Methods and compositions for gastroenteric upset therapy

FIELD: medicine.

SUBSTANCE: present invention refers to medicine and concerns methods and compositions for gastroenteric upset therapy. Substance of the invention involves purified polypeptide of 14 amino acids for gastroenteric upset and condition therapy (including, e.g. gastroenteric motor disturbance, functional gastroenteric disease, gastroesophageal reflux disease (GERD), Crohn's disease, ulcerative colitis, inflammatory bowel disease. Additionally the invention involves method of guanylate cyclase activity improvement with using peptide specified above.

EFFECT: advantage of invention consists in extended application.

9 cl, 6 ex, 10 dwg

 

The technical FIELD TO WHICH the INVENTION RELATES

The present invention relates to methods and compositions for the treatment of various disorders, including gastrointestinal disorders, obesity, congestive heart failure and benign prostatic hypertrophy.

The LEVEL of TECHNOLOGY

The irritable bowel syndrome (IBS (IBS)is a chronic bowel disorder that affects 20 to 60 million people only in the United States (Lehman Brothers Global Healthcare-Irritable bowel syndrome industry update, September 1999). IBS is the most common disorder diagnosed by gastroenterologists (28% of the surveyed patients), and is the cause of 12% of visits to General practitioners (Camilleri 2001, Gastroenterology 120:652-668). In the USA the economic contribution of IBS is evaluated as $25 billion annually through direct expenditures on health care and indirect costs of absenteeism (Talley 1995, Gastroenterology 109:1736-1741). Patients with IBS are three times more absenteeism and reports of reduced quality of life. With this may be unable or may not have a desire to attend a public event, keep a job or to travel even short distances (Drossman 1993, Dig Dis Sci 38:1569-1580). There is a huge unmet medical need in this population, as cured what I IBS, there are a few variations of the recipe.

Patients with IBS suffer from abdominal pain and disorders of the intestine. Identified three subgroups of patients with IBS on the basis of the predominant features of the functioning of the intestines: constipation (C-IBS), with the prevalence of diarrhea (d-IBS) or changing between the two (and-IBS). Assessment of people who suffer from C-IBS varies from 20-50% of patients with IBS frequently mentioned 30%. In contrast to the other two subgroups, which have the same ratio by sex, C-IBS is more common in women (ratio 3:1) (Talley et al. 1995, Am J Epidemiol 142:76-83).

The definition and diagnostic criteria for IBS officially listed in the "Rome Criteria (Drossman et al. 1999, Gut 45:Suppl II: 1-81), which is well accepted in clinical practice. However, the complex of symptoms not due to anatomical abnormalities or metabolic changes. This has led to the classification of IBS as a functional LCD disorder, which is diagnosed on the basis of the Rome criteria and a limited examination to exclude organic disease (Ringel et al. 2001, Annu Rev Med 52: 319-338). IBS is considered as a "biopsychosocial disorder, resulting from a combination of three interacting mechanisms: altered bowel mobility, increased sensitivity thin or large intestine to painful stimuli (visceral sensitivity) and psychosoc the social factors (Camilleri 2001, Gastroenterology 120:652-668). Recently, there is increasing evidence of the role of inflammation in the etiology of IBS. The reports show that subgroups of patients with IBS have a small but reliable increase in inflammatory and fat cells of the colon, increased inducible nitric oxide (NO) and synthase (iNOS) and modified the expression of inflammatory cytokines (review Talley 2000, Medscape Coverage of DDW week).

DISCLOSURE of INVENTION

The present invention includes compositions and related methods for treating IBS and other gastrointestinal disorders and conditions (e.g., disorders of gastrointestinal motility and functional gastrointestinal disorders, gastro-oesophageal reflux disease (GERD (GERD), Crohn's disease, ulcerative colitis, inflammatory bowel disease, functional heartburn, dyspepsia (including functional dyspepsia or nonulcer dyspepsia, gastroparesis, chronic small bowel pseudoobstruction (or colonic pseudoobstruction), and disorders and conditions associated with constipation, such as constipation associated with use of opiate analgesics, surgical constipation and constipation associated with neuropathic disorders as well as other conditions and disorders. The compositions are distinguished by the presence of peptides that enable the t receptor guanylate cyclase C (GC-C).

The present invention also relates to compositions and related methods for treating obesity, congestive heart failure and benign prostatic hyperplasia (BPH (BPH)).

Without regard to any particular theory, in the case of IBS and other gastrointestinal disorders peptides are applicable, as they may increase gastrointestinal motility.

Without regard to any particular theory, in the case of IBS and other gastrointestinal disorders peptides are applicable, in part, because they can reduce inflammation.

Without regard to any particular theory, in the case of IBS and other gastrointestinal disorders peptides are also applicable, as they may reduce gastrointestinal or visceral pain.

The invention provides pharmaceutical compositions comprising certain peptides that are able to activate the receptor guanylate cyclase C (GC-C). Also within the invention are pharmaceutical compositions comprising the peptide according to the invention and a composition comprising the peptide according to the invention and a second therapeutic agent, such as an agent for the treatment of constipation (such as SPI-0211; Sucampo Pharmaceuticals, Inc.; Bethesda, MD) or some other gastrointestinal disorders. Examples of second therapeutic and the enta include: agents, lowering the acidity, such as proton pump inhibitors and H2-receptor blockers, prokinetics, such as receptor agonists 5HT (for example, Zelnorm®), anti-inflammatory agents, antispasmodic, antidepressant, analgesic agents Central action, such as opioid receptor agonists, antagonists of opioid receptors, agents for the treatment of inflammatory bowel disease, Crohn's disease and ulcerative colitis (e.g., Traficet-ENTM(ChemoCentryx, Inc.; San Carlos, CA), agents that treat gastrointestinal or visceral pain and inhibitors of cGMP phosphodiesterase (motapizone, zaprinast and suldanessellar). Thus, for example, pharmaceutical compositions can include an anesthetic agent selected from the group consisting of: blockers Ca channel (for example, ziconotide), 5HT receptor antagonists (for example, antagonists of receptors, 5HT3, 5HT4 and 5HT1)agonists of opioid receptors (e.g., loperamide, fedotozine and fentanyl, naloxone, naltrexone, maternalized, nalmefene, ziprider, beta-funaltrexamine, naloxonazine, naltrindole and Nord-binaltorphimine, morphine, Diphenoxylate, Pentapeptide enkefalina, trimebutine), NK1 receptor antagonists (for example, ezlopitant and SR-14033), agonists of CCK receptor (for example, loxiglumide), NK1 receptor antagonists, NK3 receptor antagonists (for example, t is latent, osanetant (SR-142801)), inhibitors of reuptake of norepinephrine-serotonin (NSRI; for example, milnacipran), antagonists vanilloids and cannabanoid receptors (for example, arvanil), sialorphin, sialorphin-related peptides comprising the amino acid sequence QHNPR (SEQ ID NO: ) for example, VQHNPR (SEQ ID NO: ); VRQHNPR (SEQ ID NO: ); VRGQHNPR (SEQ ID NO: ); VRGPQHNPR (SEQ ID NO: ); VRGPRQHNPR (SEQ ID NO: ); VRGPRRQHNPR (SEQ ID NO: ); and RQHNPR (SEQ ID NO: ), compounds or peptides that are inhibitors neprilysin, makevalid (H-Tyr-D-Ala-Phe(F)-Phe-NH2; WO 01/019849 A1), loperamide, Tyr-Arg (kyotorphin), agonists of CCK receptor (caerulein), conotoxin peptides, peptide analogues of timeline, loxiglumide, dexloxiglumide (R-isomer loxiglumide) (WO 88/05774), and other analgesic peptides or compounds that can be used with or linked to the peptides according to the invention.

The invention includes methods for the treatment of various gastrointestinal disorders introduction peptide that acts as a partial or full agonist of the receptor HZ-C (GC-C). The peptide includes at least six cysteines, which form three disulfide bonds. In certain embodiments of the embodiment of the invention disulfide bond replaced by other covalent crosslinks, and in some cases cysteine replaced by other residues to provide an alternative covalent crosslinks. The peptides can also include the step, at least one site of cleavage by trypsin or chymotrypsin and/or carboxykinase analgesic peptide or small molecule, for example AspPhe or some other analgesic peptides. In the presence of the peptide used as an analgesic peptide or small molecule may be preceded by a site cleavage by chymotrypsin or trypsin, which allows you to release the analgesic peptide or a small molecule. Peptides and methods according to the invention are also applicable to the treatment of pain and inflammation associated with various disorders, including gastrointestinal disorders. Certain peptides include functional sites of cleavage by chymotrypsin or trypsin, positioned to allow the inactivation of the peptide upon cleavage. Certain peptides having functional sites of cleavage, are subjected to hydrolysis and gradual inactivation in the digestive tract, and it is desirable in some circumstances. In certain peptides, functional chymotrypsinogen plot changed, increasing the stability of peptidein vivo.

The invention includes methods for the treatment of other disorders such as congestive heart failure and benign prostatic hyperplasia, the introduction of a peptide or a small molecule (parenterale or oral), which acts as an agonist of the receptor HZ-C. Such agents can be used in combination with natriuretic peptides (for example, atrial natriuretic peptide, brain natriuretic peptide or natriuretic peptide C-type), a diuretic or angiotensin converting enzyme.

The invention includes methods and compositions for increasing intestinal motility. Intestinal motility includes spontaneous coordinated relaxation and contraction of the stomach, small intestine, colon and rectum for moving food through the gastrointestinal tract during digestion.

In certain embodiments of the embodiment, the peptides comprise one or two or more contiguous negatively charged amino acids (e.g., Asp or Glu), or one or two or more adjacent positively charged residues (e.g., Lys or Arg), or one or two or more adjacent positively, or negatively charged amino acids at the carboxyl end. In such embodiments, the embodiment of the invention the lateral amino acids at the carboxyl end are either positively or negatively charged. In other embodiments, embodiments of the invention carboxykinase charged amino acids preceded by Leu. For example, the following sequence of amino acids can be added to the carb is xilema the end of the peptide: Asp; Asp Lys; Lys Lys Lys Lys Lys Lys; Asp Lys Lys Lys Lys Lys Lys; Leu Lys Lys and Leu Asp. It is also possible simply to add the Leu at the carboxyl end.

In the first aspect of the invention includes a peptide containing, consisting of or mainly consisting of amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5represents Asn Ser Ser Asn Tyr or is missing, or Xaa1Xaa2Xaa3Xaa4missing In certain embodiments of the embodiment of the invention Xaa8, Xaa9, Xaa12, Xaa13, Xaa14, Xaa17and Xaa19can be any amino acid. In certain embodiments of the embodiment of the invention Xaa5represents Asn, Trp, Tyr, Asp, or Phe. In other embodiments, embodiments of the invention, Xaa5can also be Thr or Ile. In other embodiments, embodiments of the invention Xaa5represents Tyr, Asp or Trp. In some embodiments, embodiments of the invention Xaa8represents Glu, Asp, Gln, Gly or Pro. In other embodiments, embodiments of the invention Xaa8represents Glu; in some embodiments, embodiments of the invention Xaa9represents Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe, is some embodiments, embodiments of the invention Xaa 9represents Leu, Ile, Val, Lys, Arg, Trp, Tyr or Phe.

In certain embodiments of the embodiment of the invention, the amino acid may be substituted non-natural amino acid or a natural or non-natural analog of the amino acids. For example, an aromatic amino acid may be substituted 3,4-dihydroxy-L-phenylalanine, 3-iodine-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) or Nord-tyrosine (norTyr). Phg and norTyr and other amino acids, including Phe and Tyr can be substituted by, for example, halogen, -CH3, -OH, -CH2NH3, -C(O)H, -CH2CH3, -CN, -CH2CH2CH3, -SH, or another group.

In some embodiments, embodiments of the invention Xaa12represents Asn, Tyr, Asp, or Ala. In other embodiments, embodiments of the invention Xaa12represents Asn. In some embodiments, embodiments of the invention Xaa13represents Ala, Pro, or Gly, and in other embodiments, embodiments of the invention it is Pro. In some embodiments, embodiments of the invention Xaa14represents Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp, and in other embodiments, embodiments of the invention it is Ala or Gly, and in still other embodiments, embodiments of the invention it is Ala. In some embodiments, embodiments of the invention Xaa16represents Thr, Ala, Asn, Lys, Arg, Trp; Xaa17represents Gly, Pro or Ala; Xaa19choose the from Trp, Tyr, Phe, Asn and Leu, or Xaa19selected from Trp, Tyr, and Phe, or Xaa19selected from Leu, Ile and Val or Xaa19is His or Xaa19selected from Trp, Tyr, Phe, Asn, Ile, Val, His and Leu and Xaa20Xaa21represents AspPhe or is missing or Xaa20is Asn or Glu and Xaa21missing or Xaa19Xaa20Xaa21no. The invention also provides methods of treating gastrointestinal disorders (e.g., disorders of gastrointestinal motility and functional gastrointestinal disorders, gastro-oesophageal reflux disease, functional heartburn, dyspepsia, nonulcer dyspepsia, gastroparesis, chronic small bowel pseudoobstruction, colonic pseudoobstruction), obesity, congestive heart failure, benign prostatic hyperplasia introduction of the composition comprising the above-mentioned peptide.

When Xaa9represents Trp, Tyr or Phe or when Xaa16is Trp, the peptide has the potential functional site of cleavage by chymotrypsin, which is located at a position where cleavage inactivates the receptor binding HZ-C peptide. When Xaa9represents Lys or Arg or when Xaa16represents Lys or Arg, the peptide has a potentially functional site of cleavage by trypsin, which is located in the n position, where cleavage inactivates the receptor binding HZ-C peptide.

When Xaa19represents Trp, Tyr or Phe, the peptide has the site of cleavage by chymotrypsin, which is located at a position where cleavage will liberate part of the peptide carboxyl end up Xaa19. When Xaa19is Leu, Ile or Val, the peptide can have a plot of cleavage with chymotrypsin, which is located at a position where cleavage will liberate aminobenzene part of the peptide to Xaa19. At relatively high pH the same effect is observed when Xaa19is His. When Xaa19represents Lys or Arg, the peptide has the site of cleavage by trypsin, which is located at a position where cleavage will liberate carboxykinase part of the peptide to Xaa19. Therefore, when the peptide includes the analgesic peptide carboxylic to Xaa19the peptide will be released in the digestive tract when exposed to the appropriate protease. Among painkillers peptides that can be included in the peptide are: AspPhe (as Xaa20Xaa21), endomorphin-1, endomorphin 2 nistatin, dalargin, lupron and substance P and other pain relievers peptides described in this description. Such peptides can, for example, be used to replace Xaa20Xaa21.

When Xaa1or aminobenzene amino acid, pepti is and according to the invention (for example, Xaa2or Xaa3) represents Trp, Tyr or Phe, the peptide has the site of cleavage by chymotrypsin, which is located at a position where cleavage will liberate aminobenzene part of a peptide to Xaa1(or Xaa2or Xaa3together with Xaa1, Xaa2or Xaa3. When Xaa1or aminobenzene amino acid peptide of the invention (for example, Xaa2or Xaa3) represents Lys or Arg, the peptide has the site of cleavage by trypsin, which is located at a position where cleavage will liberate aminobenzene part of a peptide to Xaa1together with Xaa1, Xaa2or Xaa3). When Xaa1or aminobenzene amino acid peptide according to the invention represents Leu, Ile or Val, the peptide can have a plot of cleavage with chymotrypsin, which is located at a position where cleavage will liberate aminobenzene part of a peptide to Xaa1. At relatively high pH the same effect is observed when Xaa1is His. Therefore, for example, when the peptide includes the analgesic peptide, aminocentesis to Xaa1the peptide is released in the digestive tract when exposed to the appropriate protease. Among painkillers peptides that can be included in the peptide are: AspPhe, endomorphin-1, endomorphin 2 nistatin, dalargin, lupron and substance P and other obezbolivaet is the following peptides described in the present description.

