths, or metastatic growths.
  • they can be used for the prevention or treatment of: colorectal/local metastasized colorectal cancer, gastrointestinal tract cancer, lung cancer, cancer or pre-cancerous growths or metastatic growths of epithelial cells, polyps, breast, colorectal, lung, ovarian, pancreatic, prostatic, renal, stomach, bladder, liver, esophageal and testicular carcinoma, carcinoma (e.g., basal cell, basosquamous, Brown-Pearce, ductal carcinoma, Ehrlich tumor, Krebs, Merkel cell, small or non-small cell lung, oat cell, papillary, bronchiolar, squamous cell, transitional cell, Walker), leukemia (e.g., B-cell, T-cell, HTLV, acute or chronic lymphocytic, mast cell, myeloid), histiocyton
  • nonchroinaffin pinealoma, rhabdomyoma, rhabdomyosarcoma, Sertoli cell tumor, teratoma, theca cell tumor, and other diseases in which cells have become dysplastic, immortalized, or transformed.
  • the peptides of the invention can be used for the treatment or prevention of: Familial Adenomatous Polyposis (FAP) (autosomal dominant syndrome) that precedes colon cancer, hereditary nonpolyposis colorectal cancer (HNPCC), and inherited autosomal dominant syndrome.
  • FAP Familial Adenomatous Polyposis
  • HNPCC hereditary nonpolyposis colorectal cancer
  • the peptides can be used in combination therapy with radiation or chemotherapeutic agents, an inhibitor of a cGMP-dependent phosphodiesterase or a selective cyclooxygenase-2 inhibitor (a number of selective cyclooxygenase-2 inhibitors are described in WO02062369, hereby incorporated by reference).
  • the peptides can be for treatment or prevention of inflammation.
  • they can be used alone or in combination with inhibitor of cGMP-dependent phosphodiesterase or a selective cyclooxygenase-2 inhibitor for treatment of: organ inflammation, IBD (e.g, Crohn's disease, ulcerative colitis), asthma, nephritis, hepatitis, pancreatitis, bronchitis, cystic fibrosis, ischemic bowel diseases, intestinal inflammations/allergies, coeliac disease, proctitis, eosnophilic gastroenteritis, mastocytosis, and other inflammatory disorders.
  • IBD e.g, Crohn's disease, ulcerative colitis
  • asthma e.g, Crohn's disease, ulcerative colitis
  • nephritis hepatitis
  • pancreatitis bronchitis
  • cystic fibrosis ischemic bowel diseases
  • intestinal inflammations/allergies coeliac disease
  • the peptides can also be used to treat or prevent insulin-related disorders, for example: II diabetes mellitus, hyperglycemia, obesity, disorders associated with disturbances in glucose or electrolyte transport and insulin secretion in cells, or endocrine disorders. They can be also used in insulin resistance treatment and post-surgical and non-post surgery decrease in insulin responsiveness.
  • the peptides can be used to prevent or treat respiratory disorders, including, inhalation, ventilation and mucus secretion disorders, pulmonary hypertension, chronic obstruction of vessels and airways, and irreversible obstructions of vessels and bronchi.
  • the peptides can be used in combination therapy with a phosphodiesterase inhibitor (examples of such inhibitors can be found in U.S. Pat. No. 6,333,354, hereby incorporated by reference).
  • the peptides can also be used to prevent or treat: retinopathy, nephropathy, diabetic angiopathy, and edema formation
  • the peptides can also be used to prevent or treat neurological disorders, for example, headache, anxiety, movement disorders, aggression, psychosis, seizures, panic attacks, hysteria, sleep disorders, depression, schizoaffective disorders, sleep apnea, attention deficit syndromes, memory loss, and narcolepsy. They may also be used as a sedative.
  • neurological disorders for example, headache, anxiety, movement disorders, aggression, psychosis, seizures, panic attacks, hysteria, sleep disorders, depression, schizoaffective disorders, sleep apnea, attention deficit syndromes, memory loss, and narcolepsy. They may also be used as a sedative.
  • the peptides and detectabley labeled peptides can be used as markers to identify, detect, stage, or diagnosis diseases and conditions of the small intestine, including: Crohn's disease, colitis, inflammatory bowel disease, tumors, benign tumors, such as benign stromal tumors, adenoma, angioma, adenomatous (pedunculated and sessile) polyps, malignant, carcinoid tumors, endocrine cell tumors, lymphoma, adenocarcinoma, foregut, midgut, and hindgut carcinoma, gastroinstestinal stromal tumor (GIST), such as leiomyoma, cellular leiomyoma, leiomyoblastoma, and leiomyosarcoma, gastrointestinal autonomic nerve tumor, malabsorption syndromes, celiac diseases, diverticulosis, Meckel's diverticulum, colonic diverticula, megacolon, Hirschsprung's disease,
  • the peptides can be conjugated to another molecule (e.g, a diagnostic or therapeutic molecule) to target cells bearing the GCC receptor, e.g., cystic fibrosis lesions and specific cells lining the intestinal tract.
  • a diagnostic or therapeutic molecule e.g., cystic fibrosis lesions and specific cells lining the intestinal tract.
  • they can be used to target radioactive moieties or therapeutic moieties to the intestine to aid in imaging and diagnosing or treating colorectal/metastasized or local colorectal cancer and to deliver normal copies of the p53 tumor suppressor gene to the intestinal tract.
  • the peptides can be used alone or in combination therapy to treat erectile dysfunction.