When full folding disulfide bonds are present between Cys6and Cys11; Cys7and Cys15; Cys10Cys18. The peptides according to the invention have a sequence similarity with ST peptides. However, they include amino acid changes and/or additions that improve functionality. Such changes may, for example, to increase or decrease the activity (for example, to increase or decrease the ability of the peptide to stimulate bowel motility), change the ability to develop, stability of the peptide, the ability of the peptide to contact the receptor HZ-C and/or decrease toxicity. In some cases, the peptides may function more acceptable than ST peptides of the wild type. For example, they can limit undesirable side effects such as diarrhea and dehydration.

In some embodiments, embodiments of the invention, one or both of a member of one or more pairs of Cys residues, which normally form a disulfide bond, can be replaced by homocysteine, 3-mercaptopropanol (Kolodziej et al. 1996. Int J Pept Protein Res. 48:274); β,β-dimetiltsistein (Hunt et al. 1993. Int J Pept Protein Res. 42:249) or diaminopropionic acid (Smith et al. 1978. J Med Chem. 21:117) with the formation of an alternative internal cross-linking in the normal positions of disulfide bonds.

In addition, one is more disulfide bonds can be replaced by alternative covalent bonds, for example, amide bond, ester link, alkyl link, cyclonaire link lactamases bridge, carbamoylethyl bridge, urea link timesaving link, link phosphonate of ester, the alkyl part and alkenyl link, ether, thioester link or aminosweet. For example, Ledu et al. (Proceedings NAT'l Acad. Sci. 100:11263-78, 2003) describes how to obtain a lactam and amide crosslinks. Schafmeister et al. (J. Am. Chem. Soc. 122:5891, 2000) describes a stable, fully-carbon linkage. In some cases, the formation of such alternative crosslinks requires the substitution of Cys residues other residues, such as Lys or Glu or unnatural amino acids.

In the case of a peptide comprising the sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing and/or sequence Xaa19Xaa20Xaa21no, the peptide may still contain additional carboxykinase or aminobenzene amino acids or both. For example, the peptide may include aminobenzene sequence that contributes to the recombinant production of peptide and cleaved before in the introduction of the peptide to the patient. The peptide may also include other aminobenzene or carboxykinase amino acids. In some cases, additional amino acids protect the peptide stabilizes the peptide or modify the activity of the peptide. In some cases, some or all of these additional amino acids are removed prior to administration of the peptide to the patient. The peptide may include 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids at its aminocore or carboxylic or both. The number of lateral amino acids should not be the same. For example, it may be 10 additional amino acids on aminocore peptide and no one on carboxykinase.

In one variant embodiment of the invention, the peptide includes the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing; Xaa8is Glu; Xaa9is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn; Xaa13is Pro; Xaa14is Ala; Xaa16is Thr, Ala, Lys, Arg, Trp; Xaa17is Gly; Xaa19is Tyr or Leu, and Xaa20Xaa21is AspPhe or is missing. When Xaa20Xaa21and/or Xaa1Xaa Xaa3Xaa4Xaa5no, there may be additional lateral amino acids in some embodiments, embodiments of the invention.

In a second aspect the invention also provides a therapeutic or prophylactic method involving the introduction of a peptide comprising the amino acid sequence (I): XAA1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9

Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16XAA17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5is Asn Ser Ser Asn Tyr or is missing or Xaa1Xaa2Xaa3Xaa4absent and Xaa5is Asn, Trp, Tyr, Asp, Ile, Thr or Phe; Xaa8is Glu, Asp, Gln, Gly or Pro; Xaa9is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn, Tyr, Asp, or Ala; Xaa13is Pro or Gly; Xaa14is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp; Xaa16is Thr, Ala, Asn, Lys, Arg, Trp; Xaa17is Gly, Pro or Ala; Xaa19is Trp, Tyr, Phe or Leu; Xaa20Xaa21is AspPhe or is missing or Xaa20is Asn or Glu and Xaa21missing or Xaa19Xaa20Xaa21is missing.

In certain embodiments of the embodiment of therapeutic or prophylactic methods, the peptide includes the sequence the amino acids (I): Xaa 1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing; Xaa8is Glu; Xaa9is Leu, Ile, Lys, Arg, Trp, Tyr, or Phe; Xaa12is Asn; Xaa13is Pro;

Xaa14is Ala; Xaa16is Thr, Ala, Lys, Arg, Trp, or Xaa16is any amino acid, or Xaa16is Thr, Ala, Lys, Arg, Trp, or Xaa16is any non-aromatic amino acid; Xaa17is Gly; Xaa19is Tyr or Leu, and Xaa20Xaa21is AspPhe or is missing.

In certain embodiments of the embodiment of the invention provides a purified polypeptide comprising the amino acid sequence (II): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Asn12Pro13Ala14Cys15

Xaa16Gly17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5is Asn Ser Ser Asn Tyr or is missing or Xaa1Xaa2Xaa3Xaa4absent and Xaa5is Asn; Xaa8is Glu or Asp; Xaa9is Leu, Ile, Val, Trp, Tyr or Phe; Xaa16is Thr, Ala, Trp; Xaa 19is Trp, Tyr, Phe or Leu or absent, and Xaa20Xaa21is AspPhe.

In various preferred embodiments the embodiment of the invention provides a purified polypeptide containing the amino acid sequence (II): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Asn12Pro13Ala14Cys15Xaa16Gly17Cys18Xaa19Xaa20Xaa21where Xaa9is Leu, Ile or Val and Xaa16is Trp, Tyr or Phe Xaa9is Trp, Tyr or Phe, Xaa16is Thr or Ala; Xaa19is Trp, Tyr, Phe and Xaa20Xaa21is AspPhe and Xaa1Xaa2Xaa3

Xaa4absent and Xaa5is Asn; the peptide comprises less than 50, 40, 30 or 25 amino acids; less than five amino acids preceded by a Cys6.

The peptides can be administered in conjunction with or associated, for example, covalently linked, with any of a variety of other peptides, including analgesic peptides or analgesic compounds. For example, a therapeutic peptide according to the invention can be associated with analgesic agent selected from the group consisting of: blockers Ca channel (for example, ziconotide), complete or partial 5HT receptor antagonists (for example, antagonists of receptors, 5HT3, 5HT4 and 5HT1), full or partial agonist is in 5HT receptors, including agonists of the receptors, 5HT3, 5HT4 (for example, Tegaserod, mosapride and renzapride) and 5HT1 agonists of the receptor, CRF (NBI-34041), agonists of adrenergic receptors, β-3 agonists of opioid receptors (e.g., loperamide, fedotozine and fentanyl, naloxone, naltrexone, maternalized, nalmefene, ziprider, beta-funaltrexamine, naloxonazine, naltrindole and norbinaltorphimine, morphine, Diphenoxylate, Pentapeptide enkefalina, asimadoline, trimebutine)antagonists of NK1 receptors (for example, ezlopitant and SR-14033), agonists of CCK receptors (for example, loxiglumide), antagonists receptors NK1, NK3 receptor antagonists (e.g., talnetant, osanetant (SR-142801)), inhibitors of reuptake of norepinephrine-serotonin (NSRI; for example, milnacipran), agonists vanilloids and cannabanoid receptors (for example, arvanil), sialorphin, sialorphin-related peptides comprising the amino acid sequence QHNPR (SEQ ID NO: ) for example, VQHNPR (SEQ ID NO: ); VRQHNPR (SEQ ID NO: ); VRGQHNPR (SEQ ID NO: ); VRGPQHNPR (SEQ ID NO: ); VRGPRQHNPR (SEQ ID NO: ); VRGPRRQHNPR (SEQ ID NO: ) and RQHNPR (SEQ ID NO: ), compounds and peptides that are inhibitors neprilysin, makevalid (H-Tyr-D-Ala-Phe(F)-Phe-NH2; WO 01/019849 A1), loperamide, Tyr-Arg (kyotorphin), agonists of CCK receptors (caerulein), conotoxin peptides, peptide analogs of timeline, loxiglumide, dexloxiglumide (R-isomer loxiglumide) (WO 88/05774), and other analgesic peptides or connect the deposits can be used with or linked to the peptides according to the invention.

Amino acid, diaminotoluene, peptide and intermediate ones of the inserts can be inserted between the peptide that is an agonist of the receptor HZ-C, and a peptide that has any other biological function, such as analgesic peptide or peptide used for the treatment of obesity. The link may be such that it is split off from the side of peptidesin vivoor such, which remains associated with lateral peptidesin vivo.For example, glycine, beta-alanine, glycyl-glycine, glycyl-beta-alanine, gamma-aminobutyric acid, 6-aminocaproic acid, L-phenylalanine, L-tryptophan and glycyl-L-poured-L-phenylalanine can be used as an intermediate link (Chaltin et al. 2003. Helvetica Chimica Acta. 86:533-547; Caliceti et al. 1993. FARMCO. 48:919-32), can also glycols (Butterworth et al. 1987. J. Med. Chem. 30:1295-302) and derivatives of maleinimide (King et al. 2002. Tetrahedron Lett. 43:1987-1990). Various other links described in the literature (Nestler. 1996 Molecular Diversity. 2:35-42; Finn et al. 1984. Biochemistry. 23:2554-8; Cook et al. 1994. Tetrahedron Lett. 35:6777-80; Brokx et al. 2002. Journal of Controlled Release. 78:115-123; Griffin et al. 2003. J. Am. Chem. Soc. 125:6517-6531; Robinson et al. 1998. Proc. Natl. Acad. Sci. USA 95:5929-5934).

The peptide can include the amino acid sequence of the peptide that exists in nature in a species of vertebrates (e.g. mammals) or species of bacteria. In addition, peptides can be castignoli completely unnatural peptides. Also within the invention are peptidomimetics corresponding to the peptides according to the invention. In different embodiments of the invention, a patient suffering from a gastrointestinal disorder; the patient suffers from a disorder selected from the group consisting of: disorders of gastrointestinal motility, irritable bowel syndrome, chronic constipation, functional gastrointestinal disorders, gastro-oesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronic small bowel pseudoobstruction, Crohn's disease, ulcerative colitis, irritable bowel syndrome, colonic pseudoobstruction, obesity, congestive heart failure, or benign prostatic hyperplasia; the composition is administered orally; the peptide comprises 30 or less amino acids, the peptide comprises 20 or fewer amino acids, the peptide comprises no more than 5 amino acids before Cys6; peptide includes 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40 or 30 or less amino acids. In other embodiments, embodiments of the invention, the peptide comprises 20 or fewer amino acids. In other embodiments, embodiments of the invention, the peptide comprises no more than 20, 15, 10, or 5 peptides, followed Cys18. In certain embodiments of the embodiment of izopet is of Xaa 19is the site of cleavage by chymotrypsin or trypsin, and analgesic peptide is present directly after Xaa19.

In the third aspect of the invention provides a method for treatment of a patient suffering from constipation. Clinically accepted criteria that define constipation, ranging from the frequency of bowel movements, consistency of feces and simplicity of defecation. One conventional definition of constipation is less than three bowel movements per week. Other definitions include abnormally difficult bowel movements or defecation, which requires excessive voltage (Schiller 2001, aliment oil displayed pure Pharmacol Ther. 15:749-763). Constipation can be idiopathic (functional constipation or constipation slow passage) or secondary due to other reasons, including neurological, metabolic or endocrine disorders. Such disorders include diabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcaemia, multiple sclerosis, Parkinson's disease, spinal cord injury, neurofibromatosis, autonomic neuropathy, Chagas disease, a disease Hirschprung and cystic fibrosis. Constipation can also be the result of surgery (postoperative ileus) or due to the use of medicines, such as painkillers (opioids), antihypertensives, anticonvulsants, antidepressants, antispasmodic and antipsychotics.

The method includes introducing a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5is Asn Ser Ser Asn Tyr or is missing or Xaa1Xaa2Xaa3Xaa4absent and Xaa5is Asn, Trp, Tyr, Asp, Ile, Thr or Phe; Xaa8is Glu, Asp, Gln, Gly or Pro; Xaa9is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn, Tyr, Asp, or Ala; Xaa13is Pro or Gly; Xaa14is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp; Xaa16is Thr, Ala, Asn, Lys, Arg, Trp; Xaa17is Gly, Pro or Ala; Xaa19is Trp, Tyr, Phe or Leu; Xaa19is Lys or Arg; Xaa20Xaa21is AspPhe or is missing or Xaa20is Asn or Glu and Xaa21missing or Xaa19Xaa20Xaa21is missing.

In one variant embodiment of the method, the peptide includes the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaasub> 16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing; Xaa8is Glu; Xaa9is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn; Xaa13is Pro; Xaa14is Ala; Xaa16is Thr, Ala, Lys, Arg, Trp; Xaa17is Gly; Xaa19is Tyr or Leu; Xaa19is Lys or Arg; Xaa20Xaa21is AspPhe or is missing.

In various preferred embodiments the embodiment of the invention, constipation associated with use of therapeutic agent; constipation associated with neuropathic disorders; constipation rearranges a surgical constipation (postoperative ileus); and constipation associated with a gastrointestinal disorder; the constipation is idiopathic (functional constipation or constipation slow passage); constipation associated with neuropathic, metabolic or endocrine disorder (e.g. diabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcaemia, multiple sclerosis, Parkinson's disease, spinal cord injury, neurofibromatosis, autonomic neuropathy, Chagas disease, a disease Hirshsprung or cystic fibrosis). Constipation can also be the result of surgery (postoperative ileus) or because the drug is the means, such as pain relievers (e.g., opioids), antihypertensive drugs, anticonvulsants, antidepressants, antispasmodics and antipsychotic drugs.

In the fourth aspect of the invention provides a method for treatment of a patient suffering from a gastrointestinal disorder, the method comprises administration to a patient of a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5is Asn Ser Ser Asn Tyr or is missing or Xaa1Xaa2Xaa3Xaa4absent and Xaa5is Asn, Tip, Tyr, Asp, Ile, Thr or Phe; Xaa8is Glu, Asp, Gln, Gly or Pro; Xaa9is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn, Tyr, Asp, or Ala; Xaa13is Pro or Gly; Xaa14is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp; Xaa16is Thr, Ala, Asn, Lys, Arg, Trp; Xaa17is Gly, Pro or Ala; Xaa19is Trp, Tyr, Phe or Leu; Xaa19is Lys or Arg; Xaa20Xaa21is AspPhe or is missing or Xaa20is Asn or Glu and Xaa21missing or Xaa19Xaa20Xaa21 no.

In one variant embodiment of the method, the peptide includes the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing; Xaa8is Glu; Xaa9is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn; Xaa13is Pro; Xaa14is Ala; Xaa16is Thr, Ala, Lys, Arg, Trp; Xaa17is Gly; Xaa19is Tyr or Leu; Xaa19is Lys or Arg; Xaa20Xaa21is AspPhe or is missing.

In different embodiments of the invention, a patient suffering from a gastrointestinal disorder; the patient suffers from a disorder selected from the group consisting of: disorders of gastrointestinal motility, irritable bowel syndrome, chronic constipation, functional gastrointestinal disorders, gastro-oesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronic small bowel pseudoobstruction, Crohn's disease, ulcerative colitis, inflammatory bowel disease, colonic pseudoobstruction, obesity, congestive behold the Dechen failure, or benign prostatic hyperplasia.

In preferred embodiments, embodiments of the invention Xaa9is Leu, Ile or Val and Xaa16is Trp, Tyr or Phe; Xaa9is Trp, Tyr or Phe and Xaa16is Thr or Ala; Xaa19is Trp, Tyr, Phe; Xaa19is Lys or Arg;

Xaa20Xaa21is AspPhe; Xaa1Xaa2Xaa3Xaa4absent and Xaa5is Asn.

In the fifth aspect of the invention provides a method for increasing gastrointestinal motility in a patient; the method includes: introduction to the patient a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5is Asn Ser Ser Asn Tyr or is missing or Xaa1Xaa2Xaa3Xaa4absent and Xaa5is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa8is Glu, Asp, Gln, Gly or Pro; Xaa9is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn, Tyr, Asp, or Ala; Xaa13is Pro or Gly; Xaa14is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp; Xaa16is Thr, Ala, Asn, Lys, Arg, Trp; Xaa17is Gly, Pro or Ala; Xaa19is Trp, Tyr, Phe or Leu; Xaa19the two who is Lys or Arg; Xaa20Xaa21is AspPhe or is missing or Xaa20is Asn or Glu and Xaa21missing or Xaa19Xaa20Xaa21is missing.

In one variant embodiment of the invention, the peptide includes the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing; Xaa8is Glu; Xaa9is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn; Xaa13is Pro; Xaa14is Ala; Xaa16is Thr, Ala, Lys, Arg, Trp; Xaa17is Gly; Xaa19is Tyr or Leu; Xaa19is Lys or Arg; Xaa20Xaa21is AspPhe or is missing.

In the sixth aspect of the invention provides a method for increasing the activity of the receptor of the intestinal guanylate cyclase (GC-C) the patient; the method includes: introduction to the patient a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21, where the Xaa 1Xaa2Xaa3Xaa4Xaa5is Asn Ser Ser Asn Tyr or is missing or Xaa1Xaa2Xaa3Xaa4absent and Xaa5is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa8is Glu, Asp, Gln, Gly or Pro; Xaa9is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn, Tyr, Asp, or Ala; Xaa13is Pro or Gly; Xaa14is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp; Xaa16is Thr, Ala, Asn, Lys, Arg, Trp; Xaa17is Gly, Pro or Ala; Xaa19is Trp, Tyr, Phe or Leu; Xaa19is Lys or Arg; Xaa20Xaa21is AspPhe or is missing or Xaa20is Asn or Glu and Xaa21missing or Xaa19Xaa20Xaa21is missing.

In one variant embodiment of the invention, the peptide includes the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing; Xaa8is Glu; Xaa9is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn; Xaa13is Pro; Xaa14is Ala; Xaa16is Thr, Ala, Lys, Arg, Trp; Xaa17is Gly; Xaa19is Tyr or Leu; Xaa19is Lys or Arg; Xaa20Xaa21AspPhe or missing.

In the seventh aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of: (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5is Asn Ser Ser Asn Tyr or is missing or Xaa1Xaa2Xaa3Xaa4absent and Xaa5is Asn, Trp, Tyr, Asp, Ile, Thr or Phe; Xaa8is Glu, Asp, Gln, Gly or Pro; Xaa9is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn, Tyr, Asp, or Ala; Xaa13is Pro or Gly; Xaa14is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp; Xaa16is Thr, Ala, Asn, Lys, Arg, Trp; Xaa17is Gly, Pro or Ala; Xaa19is Trp, Tyr, Phe or Leu; Xaa19is Lys or Arg; Xaa20Xaa21is AspPhe or is missing or Xaa20is Asn or Glu and Xaa21missing or Xaa19Xaa20Xaa21is missing.

In one variant embodiment of the invention, the peptide includes the amino acid sequence (I): Xaa1Xaa2XAA3XAA4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12 Xaa13Xaa14Cys15Xaa16XAA17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing; Xaa8is Glu; Xaa9is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn; Xaa13is Pro; Xaa14is Ala; Xaa16is Thr, Ala, Lys, Arg, Trp; Xaa17is Gly; Xaa19is Tyr or Leu; Xaa19is Lys or Arg; Xaa20Xaa21is AspPhe or is missing.

In the eighth aspect of the invention provides a method for the treatment of constipation, the method includes the introduction of the receptor agonist of the intestinal guanylate cyclase (GC-C). In different embodiments of the invention, the agonist is a peptide, the peptide includes four Cys that form two disulfide bonds, and peptide includes six Cys that form three disulfide bonds.

In the ninth aspect of the invention provides a method for treatment of gastrointestinal disorders, gastrointestinal motility, irritable bowel syndrome, chronic constipation, functional gastrointestinal disorders, gastro-oesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronic small bowel pseudoobstruction, colonic pseudoobstruction, b is Lesni Crown, ulcerative colitis, inflammatory bowel disease, obesity, congestive heart failure, or benign prostatic hyperplasia, the method includes the introduction of the receptor agonist of the intestinal guanylate cyclase (GC-C) orally, by rectal suppositories, or parenterally. In different embodiments of the invention, the agonist is a peptide, the peptide includes four Cys that form two disulfide bonds, and peptide includes six Cys that form three disulfide bonds.

In the tenth aspect of the invention provides a method for treating a gastrointestinal disorder selected from the group consisting of disorders of gastrointestinal motility, irritable bowel syndrome, chronic constipation, functional gastrointestinal disorders, gastro-oesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronic small bowel pseudoobstruction, colonic pseudoobstruction, Crohn's disease, ulcerative colitis, inflammatory bowel disease, the method includes the introduction of the receptor agonist of the intestinal guanylate cyclase (GC-C). In different embodiments of the invention the composition is administered orally; the peptide comprises 30 or fewer amino acid is t, the peptide comprises 20 or fewer amino acids, and the peptide comprises no more than 5 amino acids before Cys5.

In different embodiments of the invention, the agonist is a peptide, the peptide includes four Cys that form two disulfide bonds, and peptide includes six Cys that form three disulfide bonds.

In the eleventh aspect of the invention provides a method for treating obesity, the method includes the introduction of the receptor agonist of the intestinal guanylate cyclase (GC-C). In different embodiments of the invention, the agonist is a peptide, the peptide includes four Cys that form two disulfide bonds, and peptide includes six Cys that form three disulfide bonds.

In the twelfth aspect of the invention provides a method for the treatment of obesity; this method includes the introduction of the polypeptide comprising the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5is Asn, Ser, Ser, Asn, Tyr or is missing or Xaa1Xaa2Xaa3Xaa4absent and Xaa5is Asn, Trp, Tyr, Asp, Ile, Thr or Phe; Xaa8is Glu, Asp, Gl, Gly or Pro;

Xaa9is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn, Tyr, Asp, or Ala; Xaa13is Pro or Gly; Xaa14is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp; Xaa16is Thr, Ala, Asn, Lys, Arg, Trp; Xaa17is Gly, Pro or Ala; Xaa19is Trp, Tyr, Phe or Leu and Xaa20Xaa21is AspPhe or is missing or Xaa20is Asn or Glu and Xaa21missing or Xaa19Xaa20Xaa21no. The peptide can be administered alone or in combination with another agent for treatment of obesity, such as sibutramine or other agent, for example, the agent described in the present description.

In one variant embodiment of the invention, the peptide includes the amino acid sequence (I): Xaa1XAA2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15XAA16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing; Xaa8is Glu; Xaa9is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn; Xaa13is Pro; Xaa14is Ala; Xaa16is Thr, Ala, Lys, Arg, Trp; Xaa17is Gly; Xaa19is Tyr or Leu; Xaa20Xaa21is AspPhe or is missing.

In the thirteenth aspect of the invention preduster who provides the pharmaceutical composition, comprising a polypeptide, described in the present description.

In the fourteenth aspect of the invention provides a method for the treatment of congestive heart failure; this method includes the introduction to the patient a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5is Asn, Ser, Ser, Asn, Tyr or is missing or Xaa1Xaa2Xaa3Xaa4absent and Xaa5is Asn, Trp, Tyr, Asp, Ile, Thr or Phe; Xaa8is Glu, Asp, Gln, Gly or Pro;

Xaa9is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn, Tyr, Asp, or Ala; Xaa13is Pro or Gly; Xaa14is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp; Xaa16is Thr, Ala, Asn, Lys, Arg, Trp; Xaa17is Gly, Pro or Ala; Xaa19is Trp, Tyr, Phe or Leu; Xaa20Xaa21is AspPhe or is missing or Xaa20is Asn or Glu and Xaa21or missing or Xaa19Xaa20Xaa21no. The peptide may be administered in combination with another agent for treatment of congestive heart failure, for example natriuretic peptide is a home, such as atrial natriuretic peptide, brain natriuretic peptide or natriuretic peptide C-type diuretics or angiotensin-converting enzyme inhibitor.

In one variant embodiment of the invention, the peptide includes the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing; Xaa8is Glu; Xaa9is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn; Xaa13is Pro; Xaa14is Ala; Xaa16is Thr, Ala, Lys, Arg, Trp; Xaa17is Gly; Xaa19is Tyr or Leu; Xaa19is Lys or Arg; Xaa20Xaa21is AspPhe or is missing.

In the fifteenth aspect of the invention provides a method for the treatment of benign prostatic hyperplasia, the method comprises administration to a patient of a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa 20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5is Asn Ser Ser Asn Tyr or is missing or Xaa1Xaa2Xaa3Xaa4absent and Xaa5is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa8is Glu, Asp, Gln, Gly or Pro; Xaa9is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn, Tyr, Asp, or Ala; Xaa13is Pro or Gly; Xaa14is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp; Xaa16is Thr, Ala, Asn, Lys, Arg, Trp;

Xaa17is Gly, Pro or Ala; Xaa19is Trp, Tyr, Phe or Leu; Xaa19is Lys or Arg; Xaa20Xaa21is AspPhe or is missing or Xaa20is Asn or Glu and Xaa21missing or Xaa19Xaa20Xaa21is missing.

The peptide can be introduced in combination with another agent for treatment of BPH, for example an inhibitor of 5-alpha reductase inhibitors (e.g. finasteride) or alpha-adrenergic inhibitor (eg, doxazosin).

In one variant embodiment of the invention, the peptide includes the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing; Xaa8is Glu; Xaa9is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa12is Asn; Xaa13is Pro; Xaa14is Ala; Xaa16is Thr, Ala, Lys, Arg, Trp; Xaa17is Gly; Xaa19is Tyr or Leu; Xaa20Xaa21is AspPhe or is missing.

In the sixteenth aspect of the invention provides a method for treating or reducing pain, including visceral pain, pain associated with a gastrointestinal disorder or pain associated with any other disorder, the method comprises administration to a patient of a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21for example a purified polypeptide comprising an amino acid sequence described in the present description.

In the seventeenth aspect of the invention provides a method for treating inflammation, including inflammation of the gastrointestinal tract, such as inflammation associated with a gastrointestinal disorder or infection or some other disorder, the method comprises administration to a patient of a composition comprising a purified polypeptide comprising the amino acid sequence (I): Xaa1 Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21for example a purified polypeptide comprising an amino acid sequence described in the present description.

In certain embodiments of the embodiment of the invention, the peptide includes a peptide comprising or consisting of the amino acid sequence Xaa1Xaa2Xaa3Xaa4Xaa5Cys Cys Glu Xaa9Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Xaa20Xaa21(II) (SEQ ID NO:____), where Xaa9is any amino acid, where Xaa9is any amino acid other than Leu, where Xaa9selected from Phe, Trp and Tyr; where Xaa9choose from any other natural or unnatural amino acid, where Xaa9is Tyr; where Xaa1Xaa2Xaa3Xaa4Xaa5is Asn, Ser, Ser, Asn, Tyr, where Xaa1, Xaa2, Xaa3, Xaa4and Xaa5no; where Xaa1Xaa2Xaa3and Xaa4no; where Xaa1Xaa2and Xaa3no; where Xaa1and Xaa2no; where Xaa1missing; where Xaa20Xaa21is AspPhe or is missing or Xaa20is Asn or Glu and Xaa21missing or Xaa19Xaa20Xaa21no. In the case of the peptide, including the surrounding sequence (I): Xaa 1Xaa2Xaa3Xaa4Xaa5Cys6Cys7Xaa8Xaa9Cys10Cys11Xaa12Xaa13Xaa14Cys15Xaa16Xaa17Cys18Xaa19Xaa20Xaa21where Xaa1Xaa2Xaa3Xaa4Xaa5missing and/or sequence Xaa19Xaa20Xaa21no, the peptide may contain carboxykinase or aminobenzene amino acids, or both.

Among the applicable peptides are peptides consisting of or comprising mainly the amino acid sequence of Xaa1Xaa2Xaa3Xaa4Xaa5Cys Cys Glu Xaa9Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Xaa20Xaa21(II) (SEQ ID NO:---) are the following peptides:

Gln Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Thr Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Leu Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Ile Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Ser Ser Gln Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Gln Ser Ser Gln Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Ser Ser Gln Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Ser Ser Asn Tyr Cys Cys Glu Ala Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Ser Ser Asn Tyr Cys Cys Glu Arg Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Asn Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Asp Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Cys Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Gln Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Glu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Gly Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu His Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu LEU Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Lys Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Met Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Pro Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Asn Ser Ser Asn Tyr Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Ala Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Arg Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Asn Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

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

Cys Cys Glu Cys Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Gln Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Glu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Gly Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu His Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu LEU Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Lys Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Met Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Pro Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ED NO: )

Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: )

In the eighteenth aspect, the invention provides a method for the treatment of congestive heart failure, the method includes the introduction of full or partial receptor agonist of the intestinal guanylate cyclase (GC-C). The agonist can be introduced in combination with another agent for treatment of congestive heart failure, for example natriuretic peptide, such as atrial natriuretic peptide, brain natriuretic peptide or natriuretic peptide C-type diuretic or angiotensin converting enzyme.

In the nineteenth aspect, the invention provides a method for the treatment of BPH; this method includes the introduction of full or partial receptor agonist of the intestinal guanylate cyclase (GC-C). The agonist can be introduced in combination with another agent for treatment of BPH, for example an inhibitor of 5-alpha reductase inhibitors (e.g. finasteride) or alpha adrenergic inhibitor (eg, doxazosin).