  • the peptides can be used alone or in combination therapy to treat inner ear disorders, e.g., to treat Meniere's disease, including symptoms of the disease such as vertigo, hearing loss, tinnitus, sensation of fullness in the ear, and to maintain fluid homeostasis in the inner ear.
  • the peptides can be used alone or in combination therapy to treat disorders associated with fluid and sodium retention, e.g., diseases of the electrolyte-water/electrolyte transport system within the kidney, gut and urogenital system, congestive heart failure, hypertension,

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US20050266028A1 (en) * 2004-05-25 2005-12-01 University Of Georgia Research Foundation, Inc. Compositions and methods for modulating an immune response
US20060094658A1 (en) * 2003-06-13 2006-05-04 Currie Mark G Method and compositions for the treatment of gastrointestinal disorders
US20060258593A1 (en) * 2003-01-28 2006-11-16 Currie Mark G Methods and compositions for the treatment of gastrointestinal disorders
US20060281682A1 (en) * 2003-01-28 2006-12-14 Currie Mark G Methods and compositions for the treatment of gastrointestinal disorders
US20070010450A1 (en) * 2003-06-13 2007-01-11 Microbia, Inc., A Massachusetts Corporation Methods and compositions for the treatment of gastrointestinal disorders
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ES2285417T3 (es) 2007-11-16
CN101787073A (zh) 2010-07-28
RU2763796C2 (ru) 2022-01-11
KR20110114682A (ko) 2011-10-19
DE602004007105D1 (de) 2007-08-02
JP2010222377A (ja) 2010-10-07
CY2013021I1 (el) 2020-05-29
EP1594517A2 (en) 2005-11-16
LU92200I2 (fr) 2024-03-21
DK1911763T3 (da) 2010-11-15
DE602004007105T2 (de) 2008-02-28
JP5748791B2 (ja) 2015-07-15
EP2246360A1 (en) 2010-11-03
DK1594517T3 (da) 2007-10-29
CA2514507C (en) 2012-03-20
CN1795007A (zh) 2006-06-28
BE2013C032I2 (pt) 2023-08-22
AU2009235993B2 (en) 2013-07-04
RU2014153593A3 (pt) 2018-08-06
FR13C0029I2 (fr) 2024-04-19
ES2350123T3 (es) 2011-01-18
KR20050106404A (ko) 2005-11-09
CY1111058T1 (el) 2015-06-11
SI1911763T1 (sl) 2010-11-30
CN101787073B (zh) 2013-12-25
EP2246360B1 (en) 2012-06-27
EP1911763A2 (en) 2008-04-16
IL221380A0 (en) 2012-09-24
AU2004210161A1 (en) 2004-08-19
CN100589839C (zh) 2010-02-17
BR122018074353B8 (pt) 2023-05-02
KR101227626B1 (ko) 2013-02-01
LTC1594517I2 (lt) 2020-03-10
FR13C0030I1 (fr) 2013-07-05
SG192300A1 (en) 2013-08-30
IL169863A (en) 2012-10-31
PT2246360E (pt) 2012-08-31
KR101227627B1 (ko) 2013-01-31
EP1594517A4 (en) 2006-03-29
WO2004069165A2 (en) 2004-08-19
NO20130460L (no) 2005-10-26
CY1110905T1 (el) 2015-06-10
NO336768B1 (no) 2015-11-02
AU2009235993A1 (en) 2009-11-26
US20110124583A1 (en) 2011-05-26
HK1078768A1 (en) 2006-03-24
US7704947B2 (en) 2010-04-27
JP2006516631A (ja) 2006-07-06
US20090005534A1 (en) 2009-01-01
FR13C0029I1 (fr) 2013-07-05
SI2246360T1 (sl) 2012-10-30
RU2014153593A (ru) 2016-07-20
JP2013116910A (ja) 2013-06-13
EP1594517B1 (en) 2007-06-20
NL300593I1 (nl) 2015-10-21
DK2246360T3 (da) 2012-08-13
RU2543350C2 (ru) 2015-02-27
ATE365174T1 (de) 2007-07-15
CY2013022I1 (el) 2020-05-29
NO20053864L (no) 2005-10-26
HUS1300022I1 (hu) 2019-11-28
RU2008147983A (ru) 2010-06-10
RU2005127058A (ru) 2006-03-10
SG168407A1 (en) 2011-02-28
PT1911763E (pt) 2010-11-18
CY1113217T1 (el) 2016-04-13
BR122018074353B1 (pt) 2019-07-16
NO334112B1 (no) 2013-12-09
MXPA05008097A (es) 2006-02-08
NZ570134A (en) 2010-02-26
NZ541595A (en) 2008-08-29
JP5563384B2 (ja) 2014-07-30
BRPI0407071B1 (pt) 2019-02-12
AU2004210161B2 (en) 2009-11-26
ATE477268T1 (de) 2010-08-15
LU92200I1 (fr) 2020-06-02
EP1911763B1 (en) 2010-08-11
SI1594517T1 (sl) 2007-10-31
US8080526B2 (en) 2011-12-20
PT1594517E (pt) 2007-07-20
LU92201I2 (fr) 2013-11-22
CA2514507A1 (en) 2004-08-19
EP1911763A3 (en) 2008-06-25
DE602004028678D1 (de) 2010-09-23
BRPI0407071B8 (pt) 2021-05-25
ES2387848T3 (es) 2012-10-02
BRPI0407071A (pt) 2006-01-24
RU2353383C2 (ru) 2009-04-27
WO2004069165A3 (en) 2005-03-17
JP4584911B2 (ja) 2010-11-24
NL300593I2 (nl) 2020-04-15

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