In the twentieth aspect of the invention provides a method for the treatment of obesity; this method includes the introduction of full or casticin the th receptor agonist of the intestinal guanylate cyclase (GC-C). The agonist can be introduced in combination with another agent for treatment of obesity, for example a fragment of gastrointestinal hormone peptide YY3-36(PYY3-36) (N. Engl. J. Med.349:941, 2003; ikpeapge daspeelnry yaslrhylnl vtrqry) glp-1 (like peptide-1), accendino-4 inhibitor glp-1), sibutramine, phentermine, phendimetrazine, hydrochloride benzfetamine (Didrex), orlistat (xenical), hydrochloride diethylpropion (Tenuate), fluoxetine (Prozac), bupropion, ephedra, chromium, mangosteen Cambodian, benzocaine, brown algae (focus vesiculosus), chitosan, grass hay, Galega (goat's Rue, French lilac), conjugated linoleic acid, L-carnitine, fibers (fiber Sandpiper, psyllium, guar), caffeine, dehydroepiandrosterone, Dubrovnik (teucrium chamaedrys), B-hydroxy-β-methylbutyrate and pyruvate. Peptide applicable for the treatment of obesity, can be entered as a co-therapy with peptide according to the invention in the form of individual molecules, and in the form of crosslinked protein with a peptide according to the invention. Therefore, for example, PYY3-36can be sewn with a carboxy - or aminocom.com peptide according to the invention. Such crosslinked protein may include sites of cleavage by chymotrypsin or trypsin, which can allow splitting to separate the two peptide.

Peptides and receptor agonist of the intestinal guanylate cyclase (G is-C) can be used to treat constipation or decreased intestinal motility, slow digestion or slow emptying of the stomach. The peptides can be used to facilitate one or more IBS symptoms (bloating, pain, constipation), GERD (reflux of acid into the esophagus), functional dyspepsia, or gastroparesis (nausea, vomiting, bloating, delayed gastric emptying) and other disorders described in the present description.

Details of one or more variants of embodiment of the invention installed in the accompanying description. All publications, patents and patent applications, therefore, incorporated by reference.

BRIEF DESCRIPTION of DRAWINGS

Figa depicts IHMS analysis of recombinant peptide MM-416776 and peptide MD-915.

Fig.1b and c depicts the results IHMS analysis of synthetic peptide MD-1100 and control.

Figure 2 depicts the results of the analysis of receptor activity of intestinal HZ-C synthetic peptide MM-416776, peptide MD-915 and two different peptides MD-1100.

Figa depicts the effect of recombinant peptide MM-416776 and Zelnorm® in a mouse model of gastrointestinal transit.

Fig.3b depicts the effect of synthetic peptide MD-1100 and Zelnorm® in a mouse model of acute gastrointestinal transit.

Fig.3b depicts the effect of synthetic peptide MD-1100 and Zelnorm® in a mouse model of chronic gastrointestinal transit.

Figa and 4b depict the effect of peptides MD-915, MD 1100 MM-416776 in mouse is the first model of acute gastrointestinal transit.

Figs depicts the effect of peptide MD-1100 in a murine model of chronic gastrointestinal transit.

Figa depicts the effect of peptide MM-416776 and Zelnorm® in model intestinal secretion sucking mice.

Fig.5b depicts the effects of MD-1100 and Zelnorm® in a mouse model of intestinal secretion.

Figa and 6b depict the effects of peptides MM 416776, MD 1100 and MD 915 in murine models of intestinal secretion.

Fig.7 shows the results of an experiment in which the activity of MD-1100 analyzed in the model stretching of the colon TNBS.

Figa and 8b show the effects of different doses of MD-915 and MD-1100 in the analysis of pain cramps PBQ.

Fig.9 shows the results of the analysis determine the KD using MD-1100 in the analysis of competitive binding of radioactive ligand.

Figa and 10b show data bioavailability for I/o and oral input MD-1100, as determined by ELISA and IHMS.

The IMPLEMENTATION of the INVENTION

The peptides of the invention bind to the receptor of the intestinal guanylate cyclase (GC-C), a key regulator of fluid balance and electrolytes in the intestine. During stimulation of this receptor, which is located on the apical membrane of the intestinal epithelial surface, causes an increase in intestinal epithelial cyclic GMP (cGMP). Believe that this increase in cGMP causes reduced absorption of water and sodium and increase secretion of chlorine ions is Yes and potassium, resulting in changes in the intestinal transport of fluid and electrolytes and increase intestinal motility. The intestinal receptor HZ-C has an extracellular binding site of the ligand, the transmembrane area, intracellular plot similar to protein kinase, and Zelazny catalytic domain. Proposed functions for receptor HZ-C are the homeostasis of fluid and electrolytes, regulation of proliferation of epithelial cells and induction of apoptosis (Shalubhai. 2002. Curr Opin Drug Dis Devel. 5:261-268).

In addition to expression in the intestine gastrointestinal epithelial cells, GC-C is expressed in extra-intestinal tissues, including kidneys, lungs, pancreas, pituitary gland, adrenal gland, developing liver (described in Vaandrager. 2002, Mol Cell Biochem. 230:73-83), and male and female reproductive tissues (described in Vaandrager. 2002.MolCellBiochem230:73-83)). This suggests that agonists of the receptor HZ-C can be used in the treatment of disorders outside of the LCD tract, such as congestive heart failure and benign prostatic hyperplasia.

Ghrelin, a peptide hormone, secretory stomach, is a key regulator of appetite in humans. The level of expression of ghrelin is regulated by starvation and the emptying of the stomach (Kim et al., 2003, Neuroreprt. 14:1317-20; Gualillo et al., 2003, FEBS Letts. 552: 105-9). Therefore, by increasing gastro-Kish who offered motility receptor agonists HZ-C may also be used for the regulation of obesity.

In humans, the receptor HZ-activated guanylin (Gn) (U.S. patent 596097), uroguanylin (Ugn) (U.S. patent 5140102) and lymphogranuloma (Forte et al., 1999,Endocrinology. 140:1800-1806). Interestingly, these agents are 10-100 times less powerful than the class of peptides derived from bacteria, called ST (described in Gianella. 1995. J Lab Clin Med. 125:173-181). ST peptides are considered as superagonist HZ WITH and are very resistant to proteolytic cleavage.

ST peptide is able to stimulate 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). Also reported that cGMP has antinociceptive effects in multiple pain models in animals (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). Therefore, agonists HZ-may have analgesic and anti-inflammatory effect.

The bacterial ST peptides derived from prefrably, which usually has at least 70 amino acids. Pre - and Pro-plots hatshepsuts as part of the process of secretion, and the resulting Mature protein, which typically includes less than 20 amino acids, is biologically active.

Among known bacterial ST peptides are: ST IbE.coli(Moseley et al. (1983)Infect.Immun. 39:1167), with the Mature amino acid sequence Asn Ser Ser Asn Tyr Cys Cys Glu Leu CysCys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:__); ST IaE.coli(So and McCarthy (1980)Proc.Natl.Acad.Sci. USA 77:4011), with 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:_); ST I*E.coli(Chan and Giannella (1981)J.Biol.Chem. 256:7744), with the Mature amino acid sequence Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Tyr Pro Ala Cys Ala Gly Cys Asn (SEQ ID NO:___); ST peptideC.freundii(Guarino et al. (1989)Infect.Immun. 57:649)with 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:__), ST peptidesY.enterocolitica, Y ST(Y-STa) Y-STb, and Y-STc (described in Huang et al. (1997)Microb.Pathog. 22:89)having the following amino acid sequence Pro-forms: 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:___) (and Ser-7 Leu-7 variant Y-STa (SEQ ID NO:___), (Takao et al. (1985)Eur.J.Biochem. 152:199)); Lys Ala Cys Asp Thr Gln Thr Pro Ser Pro Ser Glu Glu Asn Asp Asp Trp Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Ala Gly Cys (SEQ ID NO:___); Gln Glu Thr Ala Ser Gly Gln Val Gly Asp Val Ser Ser Ser Thr Ile Ala Thr Glu Val Ser Glu Ala Glu Cys Gly Thr Gln Ser Ala Thr Thr Gln Gly Glu Asn Asp Trp Asp Trp Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Phe Gly Cys (SEQ ED NO:___), respectively; ST peptideY.kristenseniiwith the Mature amino acid sequence Ser Asp Trp Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Ala Gly Cys (SEQ ED NO:___); not-01 ST peptide V.cholerae(Takao et al. (1985)FEBS lett. 193:250), with the Mature amino acid sequence Ile Asp Cys Cys Glu LEU Cys Cys Asn Pro Ala Cys Phe Gly Cys Leu Asn (SEQ ED NO:__); and ST peptideV.mimicus(Arita (1991) et al.FEMS Environ.Lett. 79:15), with the Mature amino acid sequence Ile Asp Cys Cys Glu LEU Cys Cys Asn Pro Ala Cys Phe Gly Cys Leu Asn (SEQ ID NO:__). The table below provides a sequence of all or part of a number of Mature ST peptides.

GenBank® directoryGenBank® GISequence
QHECIB69638NSSNYCCELCCNPACTGCY(SEQ ID NO:_)
P01559123711NTFYCCELCCNPACAGCY(SEQ ID NO:_)
AAA24653147878NTFYCCELCCNPACAPCY(SEQ ED NO:_)
P01560123707NTFYCCELCCYPACAGCN(SEQ ED NO:_)
AAA27561295439IDCCEICCNPACFGCLN(SEQ ID NO:_)
P04429123712IDCCEICCNPACFGCLN(SEQ ID NO:_)
S34671421286IDCCEICCNPACF(SEQ ID NO:_)
CAA52209395161IDCCEICCNPACFG(SEQ ID NO:_)
A54534 628844IDCCEICCNPACFGCLN(SEQ ID NO:_)
AAL0215915592919IDRCEICCNPACFGCLN(SEQ ID NO:_)
AAA18472487395DWDCCDVCCNPACAGC(SEQ ID NO:_)
S25659282047DWDCCDVCCNPACAGC(SEQ ED NO:_)
P749773913874NDDWCCEVCCNPACAGC(SEQ ID NO:__)
BAA236562662339WDWCCELCCNPACFGC(SEQ ID NO:_)
P31518399947SDWCCEVCCNPACAGC(SEQ ID NO:_)

The immature (including pre and Pro - plots) form of protein ST-1A (ST-P)E. colihas the sequence mkkmalaifisvlsfpsfsqstesldsskekitletkkcdvvknnseldcsenmrintfyccelccnpacagcy (SEQ ID NO:___; see GenBank®, catalog No. P01559 (gi:123711). Pre-sequence extends from AK 1-19. Pro-sequence extends from AK 20-54. The Mature protein extends from 55-72. The immature (including pre and Pro-plots) form of protein ST-1B (ST-H)E.colihas the sequence: mkksilfiflsvlsfspfaqdakpvesskekitleskkcniakksnksgpesmnssnyccelccnpactgcy (SEQ ID NO:___; see GenBank®, catalog No. P07965 (gi:3915589). The immature (including pre and Pro sites) form ST proteinY.enterocoliticahas posledovatel the activity: mkkivfvlvlmlssfgafgqetvsgqfsdalstpitaevykqacdpplppaevssdwdccdvccnpacagc (SEQ ID NO:___; see GenBank®, catalog No. S25659 (gi:282047).

The peptides according to the invention, as bacterial ST peptides are six Cys residues. These six Cys residues forming three disulfide bonds in the Mature and active form of the peptide. If identify six Cys residues from the amino to carboxylic peptide as A, B, C, D, E and F, then the disulfide bonds are formed, as indicated below: A-D, B-E and C-F. Believe that the formation of these linkages is important for binding of the receptor HZ-C. Certain peptides of the invention include potentially functional sites of cleavage by chymotrypsin, for example, a Trp, Tyr or Phe, located between or Cys B and Cys D or between Cys E and Cys F. Splitting in any site of cleavage by chymotrypsin reduces or eliminates the ability of the peptide to contact the receptor HZ-C.

In the human body is the inactive form of chymotrypsin, chymotrypsinogen, is produced in the pancreas. When this inactive enzyme reaches the small intestine, it is converted to active disease by cutting two di-peptides. Active disease can potentially cleave peptides by peptide bond on carboxykinase side Trp, Tyr or Phe. The presence of active disease in the intestinal tract can potentially lead to the splitting of certain peptides according to the invention, having certain way located options the regional sites of cleavage by chymotrypsin. Expect cleavage by chymotrypsin will regulate the action of the peptide according to the invention, having certain way located the site of cleavage by chymotrypsin when the peptide passes through the intestinal tract.

Trypsinogen as 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 with Lys or Arg. The presence of active trypsin in the intestinal tract can lead to the breakdown of certain of the peptides according to the invention, having appropriately located functional site of cleavage by trypsin. Expect cleavage by chymotrypsin will regulate the action of the peptide according to the invention, having appropriately located land cleavage by trypsin, when the peptide passes through the intestinal tract.

Many gastrointestinal disorders, including IBS, associated with abdominal or visceral pain. Some of the peptides according to the invention include analgesic or antinociceptive ends, such as carboxykinase sequence AspPhe directly after Trp, Tyr or Phe, which creates a functional site of cleavage by chymotrypsin, or after Lys or Arg, which creates a functional site of cleavage by trypsin. Himot Ipsen in the intestinal tract can potentially break down these peptides directly carboxyterminal in relation to the residue Trp, Phe or Tyr, releasing dipeptide, AspPhe. It was shown that this dipeptide has analgetic activity in animal models (Abdikkahi et al. 2001, Fundam Clin Pharmacol. 15:117-23; Nikfar et al 1997, 29:583-6; Edmundson et al. 1998, Clin Pharmacol Ther. 63:580-93). Thus, such peptides can treat pain and inflammation. Other analgesic peptides may be present on carboxylic peptide (following functional cleavage site), including: endomorphin-1, endomorphin 2 nistatin, dalargin, lupron and substance P. the applicable Number of peptides based on the core sequence: Cys Cys GluLeuCys Cys Asn Pro Ala CysThrGly Cys Tyr. To create variants with potential functional site of cleavage by chymotrypsin, is able to inactivate the peptide, or Leu (underlined), or Thr (underlined) can be replaced by Trp, Phe or Tyr, or both Leu and Thr may be replaced by (independently) Trp, Phe or Tyr. To create a variant with anesthetic di-peptide, the core sequence is followed by Asp Phe. Carboxykinase Tyr in the core sequence can afford the dipeptide Asp Phe to be released by chymotrypsin in the digestive tract. The core sequence may not necessarily precede Asn Ser Ser Asn Tyr or Asn.

Therefore, the applicable options, based on the core sequence, include:

Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro AlaCys Thr Gly Cys Tyr (SEQ ID NO:---; MM-416776)

Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Trp Gly Cys Tyr (SEQ ID NO:---)

Asn Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---; MD 915)

Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---; MM416774)

Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Trp Gly Cys Tyr (SEQ ID NO:---)

Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---; MD-1100)

Asn Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Trp Gly Cys Tyr (SEQ ED NO:---)

Asn Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Cys Cys Glu Arg Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Cys Cys Glu Lys Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---)

Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Trp Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Ser Ser Asn Tyr Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Ser Ser Asn Tyr Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Ser Ser Asn Tyr Cys Cys Glu Arg Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Ser Ser Asn Tyr Cys Cys Glu Lys Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Trp Gly Cys Tyr Asp Phe (SEQ ID NO:-)

Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Cys Cys Glu Arg Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Cys Cys Glu Lys Cys Cys Asn Pro Ala Cys ThrGly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Trp Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Cys Cys Glu Arg Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

Asn Cys Cys Glu Lys Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO:---)

In some cases, the peptides according to the invention are in the form of preproblem, which include aminobenzene leading sequence:

mkksilfiflsvlsfspfaqdakpvesskekitleskkcniakksnksgpesmn. When the peptide is produced by a bacterial cell, for example,E.colithe above-mentioned leading sequence is cleaved, and the Mature peptide is efficiently secreted from the bacterial cells. U.S. patent No. 5395490 describes vectors, expression systems and methods for efficient production of ST peptides in bacterial cells and methods to achieve efficient secretion of Mature ST peptides. Vectors, expression systems, and methods described in U.S. patent No. 5395490, can be used to obtain the ST peptides and alternative ST peptides of the present invention.

Alternative peptides

The invention includes alternative peptides, which may include one, two, three, four, five, six, seven, eight, nine or ten (in some embodiments, embodiments of izobreteny is less than 5, or less than 3, or 2, or less) substitutions of amino acids compared to SEQ ID NO:do. Substitution(I) may be conservative or non-conservative. Natural amino acids can be substituted for D-isomers of any of the amino acids, unnatural amino acids and other groups. Conservative amino acid substitution changes the amino acid at amino acid for such actions or amino acid of similar charge, polarity, or hydrophobicity. In some States, even a conservative substitution of amino acids can reduce the activity of the peptide. Among the substitution of natural amino acids, generally regarded as conservative, are:

For amino acidsCodeSubstitution of any of
Alanine
Arginine
Ala
Arg
Gly, Cys, Ser
Lys, His,
AsparagineAsnAsp, Glu, Gln,
Aspartic acidAspAsn, Glu, Gln
CysteineCysMet, Thr, Ser
CH is the drug of choice GlnAsn, Glu, Asp
Glutamina acidGluAsp, Asn, Gln
GlycineGlyAla
HistidineHisLys, Arg
IsoleucineIleVal, Leu, Met
LeucineLeuVal, Ile, Met
LysineLysArg, His,
MethionineMetIle, Leu, Val
PhenylalaninePheTyr, His, Trp
ProlinePro
SerineSerThr, Cys, Ala
ThreonineThrSer, Met, Val
TryptophanTrp Phe, Tyr
TyrosineTyrPhe, His,
ValineValLeu, Ile, Met

In some circumstances, it may be desirable to treat patients with variant peptide that binds to and activates the receptor of Escherichia HZ, but is less active than the variant form of the peptide. This reduced activity may result from a reduced affinity to the receptor or reduced ability to activate the receptor upon binding or reduced stability of the peptide.

Some peptides pairs of Cys residues, which usually form disulfide bonds, one or both members of a pair can be replaced by homocysteine, 3-mercaptopropanol (Kolodziej et al. 1996. Int J Pept Protein Res. 48:274); β,β-dimetiltsistein (Hunt et al. 1993. Int J Pept Protein Res. 42:249) or diaminopropionic acid (Smith et al. 1978. J Med Chem. 21:117) with the formation of an alternative internal links within the provisions of the normal disulfide bonds.

Production of peptides

Applicable peptides can be obtained as in bacteria, including, without limitation,E. coliand in other existing systems to obtain a peptide or protein (e.g.,Bacillussubtilisthe system is the baculovirus expression using Drosophila cells Sf9, si is theme of expression of yeast or threadlike fungi, expression systems in mammalian cells), or they can be chemically synthesized.

If the peptide or variant peptide must be received in bacteria, such asE.colithe nucleic acid molecule encoding a peptide, preferably also encodes a leading sequence, which allows secretion of the Mature peptide from the cell. Therefore, the sequence encoding the peptide may include pre-sequence and Pro-sequence, for example, natural bacterial ST peptides. Secreted, Mature peptide can be purified from the culture medium.

The sequence encoding the peptide according to the invention is preferably inserted into a vector capable of delivering and save the nucleic acid molecule in a bacterial cell. The DNA molecule can be inserted in the Autonomous vector can replicate (suitable vectors include, for example, pGEM3Z and pcDNA3 and their derivatives). The nucleic acid vector may be a DNA bacteria or bacteriophage, such as the bacteriophage lambda or M13 and their derivatives. Create a vector containing a nucleic acid described in the present description, may be followed by transformation of the host cell, such as bacteria. Suitable bacterial hosts include, but are not limited to,E.coli,B.subtilis,Pseudomonas,Salmonell . Genetic structure also includes, in addition to the coding nucleic acid molecule, the elements that make possible the expression, such as promoter and regulatory sequences. The expression vectors may contain transcriptional regulatory sequences that regulate the initiation of transcription, such as sequence, promoter, enhancer, operator and repressor. Many transcriptional regulatory sequences are well known to specialists in the field of technology. The expression vector may also include a regulatory sequence broadcast (e.g., untranslated 5'sequence, untranslated 3'sequence or internal input site of the ribosome). The vector may be capable of Autonomous replication or may be integrated into the DNA of the host cell to ensure stability during the production of the protein.

Sequence encoding a protein, which comprises the peptide according to the invention can also be sewn to a nucleic acid that encodes a polypeptide affinity end, such as glutathione S-transferase (GST), protein a, maltose binding F, protein A, FLAG the end, hexa-histidine, myc end or the end of the HA of influenza, to facilitate cleaning. Affine end or alloy reporter combines reading frame of interest of the peptide with what amcol read gene, coding affine end, so that a translational cross-linking.

The stitched expression of the gene leads to broadcast a single polypeptide, which comprises the peptide of interest and affine end. In some cases, when using an affine ends, the DNA sequence encoding the site of the protease recognition made between reading frames with affine end and a peptide of interest.

The genetic constructs and methods suitable for obtaining immature and Mature forms of the peptides and variants according to the invention in the system of the expression of the protein, other than bacteria, and well-known specialist in the field of technology can also be used to produce peptides in a biological system.

Mature peptides and their variants can be synthesized by a solid phase method using an automatic peptide synthesizer. For example, the peptide can be synthesized on Cyc(4-CH2Bxl)-OCH2-4-(oxymethyl)-phenylacetylamino the resin using a double bind. The protective group should be used accordingly to create the right system disulfide bonds. For example, the following protective groups can be used: t-butyloxycarbonyl (alpha-amino group); atsetamidometil (thiol groups of Cys residues B and E); 4-methylbenzyl (thiol groups of Cys residues C and F); b is nil (y-carboxyl glutaminol acid and the hydroxyl group of threonine, if present) and bromanil (phenolic group of tyrosine, if present). The binding is made with symmetrical anhydride of the t-butoxycarbonylamino or hydroxybenzotriazole esters (for residues of asparagine or glutamine), and peptide deprotected and cleaved from the solid Foundation in formotorola, dimethyl sulfide, anisole and p-thiocresol using the ratio 8/1/1/0,5 (about./about./about./wt.) at 0°C for 60 minutes After removal of pterodroma and dimethyl sulfide reduced pressure and anisole and p-thiocresol extraction with ethyl ether and ethyl acetate successively crude peptides were extracted with a mixture of 0,5M sodium phosphate buffer, pH 8.0 and N,N-dimethylformamide using a ratio of 1/1, vol/about. Disulfide bonds to residues Cys B and E were formed using dimethyl sulfoxide (Tam et al. (1991)J.Am.Chem.Soc. 113:6657-62).The resulting peptide was purified by chromatography with reversed phase. Disulfide bonds between Cys residues C and F was formed by first dissolving the peptide in 50% acetic acid in water. Was added a saturated solution of iodine in glacial acetic acid (1 ml of iodine solution per 100 ml of solution). After incubation at room temperature for 2 days in a closed glass container, the solution was diluted five times with deionized water and was extracted with ethyl shall firom four times to remove unreacted iodine. After removal of the residual amount of ethyl ether rotary evaporation of the solution of crude product liofilizirovanny and was purified further by chromatography with reversed phase.

Analysis of the binding of the receptor intestinal HZ WITH

The ability of peptides and other agents to contact with the intestinal receptor HZ-may be studied as follows. Cells cell line of human colon carcinoma T84 colon (American Type Culture Collection (Bethesda, Md.) were grown to confluence in 24-hole culture tablets with a mixture of 1:1 environment Ham's F12 and modified Dulbecco medium Eagle's (DMEM)with 5% fetal serum of calves. Cells used in the analysis, which typically were between passages 54-60. Briefly, monolayers of T84 cells in 24-hole tablets were washed twice with 1 ml binding buffer (DMEM containing 0.05% bovine serum albumin and 25 mM HEPES, pH of 7.2), and then incubated for 30 min at 37°C in the presence of Mature, labeled with a radioactive label ST peptideE.coliand a test substance in various concentrations. Then cells were washed four times with 1 ml DMEM and was dissolved with 0.5 ml/cell 1H. NaOH. The level of radioactivity in the dissolved substance is then determined using standard methods.

Example 1: Getting ST variant peptides and ST peptide of wild-type

1a: Getting rekombinatsionnykh ST peptides and ST peptide of wild-type

Variant ST peptide, referred to as MD-915, reproduced recombinante and investigated in animal models. 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:-). Also created a peptide having the sequence ST peptide of wild type (MM-416776).

Peptides MD-915 and MM-416776 received in the form of preproblem using vectors obtained as follows. The sequence encoding preprofessionals heat-resistant enterotoxin, amplified from pGK51/pGSK51 (ATCC 67728) using the oligonucleotide MO3514 (5' CACACCATATGAAGAAATCAATATTATTTATTTTTCTTTCTG 3' (SEG ID NO: )) and the oligonucleotide MO3515 (5' CACACCTCGAGTTAGGTCTCCATGCTTTCAGGACCACTTTTATTAC 3' (SEQ ID NO:__)). Fragment amplication product was absorbed with NdeI/XhoI and was associated with the expression vector T7, pET26b(+) (Novagen) was absorbed with NdeI/XhoI, thereby creating plasmid MB3976. Plot encoding of prepublic, sequenced and found that it encodes an amino acid sequence:

mkksilfiflsvlsfspfaqdakpagsskekitleskkcnivkksnksgpesm (SEQ ID NO:__), which differed from the amino acid sequence of the precursor thermostable enterotoxin a2 (sta2; mkksilfiflsvlsfspfaqdakpagsskekitleskkcnivkknnesspesm (SEQ ID NO:___); GenBank®, catalog No. Q47185, GI: 3913876) in three positions (indicated with an underline and bold text) about C-Terminus. To generate expression vectors with preprofessionals the Yu complementary oligonucleotides, encoding each variant of the ST peptide or ST peptide of wild-type, were annealed and cloned into the expression vector MB3976. To create MB3984 (MM coding-416776 peptide full length ST of the wild-type peptide in the form of prefrably), containing an amino acid sequence that NSSNYCCELCCNPACTGCY (SEQ ID NO:___), was made lower than preprofessionals, MB 3976 absorbed with BsaI/XhoI and was associated with the annealed oligonucleotides MO3621 (5' GCATGAATAGTAGCAATTACTGCTGTGAATTGTGTTGTAATCCTGCTTGTAC CGGGTGCTATTAATAAC 3' (SEQ ED NO:_)and MO3622 (5' TCGAGTTATTAATAGCACCCGGTACAAGCAGGATTACAACACAATTCACAGCAGTAATTGCTACTATTC 3'(SEQ ID NO:_)). To create MB3985 (encoding MD-915 as prefrably)containing the following amino acid sequence that is NSSNYCCEYCCNPACTGCY sewn below preprofessionals, MB 3976 absorbed with BsaI/XhoI and was associated with the annealed oligonucleotides MO3529 (5' GCATGAATAGTAGCAATTACTGCTGTGAATATTGTTGTAATCCTGCTTGTACCGGGTGCTATTAATAAC 3' (SEQ ID NO:_)and MO3530 (5' TCGAGTTATTAATAGCACCCGGTACAAGCAGGATTACAACAATATTCACAGCAGTAATTGCTACTATTC 3'(SEQ ID NO:_)).

Peptide MD-915 and peptide MM-416776 received, as listed below. The expression vectors were transformed into bacterial host cellsE.coliBL21γDE3 (Invitrogen). One colony was inoculable and were grown with shaking overnight at 30°C in a nutrient medium L + 25 mg/l kanamycin. Overnight culture was added to 3.2 l batch medium (glucose 25 g/l, Kazarinova acid 5 g/l, yeast extract 5 g/l, KH2PO413.3 g/l (NH4)2HPO44 g/l, MgSO4·7H2O 1.2 g/is, citric acid 1.7 g/l, EDTA 8,4 mg/l CoCl2·6H2O 2.5 mg/l MnCl2·4H2O 15 mg/l CuCl2·4H2O 1.5 mg/l, H3BO33 mg/l, Na2MoO4·2H2O 2.5 mg/l, acetate Zn·2H2O 13 mg/l, iron citrate 100 mg/l kanamycin 25 mg/l, antifoam DF2O41 ml/l) and was fermentatively using the following process parameters: pH of 6.7 - control-only basis (28% NH4OH), 30°C, oxygenation: 5 liters per minute. After the initial consumption of the batch glucose (based on monitoring the level of dissolved oxygen (DO)) was added to 1.5 l of culture medium (glucose 700 g/l, Kazarinova acid 10 g/l, yeast extract 10 g/l, MgSO4·7H2O 4 g/l, EDTA 13 mg/l CoCl2·6H2O 4 mg/l MnCl2·4H2O 23,5 mg/l CuCl2·4H2About 2.5 mg/l, H3BO35 mg/l, Na2MoO4·2H2O 4 mg/l, acetate Zn·2H2About 16 mg/l, iron citrate 40 mg/l, antifoam DF2041 ml/l) at a feed speed, adjusting to maintain 20% DO. Was added to 0.2 mm IPTG after 2 hours after the start of power supply. Total time was approximately 40-45 hours (up to no power).

The cells were collected by centrifugation at 5000g for 10 minutes. The precipitated cells were discarded and the supernatant was passed through a 50 KD-section for ultrafiltration. 50 CD filtrate (0.6-liter) were loaded on 110 ml to Lanco fast current Q-sepharose (Amersham Pharmacia, balanced 20 mm Tris-HCl pH 7.5) at a flow rate of 400 ml/hour. The column was washed with six volumes of 20 mm Tris-HCl pH 7.5 and proteins were suirable 50 mm acetic acid, collecting fractions of 50 ml. Fractions containing ST variant peptide or ST the wild-type peptide was collected and the solvent was removed with rotary evaporation. Dry whites resuspendable in 10 ml of 8% acetic acid, 0.1% of triperoxonane acid (TFU) and were loaded on the column Varian Polaris C18-A (250×of 21.2 mm 10 micron, equilibrated in the same buffer) at a flow rate of 20 ml/min the Column was washed with 100 ml of 8% methanol, 0.1% of TFU and treated with a gradient (300 ml) for 24 to 48% methanol, 0.1% of TFU, collecting fractions of 5 ml Fractions containing the peptide were collected and the solvent was removed with rotary evaporation. The peptides were dissolved in 0.1% TFU and liofilizirovanny.

The fraction of peptide MD-915 and peptide MM-416776 were analyzed by standard IHMS and HPLC. IHMS-analysis found that MD-915 is more homogeneous than MM-416776 (see Figa; note that the peptide MD-915 shows smaller peaks (panel B)than MM-416776 (panel A)).

1b: Obtain synthetically ST variant peptides and ST peptide of wild-type

The peptides are chemically synthesized by a commercial company of peptide synthesis. Changing the outputs of the peptides were obtained depending on the efficiency of chemical synthesis. Therefore, four peptides, in decreasing order of output were: ys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:---; MD-1100), 10-20% yield; Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:-; MM416774); Asn Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:-; MD-915); Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:-MM-416776), <5% output.

Thus, the specific amino acid changes introduced into the peptides can create improved performance properties.

Fig. 1b shows the General profile of ion chromatography synthetically derived MD-1100. Fig. 1c shows the General profile of ion chromatography net control sample. There is one main peak is present in the sample MD-1100, which was not present in the sample. Quantitative analysis revealed that the MD-1100 is >98% pure.

Example 2: Activation of the receptor intestinal HZ-ST variant peptide and ST peptide

The ability of MD-915, MM-416776 and MD-1100 activate the receptor intestinal HZ-evaluated in the analysis using the cell line of human colon carcinoma T84 colon (American type culture collection (Bethesda, Md.)). For analysis, cells were grown to confluence in 24-hole tablets with a mixture of 1:1 environment Ham's F12 and modified Dulbecco medium Eagle's (DMEM)with 5% fetal serum of calves, and used between passages 54 and 60.

Briefly, monolayers of T84 cells in 24-hole tablets were washed twice ml/cell in DMEM, then incubated at 37°C for 10 min with 0.45 ml of DMEM, tereasa 1 mm isobutylmethylxanthine (IBMX), inhibitor of cyclic nucleotidyltransferase. The tested peptides (50 μl) was then added and incubated for 30 minutes at 37°C. the Medium aspirated and the reaction was then terminali the addition of ice-cold 0.5 ml of 0.1 n HCl. Samples were kept on ice for 20 minutes and then evaporated to dryness using inkjet drying apparatus or vacuum centrifugation. Dry samples resuspendable in 0.5 ml phosphate buffer provided in the kit Cayman Chemical Cyclic GMP EIA (Cayman Chemical, Ann Arbor, MI). Cyclic GMP was measured by EIA in accordance with the method described in the kit Cayman Chemical Cyclic GMP EIA.

Figure 2 shows the activity of chemically synthesized peptide variants in this analysis the activity of the receptor HZ-C. In this analysis MM-416776 and two different peptide MD-1100 (MD-1100(a) and MD-1100(b), synthesized by two different methods), had activity comparable to MM-416776. Peptides MD-915 and MM-416776 were chemically synthesized manner identical to that of MD-1100(b).

Example 3: MD-915 and MM-416776 increase intestinal transit in mice

In order to determine the increase does peptides rate of gastrointestinal transit peptides and the control were investigated using analysis of murine gastrointestinal transit (GIT) (Moon et al.InfectionandImmunity, 25:127, 1979). In this analysis of activated carbon, which is easily visualized in docno-intestinal tract, injected mice after administration of test compounds. The distance traveled by activated carbon was measured and expressed as a percentage of the total length of the colon.

Mice were fasted with free access to water for 12 to 16 hours before treatment with peptide or control buffer. The peptides were administered orally at a dose of 1 mcg/kg -1 mg/kg of peptide in buffer (20 mm Tris pH 7.5) for 7 minutes to obtain oral dose of 5% activated charcoal (Aldrich 242276-250G). Control mice were administered only the buffer before receiving doses of activated charcoal. After 15 minutes, mice were killed and then dissected the small intestine from the stomach to the cecum. The total length of the small intestine, as well as the distance traveled from the stomach to the front line of activated carbon were measured for each animal and the results were expressed as a percentage of the total length of the small intestine, passed the front line of activated charcoal. All results were presented as the mean of 10 mice±standard deviation. Comparison of the distance traveled activated carbon between the mice that received the peptide, compared to mice that received only the solvent was performed using t-student test and a statistically significant difference was considered for P<0,05. P-values were calculated using two-sided T-test, assuming unequal variance.

As is can be seen in Fig. 3a, b, ST peptide of wild type (MM-416776, (Sigma-Aldrich, St Louis, MO; 0.1 mg/kg), a synthetically derived MD-1100 and Zelnorm® (0.1 mg/kg), a drug approved for IBS, which is an agonist of the receptor of serotonin 5HT4, increased the rate of gastrointestinal transit in this model. Figa shows the result of a study showing that the rate of intestinal transit increases with dosage and recombinante synthesized MM-416776 or MD-915. Fig. 4b shows the results of a study showing that both chemically synthesized peptide MM-416776 or MD-1100 peptide increases the speed of intestinal transit more than as a buffer Tris separately or equivalent dose of Zelnorm®.

A similar experiment was carried out to determine whether MD-1100 effective in chronic regimen. Briefly, 8-week old female CD1 mice were given orally once daily for 5 days MD-1100 (0.06 mg/kg or 0.25 mg/kg in 20 mm Tris pH 7.5) or one solvent (20 mm Tris pH 7.5). On the 5th day of the GIT analysis, similar to that above, except that introduced 200 μl of 10% solution of activated charcoal. Fig. 4c shows the results of a study showing that both chemically synthesized MD-1100 or Zelnorm® were effective in the analysis of the murine gastrointestinal motility in chronic introduction (daily for 5 days). The results show the us next with acute dosing (day 1).

Example 4: the Peptide MD-915 and peptide MM-416776 increase intestinal secretion in sucking mice (analysis of SuMi)

Peptide MM-416776 and peptide MD-915 tested for their ability to increase intestinal secretion using a model of intestinal secretion sucking mice. In this model, the test compound was administered to suckling mice, who were aged between 7 and 9 days. After killing the mice gastrointestinal tract from the stomach to the caecum was dissected ("digestive channels"). The remaining ("body"), as well as the digestive channels weighed and calculated the ratio of the digestive canal to the body. If the ratio was above 0,09 was concluded that the test compound increases intestinal secretion. Figa curve shows the dependence of the dose-effect for ST peptide of wild type (MM-416776) in this model. Fig.5b curve shows the dependence of the dose-effect for peptide MD-1100 in this model. These data show that ST the wild-type peptide (purchased from TDT, Inc. West Chester, PA) and peptide MD-1100 increase intestinal secretion. The effect of Zelnorm® also investigated. As can be seen from Figure 5, Zelnorm® in a dose of 0.2 mg/kg did not increase intestinal secretion in this model. Figa curve shows the dependence of the dose-effect for recombinant peptide MM-416776 described above, and recombinant peptide MD-915, described above. As can be seen from Tiga, both peptidoglycan intestinal secretion in this model. Similarly Fig.6b curve shows the dependence of the dose-effect for chemically synthesized MD-915, MD 1100 MM-416776 and ST of the wild-type peptide (purchased from Sigma-Aldrich, St Louis, MO).

The animal model of hyperalgesia colon

Hypersensitivity to colorectal distension region is common in patients with IBS and may be responsible for the primary symptom of pain. Were developed as inflammatory and non-inflammatory animal models of visceral hyperalgesia in tension to study the effect of compounds on visceral pain in IBS.

I. Model induced trinitrobenzenesulfonic acid(TNBS) rectal-allodynia

Male Wistar rats (220-250 g) were premedication with 0.5 mg/kg acepromazine, injected intraperitoneally (WB), and anestesiologi intramuscular injection of 100 mg/kg of ketamine. A pair of electrodes of nichrome wire (60 cm long and 80 μm in diameter) were implanted in the striated muscle of the abdomen, 2 cm lies lateral to the white line. The free ends of the electrodes are taken out on the back of the neck and protected by a plastic tube, attached to the skin. Electromyographic recordings (EMG) was started 5 days after surgery. The electrical activity of the striated muscles of the abdomen were recorded by means of the apparatus electroencephal the graphy (Mini VIII, Alvar, Paris, France) using a constant short period of time (0,03) to remove low-frequency signal (<3 Hz).

Ten days after surgical implantation was introduced trinitrobenzenesulfonic acid (TNBS) to induce inflammation of the rectum. TNBS (80 mg kg-1in 0.3 ml of 50% ethanol) was administered intrarectally through a catheter made of silicone rubber, held on 3 cm from the anus under light diethylamino anesthesia, as described Morteau et al. 1994. Dig Dis. Sci 39:1239. After administration of TNBS rats were placed in plastic channels, where they were somewhat limited in mobility for several days prior to colorectal distension zone (CRD). Experimental the compound was administered one hour before CRD that was inserted into the rectum at 1 cm from the anus of a cylinder of length 4 cm, made of latex condom (Gue et al., 1997.Neurogastroenterol. Motil.9:271). The cylinder was fixed on a rigid catheter, obtained from the probe to embolectomy (Fogarty). The balloon attached to the catheter, recorded at the base of the tail. The balloon is attached to barostat, steadily inflated in increments of 15 mm RT. Art. from 0 to 60 mm RT. Art., each stage of inflation lasted 5 minutes Study rectal sensitivity, as measured by EMG, conducted before (1-2 days) and 3 days after rectal instillation of TNBS.

The number of pulses of potentials of action which I, which corresponded to the contractions of the stomach, was determined in 5-minute periods. Statistical analysis the number of contractions of the stomach and the assessment of the relationship dose-effect conducted one-way analysis of variance (ANOVA) followed by pairwise comparisons (criteria t-test or Dunnet) and regression analysis for ED50if necessary.

Fig.7 shows the results of an experiment in which the activity of MD-1100 analyzed in colorectal TNBS model. A significant reduction of abdominal response was observed at 0.3 mg/kg and 3 mg/kg MD-1100. The results show that MD-1100 reduces the pain associated with stretching of the colorectal region in this animal model.

II. The model of hyperalgesia induced by stress

Male Wistar rats (200-250 g) were surgically implanted electrodes from nichrome wire, as in the TNBS model. Ten days after surgical implantation was performed stress partial crushing (PRS), as described by Williams et al., within two hours (Williams et al. 1988. Gastroenterology. 64:611). Briefly, under light anesthesia with ethyl ether nasal cheekbones, upper limbs and chest wrapped in a limiting straps from paper tape to restrict, but not prevent movement of the body. Control simulated stress animals were anestesiology, but not wrapped. For tricca the ü minutes before the end of the session PRS animals were injected with test compound or solvent. In thirty minutes-one hour after completion of the PRS were stretching technique CRD, as described above for the model of TNBS, barostat at pressures of 15, 30, 45 and 60 mm RT. senior Statistical analysis of the number of bursts was determined and analyzed, as in the model of TNBS above.

The model of painful cramps induced by phenylbenzophenone

The model of painful cramps, PBQ-induced, can be used to evaluate the activity of the regulation pain peptides and agonists of the receptor HZ WITH the invention. This model is described by Siegmund et al. (1957. Proc. Soc. Exp. Bio. Med. 95:729-731). Briefly, within one hour after oral administration of test compounds, such as peptides, morphine or solvent, 0.02% solution of phenylbenzophenone (PBQ) (12.5 ml/kg) was administered to mice by intraperitoneal. The number of vytjagivanii and cramps were recorded from 5-th to 10-th minute after injection PBQ, and also can be counted between the 35th and the 40th minute, and between the 60th and 65th minute for dynamic evaluation. The results are expressed as the number of sprains and cramps (mean±SEM) and percentage variation nociceptive threshold, calculated from the average value of the group treated with the solvent. Statistical significance of any differences between the treated groups and the control group was determined by the criterion Danetta using the residual variance after one-way analysis of variance (P<,05) using the software SigmaStat.

Figa and 8b show the effect of different doses of MD-915 and MD-1100 in the study of writhing PBQ. Indomethacin, a NSAID (non-steroidal anti-inflammatory agent) with known activity regulation of pain, was used as positive control in the assay. Significant reduction of writhing was observed for MD-915 (dose 1 mg/kg) and MD-1100 (dose 2.5 mg/kg) compared to the control solvent. The loss of efficiency at the highest tested dose was also observed for many other compounds (such as antagonists 5HT-3), tested in similar assays. The results of this study suggest that both of the MD-915 and MD-1100 have antinociceptive effects in this model of visceral pain, comparable with intermediate doses of indomethacin.

Example 5: Determination of KD MD-1100

To determine the affinity of MD-1100 to receptors HZ, detected in the intestinal mucosa of rats, conducted the analysis of competitive binding using intestinal epithelial cells of rats. Epithelial cells from the small intestine of rats were obtained as described by Kessler et al. (J.Biol. Chem.245: 5281-5288 (1970)). Briefly, animals were killed and opened their abdominal cavity. The small intestine was washed with 300 ml of ice-cold saline or PBS. 10 cm of the small intestine, measured at 10 cm from the pylorus, was removed and cut into segments 1 inch. The mucous membrane of the intestine vyd is poured from the intestines accurate pressure between the piece of parafilm and the tip of the pipette P-1000. Intestinal epithelial cells were placed in 2 ml of PBS and gets up and down using a 5 ml pipette to obtain a cell suspension. The protein concentration in the suspension was measured using the Bradford method(Anal.Biochem. 72: 248-254 (1976)).

Analysis of competitive binding was carried out on the basis of method of Giannella et al.(Am.J.Physiol. 245:G492-G498) [125I] - labeled MM-416776 and MD-1100. The analyzed mixture contained: 0.5 ml of DME with 20 mm HEPES-KOH pH 7.0, 0.9 mg of cell suspension above 21,4 fmol [125I]-MM-416776 (42,8 PM) and various concentrations of competitor MD-1100 (from 0.01 to 1000 nm). The mixture is incubated at room temperature for 1 hour and the reaction was stopped by applying the mixture on the filters glass fiber GF/B (Whatman). Filters were washed in 5 ml ice-cold PBS and measured the radioactivity. Fig. 9 shows that the KD for MD-1100 in this analysis was 4.5 nm. %B/Bo represents a percentage of the radioactivity absorbed in each sample (B), compared with the radioactivity retained in the control sample without cold competitor (Bo). Giannella et al.(Am.J.Physiol.245: G492-G498) observed that the KD for ST peptide of wild type in the same analysis was ~13 nm.

Example 6: the Pharmacokinetic properties of MD-1100

To study the pharmacokinetics of MD-1100 conducted research absorptive capacity in mice by the introduction of MD-1100 intravenous pose the CTB injection into the tail vein or orally by probe 8-week old CD1 mice. Serum was obtained from animals at different time points and examined for the presence of MD-1100 using a competitive enzyme immunoperoxidase analysis (Oxoid, the set ST EIA, Cat#TD0700). In the analysis used monoclonal antibodies to the ST peptide (antibody provided in the kit Oxoid) and synthetically derived MD-1100. Fig. 10a shows the data acquisitions for intravenous and oral MD-1100, as determined by ELISA. Apparently, MD 1100 minimally systemically absorbed and is <2,2% bioavailable.

Did a similar study of bioavailability, which used IHMS instead of ELISA for determination of MD-1100. Source, serum samples were extracted from whole blood exposed and control mice, then injected directly (10 ml) to the built-column solid phase extraction (SPE) (Waters Oasis HLB column 25 mm to 2.0×15 mm, direct link) without further processing. The sample on the SPE column was washed with 5% methanol, 95% solution dH2O (2,1 ml/min, 1.0 minute), then loaded onto the analytical column using a valve switch that places the SPE column into a reverse current path on an analytical column (Waters Xterra MS C8 column (5 mm IS, of 2.1×20 mm). The sample was suirable of the analytical column by gradient reversed-phase (mobile phase A: 10 mm ammonium hydroxide in dH2O mobile phase is B: 10 mm ammonium hydroxide in 80% acetonitrile and 20% methanol; 20% B for the first 3 minutes, then move up to 95% B for 4 min and hold for 2 min, all at the speed of a current of 0.4 ml/min). 9.1 minutes the gradient is returned to the initial conditions 20% B for 1 min MD-1100 was suirable from the analytical column 1.45 minutes, and was determined by triple-quadruple mass spectrometry (MRM, 764(+2 charge status)>182 (+1 charge state) Yes; the potential funnels=30 V; collision=20 eV; source resolution=2 Yes on the main peak; the child resolution=2 Yes on the main peak). The instrumental response was converted into units of concentration by comparison with a standard curve using known quantities of chemically synthesized MD-1100 received and entered in mouse serum using the same methods.

Fig.10b shows the data acquisitions for the input/oral MD-1100, as determined IHMS. In this analysis of MD-1100 is manifested in a similar way minimally systemically absorbed and is <0,11% bioavailable.

The introduction of the peptides and agonists of the receptor HZ WITH

For treatment of gastrointestinal disorders peptides and agonists of the invention are preferably administered orally, for example in the form of tablets, gel, paste, slurry, liquid, powder, or in some other form. Oral input composition may include binding agents, flavouring agents and Simachev the matter of funds. The peptides and agonists can be co-injected with other agents used to treat gastrointestinal disorders, including, but not limited to, agents that inhibit acidity, such as agonists of the receptors of histamine-2 (H2A) and a proton pump inhibitor (PPI). The peptides and agonists can also be administered rectally by suppository. For the treatment of disorders outside the gastrointestinal tract, such as congestive heart failure and benign prostatic hyperplasia, peptides and agonists preferably administered parenterally or orally.

The peptides described in the present description, can be used alone or in combination with other agents. For example, the peptides can be administered together with analgesic peptide or connection. Analgesic peptide or compound can be covalently attached to the peptide described herein, or may be a single agent that is administered together with or in series with the peptide described in the present description, in combination therapy.

Combination therapy can be achieved by the introduction of two or more agents, such as peptide described herein, and analgesic peptide or compound, each of which receptionen and introduced separately or introduction of two or more agent is in a composition. Other combinations are also enclosed in combination therapy. For example, two agents can be receptionby together and be entered in conjunction with a separate composition comprising a third agent. While two or more agents in combination therapy may be administered simultaneously, they should not. For example, the introduction of the first agent (or combination of agents) may precede the introduction of a second agent (or combination of agents) for minutes, hours, days or weeks. Therefore, two or more of the agent 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 from each other, or within 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks from each other. In some cases even longer intervals. While in many cases it is desirable that two or more agent used in combination therapy, were present in the patient's body at the same time, it should not be so.

Combination therapy may also include two or more injections of the one or more agents used in combination. For example, if agent X and agent Y are used in combination, they can be administered sequentially in any combination one or more times, for example, in the order X-Y-X-X-X-Y, Y-X-Y, Y-Y-X-X-X-Y-Y and other

Agents, separately or in combination, can be mixed with any the m pharmaceutically acceptable carrier or medium. Therefore, they can be combined with substances which do not produce adverse, allergic or other adverse reactions when administered to the patient. Used media or environment may include solvents, dispersing agents, membrane, agents that enhance the absorption, agents, controlled release and other

Agents in the form of their free form or in salt form can be mixed with a polymer, such as polylactic-glycolic acid (PLGA), poly(I)-lactic-glycolic-tartaric acid (P(I)LGT) (WO 01/12233), polyglycolic acid (U.S. patent 3773919), polylactic acid (U.S. patent 4767628); poly(ε-caprolacton) to create a composition of extended release. Such compositions can be used in the implant, which releases the peptide or other agent within a period of a few days, several weeks or several months depending on the polymer, the particle size of the polymer and the size of the implant (see, for example, U.S. patent 6620422). Other songs prolonged release described in EP 0 467389 A2, WO 93/241150, U.S. patent 5612052; WO 97/40085, WO 94/155587, U.S. patent 5672659, U.S. patent 5893985, U.S. patent 5134122, U.S. patent 5192741 U.S. patent 5192741, and U.S. patent 5445832. In such compositions prolonged-release microparticles of the peptide is mixed with microparticles of the polymer. One or Bo is her implants with long release can be placed in the colon, the small intestine or both.

The agents may be administered, for example, intravenous injection, intramuscular injection, subcutaneous injection, or in other ways. The agents may be administered orally, for example in the form of tablets, gel, paste, slurry, liquid, powder or in any other form. Oral input composition may contain binding agents, flavouring agents and wetting agents. Agents can be included in toothpaste or rinse for the mouth. Therefore, the oral compositions can include abrasives and foaming agents. Agents can also be administered transdermal or in the form of a suppository.

Agents can be a free acid or base or pharmacologically acceptable salt. Solids can be dissolved or dispersed directly before introduction or earlier. In some circumstances, the drugs include a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injection may include sterile aqueous or organic solutions or dispersions, which include, for example, water, alcohol, organic solvent, oil, or other solvent or dispersing agent (for example, glycerol, propylene glycol, polyethylene glycol and vegetable oil). Pharmaceutical agents can be sterilize the Ana filtration sterilization, or other suitable means.

Suitable pharmaceutical compositions in accordance with the invention typically include a number of active compound(s) with an acceptable pharmaceutical diluent or auxiliary substance, such as a sterile aqueous solution, to obtain a range of final concentrations, depending on the intended purpose. The method generally well known in the art as illustrated Remington''s Pharmaceutical Sciences (18th Edition, Mack Publishing Company, 1995).

The agents described in the present description, agents, and combination therapy may be packaged as a kit that includes one or more doses of two or more agents, each Packed or receptionen separately, or one or more doses of two or more agents, Packed or receptionand in combination. Therefore, one or more agents may be present in the first container, and the kit can optionally include one or more agents in the second container. The container or containers are placed in the package, and the package may optionally include instructions for introducing or dosing. The kit may include additional components, such as syringes or other means for the introduction of agents, diluents, or other means to receptionarea.

Anaesthetic agents

The peptides described in this op is sanija, can be used in combination therapy with an analgesic agent, such as analgesic compound or analgesic peptide. Anesthetic agent may optionally be covalently attached to the peptide described in the present description. Among the applicable anesthetic agents are: blockers Ca channel, 5HT receptor antagonists(for example, antagonists of receptors, 5HT3, 5HT4 and 5HT1)agonists of opioid receptors (loperamide, fedotozine and fentanyl), NK1 receptor antagonists, agonists of CCK receptor (for example, loxiglumide), NK1 receptor antagonists, NK3 receptor antagonists, inhibitors of reuptake of norepinephrine-serotonin (NSRI), agonists vanilloids and cannabanoid receptors and sialorphin. Anaesthetic agents in different classes described in the literature.

Among the applicable painkillers peptides are sialorphin-related peptides, including those comprising the amino acid sequence QHNPR (SEQ ID NO: ), including: VQHNPR (SEQ ID NO: ); VRQHNPR (SEQ ID NO: ); VRGQHNPR (SEQ ID NO: ); VRGPQHNPR (SEQ ID NO: ); VRGPRQHNPR (SEQ ID NO: ); VRGPRRQHNPR(SEQ ID NO: ) and RQHNPR (SEQ ID NO: ). Sialorphin-related peptides associated with neprilysin and inhibit mediated neprilysin the collapse of substance P and Met-enkefalina. Therefore, compounds or peptides that are inhibitors neprilysin are applicable anesthetic agents, to the which can be entered with the peptides according to the invention in joint therapy or related peptides according to the invention, for example, through a covalent bond. Sialorphin and related peptides described in U.S. patent 6589750; U.S. patent 20030078200 A1 and WO 02/051435 A2.

Antagonists and agonists of opioid receptors may be injected with the peptides according to the invention or in joint therapy or related peptide according to the invention, for example, the covalent bond. For example, antagonists of opioid receptors, such as naloxone, naltrexone, maternalized, nalmefene, ziprider, beta-funaltrexamine, naloxonazine, naltrindole and Nord-binaltorphimine, are useful in the treatment of IBS. May be applicable to receptivity opioid antagonists such compositions with delayed or prolonged release, so that the original release of the antagonist is in the mid to distal small intestine and/or the ascending colon. Such antagonists are described in WO 01/32180 A2. Pentapeptide enkephalin (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine) is an agonist of mu and Delta opioid receptors and is considered appropriate to increase intestinal motility(Eur.J.Pharm. 219:445, 1992), and this peptide may be used in connection with the peptides according to the invention. Also applicable is trimebutine, which is that associated with the mu/Delta/Kappa opioid receptors and activates the release of motilin and regulates the release of gastrin, vasoactive and destinating peptide, gastrin and glucagon. Agonists opioid receptor Kappa, such as fedotozine, catallozzi and compounds described in WO 03/097051 A2, can be used with or linked to the peptides according to the invention. In addition, agonists of opioid receptors mu, such as morphine, Diphenoxylate, makevalid (H-Tyr-D-Ala-Phe(F)-Phe-NH2; WO 01/019849 A1) and loperamide can be used.

Tyr-Arg (kyotorphin) is a dipeptide, which acts by stimulating the release of met-enkephalins to retrieve the anesthetic effect (J.Biol. Chem262:8165, 1987). Kyotorphin can be used with or associated with the peptides according to the invention.

Agonists of CCK receptor, such as cerulein from amphibians and other species, are applicable analgesic agents that can be used with or linked to the peptides according to the invention.

Conotoxin peptides represent a large class of painkillers peptides that act on the potential-dependent Ca channels, NMDA receptors or nicotinic receptors. Such peptides can be used with or linked to the peptides according to the invention.

Peptide analogues of timeline (French application 2830451) can have analgetic activity and can be used with or linked to the peptides according to the invention.

The CCK receptor antagonists (CCKa or CCKb), including loxiglumide and the code is loxiglumide (R-isomer loxiglumide) (WO 88/05774), may have analgetic activity and can be used with or linked to the peptides according to the invention.

Other applicable anesthetic agents include agonists of the 5-HT4, such as Tegaserod/zelnorm and lyricsare. Such agonists are described in: EP 1321142 A1, WO 03/053432A1, EP 505322 A1, EP 505322 B1, US 5510353, EP 507672 A1, EP 507672 B1 and US 5273983.

Calcium channel blockers such as sinomatic and related compounds described in, for example, EP 625162B1, US 5364842, US 5587454, US 5824645, US 5859186, US 5994305, US 6087091, US 6136786, WO 93/13128 A1, EP 1336409 A1, EP 835126 A1, EP 835126 B1, US 5795864, US 5891849, US 6054429, WO 97/01351 A1, can be used with or linked to the peptides according to the invention.

Various receptor antagonists (NK-1, NK-2 and NK-3 (for a review, see Giardina et al. 2003.Drugs. 6:758) can be used with or linked to the peptides according to the invention.

Antagonists of the NK1 receptor, such as either aprepitant (Merck & Co Inc), popopirat, 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 according to the invention.

Antagonists of the receptor NK-2, 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 according to the invention.

The NK3 receptor antagonists, such as osanetant (Sanofi-Synthelabo), talnetant and related compounds described the in, for example, WO 02/094187 A2, EP 876347 A1, WO 97/21680 A1, US 6277862, WO 98/11090, WO 95/28418, WO 97/19927 and Boden et al.(JMedChem. 39:1664-75, 1996), can be used or linked to the peptides according to the invention.

Inhibitors of reuptake of norepinephrine-serotonin, such as milnacipran and related compounds described in WO 03/077897 A1, can be used with or linked to the peptides according to the invention.

Antagonists vanilloid receptors, such as arvanil and related compounds described in WO 01/64212 A1, can be used with or linked to the peptides according to the invention.

When the analgesic agent is a peptide, and it is covalently linked to the peptide described in the present description, the resulting peptide may also include at least one site of cleavage by trypsin or chymotrypsin. When present in the peptide, used as an analgesic peptide may precede (if he is carboxykinase) or follow it (if it is aminocore) site cleavage by chymotrypsin or trypsin, which allows you to release the analgesic peptide.

In addition to sialorphin-related peptides, analgesic peptides include: AspPhe, endomorphin-1, endomorphin 2 nistatin, dalargin, lupron, sicknote and substance P.

Treatment

The peptides according to the invention can be used to treat or Ave is in preventing cancer, precancerous tumors or metastatic tumors. For example, they can be used to prevent or treat colorectal/local metastatic colorectal cancer, cancer of the gastrointestinal tract, lung cancer, or precancerous tumors or metastatic tumors of epithelial cells, polyps, carcinoma of breast, colorectal, lung, ovarian, pancreatic, prostate, kidney, stomach, bladder, liver, esophageal and testicular carcinoma, carcinomas (e.g., basal cell, basosquamous, brown-Pearce carcinoma, ductal, Ehrlich tumor, Krebs, Merkel cells, small or non-small cell lung asanovich cells, papillary, bronchiolar, squamous cells, intermediate cells, Walker), leukemia (e.g., B-cell, T-cell, HTLV, acute or chronic lymphocytic, mast cells, myeloid), histiocytoma, histiocytosis, Hodgkin's disease, non-Jackinsky lymphoma, plasmacytoma, reticuloendotheliosis, adenoma, adenocarcinoma, adenofibroma, adenolymphoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, sclerosing angioma, angiomatosis, apudomy, Brahimi, malignant carcinoid syndrome, carcinoid heart disease, carcinosarcoma, cementoma, cholangioma, cholesteatoma, chondrosarcoma, x is androblastoma, chondrosarcoma, chordoma, haristoy, craniopharyngioma, chondroma, cylindroma, cystadenocarcinoma, cystadenoma, cystosarcoma of villadia, dysgerminoma, ependymomas, abnormal Ewing sarcoma, fibroma, fibrosarcoma, s IV is coming-cell tumors, ganglioneuroma, glioblastomas, hamangioma, granulosa cell tumors, gigantomastia, hamartomas, hemangioendothelioma, hemangioma, hemangio-pericytoma, hemangiosarcoma, hepatoma, Ostroukhova cell tumors, Kaposi's sarcoma, leiomyoma, leiomyosarcoma, leucosarcia, tumors of the Leydig cells, lipoma, liposarcoma, lymphangioma, lymphangioma, lymphangiosarcoma, Protocol, meningiomas, mesenchymal, mesonephroma, mesothelioma, myoblastoma, fibroids, myosarcoma, myxoma, myxosarcoma, neurinomas, neuroma, neuroblastoma, neuroepithelioma, neurofibromas, neurofibromatosis, odontoma, osteomas, osteosarcoma, papilloma, paraganglioma, achromatin paraganglion, pinealoma, rhabdomyomas, rhabdomyosarcoma, tumors of Sertoli cells, teratoid tumors, tech cell tumors and other diseases in which cells become dysplastic, immortal or transformed.

The peptides according to the invention can be used for the treatment or prevention of familial adenomatous polyposis (FAP) (autosomal Dominator syndrome), which precedes cancer of the colon, nakedest the military nonpolyposis colorectal cancer (HNPCC) and hereditary autosomal Dominator syndrome.

For the treatment or prevention of cancer, pre-cancerous tumors and metastatic tumors peptides can be used in combination therapy with radiation or chemotherapeutic agents, an inhibitor of cGMP-dependent phosphodiesterase or a selective inhibitor of cyclooxygenase-2 (the number of selective inhibitors of cyclooxygenase-2 is described in WO02062369 thus incorporated by reference).

The peptides can be for the treatment or prevention of inflammation. Therefore, they can be used alone or in combination with an inhibitor of cGMP-dependent phosphodiesterase or a selective inhibitor of cyclooxygenase-2 for the treatment of organ inflammation, INSTITUTE (for example, Crohn's disease, ulcerative colitis), asthma, nephritis, hepatitis, pancreatitis, bronchitis, cystic fibrosis, ischemic bowel disease, intestinal inflammations/allergies, abdominal diseases, proctitis, eosinophilic gastroenteritis, mastocytosis and other inflammatory disorders.

The peptides can also be used to treat or prevent insulin-related disorders, for example: II diabetes, hyperglycemia, obesity, disorders associated with disturbances in the transport of glucose and electrolytes, and insulin secretion in the cells, or endocrine disorders. They can also be used in treating insulinresistant the activity and postherpetic and not surgical reduction of insulin sensitivity.

The peptides can be used to prevent or treat respiratory disorders, including disorders of breathing, ventilation and mucus secretion, pulmonary hypertension, chronic vascular obstruction and respiratory tract and irreversible obstruction 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. patent No. 6333354 thus incorporated by reference).

The peptides can also be used to prevent or treat retinopathy, nephropathy, diabetic angiopathy and formation of edema.

The peptides can also be used to prevent or treat neurological disorders, such as headache, anxiety, movement disorders, aggression, psychosis, seizures, panic attacks, hysteria, sleep disorders, depression, schizoaffective disorders, sleep apnea, syndromes attention deficit, memory loss, and narcolepsy. They can also be used as sedatives.

Peptides and detectable labeled peptides can be used as markers for detection, identification, staging or diagnosis of diseases and conditions of the small intestine, including Crohn's disease, colitis, inflammatory bowel disease, tumors and, benign tumors, such as benign stromal tumor, adenoma, angioma, adenomatous (on the leg and without legs) polyps, malignant carcinoid tumors, tumors of endocrine cells, lymphoma, adenocarcinoma, carcinoma of the upper, middle and lower sections of the gastro-intestinal tract, gastrointestinal stromal tumor (GIST), such as leiomyoma, cell leiomyoma, leiomyoblastoma and leiomyosarcoma, tumors of the gastrointestinal autonomic nerve syndromes of malabsorption, coeliac disease, diverticulosis, Meckel's diverticulum, diverticulum of the colon, megacolon, disease Hirschprung, irritable bowel syndrome, mesenteric ischemia, ischemic colitis, colorectal cancer, non-polyposis colon cancer syndrome polyps, intestinal adenocarcinoma syndrome Liddle, Brody myopathy, infant convulsions and choreoathetosis.

The peptides can be soedinenii with another molecule (e.g., diagnostic or therapeutic molecule) to target cells bearing the receptor GDS, for example cystic fibrosis lesions and specific cells lining the intestinal tract. Therefore, they can be used for targeting radioactive components or therapeutic components in the intestine to aid in the visualization and diagnosis or treatment of colorectal/METAS Airbuses or local colorectal cancer and to deliver normal copies of the gene tumor suppressor p53 in the intestinal tract.

The peptides can be used alone or in combination therapy for the treatment of erectile dysfunction.

The peptides can be used alone or in combination therapy for the treatment of diseases of the inner ear, for example for the treatment of Meniere's disease, including symptoms such as dizziness, 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 for the treatment of disorders associated with fluid retention and sodium, for example diseases of transport systems electrolytes-water/electrolytes in the kidney, gut and urogenital system, congestive heart failure, hypertension, hypotension, liver cirrhosis and nephrotic syndrome. In addition, they can be used to enhance diuresis or regulation of intestinal fluid.

The peptides can be used alone or in combination therapy to treat disorders associated with the secretion of bicarbonate, such as cystic fibrosis.

The peptides can be used alone or in combination therapy to treat disorders associated with the regeneration of liver cells.

1. The purified polypeptide comprising the amino acid sequence: Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr.

2. The purified polypeptide according to claim 1, where the polypeptide consists of the amino acid sequence: Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr.

3. Pharmaceutical composition for the treatment of infection is a rule-intestinal disorders, containing the polypeptide according to claim 1 or 2.

4. A method of treating a gastrointestinal disorder in a patient, comprising the introduction of a pharmaceutical composition according to claim 3.

5. The method according to claim 4, where the gastrointestinal disorder is a disorder of gastrointestinal motility.

6. The method according to claim 4, where gastrointestinal disorder selected from the group consisting of: irritable bowel syndrome, chronic constipation, functional gastrointestinal disorders, gastro-oesophageal reflux disease, functional heartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronic small bowel pseudoobstruction, colonic pseudoobstruction, Crohn's disease, ulcerative colitis and inflammatory bowel disease.

7. The method according to claim 6, where the gastrointestinal disorder is irritable bowel syndrome.

8. The method of increasing the activity of the receptor guanylate cyclase (GC-C) in a patient, comprising the introduction of a pharmaceutical composition according to claim 3.

9. The method according to any of claims 4 to 8, where the pharmaceutical composition is administered orally.



 

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FIELD: chemistry.

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EFFECT: methods of obtaining the new depsipeptide compounds and intermediary products applied in obtaining these compounds.

31 cl, 3 tbl, 25 ex

FIELD: chemistry.

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EFFECT: increasing the output of the end product and simplification of the process.

3 cl, 2 ex

FIELD: medicine; pharmacology.

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EFFECT: scope expansion.

4 cl, 1 dwg, 4 tbl

FIELD: chemistry.

SUBSTANCE: invention concerns biologically active compounds with inhibition effect on binding antigen with II class MHC molecules, particularly with II class MHC molecule HLA-DR2. Also, the invention proposes pharmaceutical compositions containing such compounds, and their application in obtaining medications for treatment or prevention of diseases connected to T-cell proliferation, such as autoimmune diseases and disorders of inhibition of binding II class MHC molecule HLA-DR2.

EFFECT: obtaining pharmaceutical composition for treatment or prevention of diseases connected with T-cell proliferation.

17 cl, 4 tbl, 6 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention refers to methods of self-specific T-cell vaccine production. Self-specific T-cell vaccine for disseminated sclerosis treatment includes attenuated T-cells which are reactive relative to one or several epitopes, yet SEQ ID NOS: 1-6 contain these epitopes. Invention is intended for treatment of autoimmune diseases, such as disseminated sclerosis or rheumatoid arthritis using self-specific T-cell vaccines. Besides, invention provides diagnostics of diseases associated with T-cells. Advantage of this invention implies that it can be applied for production of T-call vaccines with heterogenous gene VR-Dp-JR to take into consideration clonal shift if self-reactive T-cells.

EFFECT: method has improved efficiency.

10 cl, 7 ex, 2 dwg

FIELD: medicine, biotechnology.

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EFFECT: valuable medicinal properties of antibodies.

24 cl, 16 dwg, 5 tbl, 20 ex

FIELD: organic chemistry, pharmacy.

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EFFECT: valuable medicinal properties of conjugates.

30 cl, 3 tbl, 4 dwg, 30 ex

FIELD: medicine, oncology, immunology.

SUBSTANCE: invention relates to antibodies directed on EGF-receptor (HER1) for designation to human, in particularly, for therapeutic using in tumors. Antibodies represent modified antibodies wherein their modification results to reduced tendency to induce immune response after their administration in human. In particular, invention relates to modification of anti-EGER antibody 425 in its different forms and their fragments that results to preparing variants of Mab 425 that are essentially not immunogenic or less immunogenic as compared with any their unmodified equivalent in using in vivo. The advantage of invention involves reducing immunogenicity of antibody.

EFFECT: improved and valuable properties of antibodies.

10 cl, 5 tbl, 1 ex

FIELD: medicine, polypeptides.

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EFFECT: enhanced pharmacokinetic properties of polypeptides.

52 cl, 18 dwg, 14 tbl, 11 ex

FIELD: biotechnology, medicine, oncology.

SUBSTANCE: invention relates to peptides found by selection of phage peptide library by a feature for selective binding with target-cells. These peptides are able to accumulate in lung adenocarcinoma cells and show the following amino acid sequences: RNVPPIFNDVYWIAF, SVAILPRSFSPFXVG, PFARAPVEHHDVVGL. New tumor-targeted peptides allow carrying out the purposeful delivery of preparation of different designation to lung adenocarcinoma cells. Invention can be used in medicine in treatment and diagnosis of lung adenocarcinoma and its metastasis.

EFFECT: valuable medicinal properties of peptides.

2 dwg, 2 ex

FIELD: medicine.

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EFFECT: when prescribed, the medicinal agent containing bifidus bacteria allows for direct connective metabolism control thus lowering and improving activity of the antioxidant systems.

6 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to balneology. The mode includes the therapy by means of nitrosiliceous thermal water of Annensky spring from bores №2 and №21 Olcha region of the Khabarovsk Territory. It is ensured by mineral baths by exposition 8-15 minutes at temperature 36C°. The course is 10-12 baths daily, or every second day. The therapy also includes daily mineral water douches for 5-10 minutes. The course is 8-12 procedures. Besides it is combined with mineral water inhalations within 5 to 15 minutes. The course of treatment is 10-15 inhalations. Mineral water drinking is prescribed at 3 ml/kg of body weight with single dose no more than 200 ml, 4 times a day. The course of treatment is 18 days.

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3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to neurology, and can be used for regional upper-limp pain syndrome management ensured by autoblood 2-4 ml sampled in a syringe prefilled with dexamethasone 0.25-0.5 ml and 1% lidocaine solution 8-10 ml. Autoblood is sampled from spongy tissues of mesoscapula. The mixture is injected intraosseously in autoblood sampling point.

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1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to gastroenterology, and concerns gastric ulcer and duodenal treatment that is ensured by dosing the colloidal silver solution in the volume V specified from the range 50≤V≤600 ml and concentration C within 3≤C≤40 mg/l introduced into the human stomach in number N doses for each session as specified from 1≤N≤25 thus making albation cycle with the interrelated inequality 1.04≤(Vi+V)/V≤2 of the volume Vj ratio, herewith 1≤i≤n in each introduced solution dose in specified number N, and the maximal solution volume V provided V1+V2…+Vi+…+Vn-1+Vn=V. Then intragastric electrophoresis in achlorhydria is preceded with oral introduction: 8-10 hours prior to electrophoresis - preparation of histamine H2-blocker group - Ranitidine dosed 300 mg or Quamatel dosed 40 mg, and 2-3 hours prior to electrophoresis - preparation from proton pump blocker group - Omeprazole or Pariet dosed 20 mg.

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3 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and pharmaceutics and concerns applications of compound of formula II to make medical product for adaptive colitis or functional dyspepsia treatment.

EFFECT: invention ensures high treatment effectiveness.

2 cl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and pharmacology, namely to pharmaceutical composition applied for external and local treatment of pyodestructive processes in skin and mucous membranes and characterised by that it contains Tilorone and at least one end additive chosen from: agent with antibacterial activity to gram-positive and/or gram-negative bacteria, antimicotic, local anaesthetic agent, reparative stimulator, hypooxidant, biogenic elements, a base or their mixture in certain ratio.

EFFECT: invention ensures extended application of the known active agent Tilorone, restored local immunity and biocoenosis of skin and mucous membranes in treatment of pyodestructive processes, anaesthetic effect; additional reparative stimulation; improved microcirculation in lesion focus, reduced inflammatory reactions in skin and mucous membranes, decreased secondary tissue neurosis of the affected areas, improved microcirculation in lesion focus, reduced itching, maintained high therapeutic activity and usability, more rapid and improved healing.

10 cl, 19 ex

Arthronosos cure // 2353349

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to pharmaceutics and can be used for making the preparation referred to chondroprotective agents used for curing degenerative atrhronosos and spinal disorders. The medical product contains chondroitin sulphate; dimethyl sulfoxide; preserving agent, emulgel base. The preserving agent is mixed Nipagin and Nipasol. The emulgel base is mixed thickening agent, solvent and softener. The thickening agent of the emulgel base is acrylic and methacrylic acid copolymers as Carbomer; the solvent is water, and the softener is mineral or vegetable oil. Additionally is contains: Carbomer neutraliser as an inorganic or organic base; coemulsifier as a nonionic surface-active substance, namely oleic acid monoester and polyoxyethylated sorbitan (TWIN 80). Optionally it contains essential oil in ratio, wt %: chondroitin sulphate 1-10 dimethyl sulfoxide 5-15 Nipagin 0.1-0.3; Nipasol 0.05-0.15; Carbomer 1-5; Mineral or vegetable oil 5-15; Twin 80 1-15; Inorganic or organic base 1-5; Essential oil 0.05-0.25; Treated water 100.0%. In certain cases the medical product can contain olive oil as a softener, inorganic base, namely sodium hydroxide or sodium bicarbonate as Carbomer neutraliser. Besides as Carbomer neutraliser it can contain organic base, namely triethanolamine. The medical product is to contain neroli oil as the essential oil preferentially.

EFFECT: invention ensures softening action, provides formulation stability and optimum fluidity, improves adhesiveness of the preparation, accelerates penetration of the active substances through skin barrier, and intensifies anti-inflammatory action.

6 cl, 3 ex, 6 tbl

FIELD: medicine.

SUBSTANCE: invention refers to chemical-pharmaceutical industry, particularly to preparation of dermatovenerologic medicines. Ointment preparation contains dry water-soluble medicinal tea extract, vaseline, penthol, beeswax, Nipagin-Nipasol in ratio 7.5:2.5, treated water in certain ratio.

EFFECT: ointment ensures the evident therapeutic outcome of various allergic skin manifestations treatment, extends a wealth of external antiallergenic preparations.

3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, urology, physiotherapy and to be used in the patients suffering from chronic pyelonephritis accompanied with chronic prostatitis and chronic cystitis. The method involves taking the mineral water of the Slavjanovsky source dosed 3 - 5 ml per kg of body weight, combined with carbonic mineral baths 35-37°C for 15 minutes 3 times a day, № 8-10, mud "panties" at 40-42°C within 30 minutes to 1.5 hours, prostate massage № 8-10, additionally 50-60 minutes later followed with electropulse therapy combined with exposure to laser magnetic rays. Electropulse therapy involves low-frequency, low-voltage pulse current with a spherical unipolar electrode. Exposure is contact, labile, clockwise and located at skin projection of ascending, transverse and descending large intestine at frequency 30.2 Hz within 8-10 minutes. The course of treatment is 10-12 procedures.

EFFECT: method improves life quality of the patients both in latent phase of inflammatory process, and in mild and moderate active chronic process.

2 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to novel atropoisomers of formula , in which R and R1 each independently represents hydrogen or methyl; R2, R3 and R4 each independently represents hydrogen or trifluoromethyl, on condition that R2, R3 and R4 all do not represent hydrogen; and R5 represents bromine, chlorine; or to its non-toxic pharmaceutically acceptable salt, solvate. Invention also relates to pharmaceutical composition, as well as to method of treatment.

EFFECT: obtaining novel biologically active compounds, possessing properties which open calcium-activated potassium channels of high conductivity.

12 cl, 6 ex, 2 tbl, 7 dwg

FIELD: medicine.

SUBSTANCE: method relates to medicine, particularly to dermatology, and can be applied in treatment of psoriasis vulgaris. It involves administration of delta sleep-inducing peptide in the following pattern: 1 ampoule in the morning and evening of 1st and 2nd day of therapy, 1 ampoule in the morning of 3rd, 4th and 5th day. The same dosage is given over a day. Delta sleep-inducing peptide is dissolved before use by boiled water of room temperature in amount of 1 ml. Further one or two drops are instilled to mid-part of nose, to olfactory nerve ending bifurcation zone over 10-20 minutes interval for 21 day.

EFFECT: improved treatment efficiency due to reduced exacerbation duration and significant prolongation of remission period.

2 ex, 1 tbl

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