Methods for attenuating release of inflammatory mediators and peptides useful therein

Abstract

The present invention includes methods of inhibiting or suppressing cellular secretory processes. More specifically the present invention relates to inhibiting or reducing the release of inflammatory mediators from inflammatory cells by inhibiting the mechanism associated with the release of inflammatory mediators from granules in inflammatory cells. In this regard, the present invention discloses an intracellular signaling mechanism that illustrates several novel intracellular targets for pharmacological intervention in disorders involving secretion of inflammatory mediators from vesicles in inflammatory cells. Peptide fragments and variants thereof of MANS peptide as disclosed in the present invention are useful in such methods.

I claim: 
     
         1 . A method of inhibiting the release of at least one inflammatory mediator from a granule in at least one inflammatory cell in a tissue and/or fluid of a subject comprising:
 administration to said tissue and/or fluid a therapeutically effective amount of a pharmaceutical composition comprising at least one peptide having an amino acid sequence selected from the group consisting of:   (a) an amino acid sequence having from 4 to 23 contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1);   (b) an amino acid sequence having the sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1); and   (c) an amino acid sequence substantially identical to the sequence defined in (a),   wherein the C-terminal amino acid of the peptide is optionally independently chemically modified, and the N-terminal amino acid of the peptide is independently chemically modified by acylation with a carboxylic acid selected from the group consisting of a C2 to C13 saturated or unsaturated aliphatic carboxylic acid, a C14 saturated or unsaturated aliphatic carboxylic acid, a C15 to C24 saturated or unsaturated aliphatic carboxylic acid, and trifluoroacetic acid, or is not chemically modified, with the proviso that said peptide is modified by acylation when its amino acid sequence begins with the sequence GAQF of the reference sequence by acylation only with a carboxylic acid selected from the group consisting of a C2 to C13 saturated or unsaturated aliphatic carboxylic acid, a C14 unsaturated aliphatic carboxylic acid, a C15 to C24 saturated or unsaturated aliphatic carboxylic acid, and trifluoroacetic acid, or is not chemically modified, wherein said peptide, optionally combined with a pharmaceutically acceptable carrier, and in a therapeutically effective inflammatory mediator release-reducing amount to reduce the release of said inflammatory mediator from at least one inflammatory cell as compared to release of said inflammatory mediator from at least one of the same type of inflammatory cell that would occur in the absence of said at least one peptide.   
     
     
         2 . The method according to  claim 1 , wherein said peptide is acetylated at the alpha N-terminal amino acid. 
     
     
         3 . The method according to  claim 2 , wherein said peptide consists of at least ten contiguous amino acid residues. 
     
     
         4 . The method according to  claim 3 , wherein said peptide consists of acetyl-peptide 106 (SEQ ID NO: 106). 
     
     
         5 . The method according to  claim 1 , wherein said peptide consists of at least four contiguous amino acid residues. 
     
     
         6 . The method according to  claim 1 , wherein said peptide consists of at least six contiguous amino acid residues. 
     
     
         7 . The method according to  claim 1 , wherein said peptide is myristoylated at the alpha N-terminal amino acid. 
     
     
         8 . The method according to  claim 1 , wherein said peptide is amidated with ammonia at the alpha C-terminal amino acid. 
     
     
         9 . The method according to  claim 1 , wherein the peptide comprises an amino acid sequence of (a) wherein the N-terminal amino acid of the amino acid sequence of (a) is selected from amino acid position 2 to 21 of the reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1). 
     
     
         10 . The method according to  claim 9 , wherein said peptide is myristoylated or acetylated at the alpha N-terminal amino acid. 
     
     
         11 . The method according to  claim 9 , wherein said peptide is amidated with ammonia at the alpha C-terminal amino acid. 
     
     
         12 . The method according to  claim 1 , wherein said reducing the release of an inflammatory mediator comprises blocking or inhibiting the mechanism that releases an inflammatory mediator from said inflammatory cell in said subject. 
     
     
         13 . The method according to any one of  claims 1 - 12 , wherein said peptide further comprises a pharmaceutically acceptable carrier to form a pharmaceutical composition. 
     
     
         14 . The method according to  claim 12 , wherein said inflammatory cell is a leukocyte. 
     
     
         15 . The method according to  claim 12 , wherein said inflammatory cell is a granulocyte. 
     
     
         16 . The method according to  claim 12 , wherein said inflammatory cell is selected from the group consisting of a neutrophil, a basophil, an eosinophil and a combination thereof. 
     
     
         17 . The method according to  claim 12 , wherein said inflammatory cell is a monocyte or macrophage. 
     
     
         18 . The method according to  claim 12 , wherein said inflammatory mediator is selected from the group consisting of myeloperoxidase (MPO), eosinophil peroxidase (EPO), major basic protein [MBP], lysozyme, granzyme, histamine, proteoglycan, protease, a chemotactic factor, cytokine, a metabolite of arachidonic acid, defensin, bactericidal permeability-increasing protein (BPI), elastase, cathepsin G, cathepsin B, cathepsin D, beta-D-glucuronidase, alpha-mannosidase, phospholipase A2, chondroitin-4-sulphate, proteinase 3, lactoferrin, collagenase, complement activator, complement receptor, N-formylmethionyl-leucyl-phenylalanine (FMLP) receptor, laminin receptor, cytochrome b558, monocyte-chemotactic factor, histaminase, vitamin B12 binding protein, gelatinase, plasminogen activator, beta-D-glucuronidase, and a combination thereof. 
     
     
         19 . The method according to  claim 12 , wherein said inflammatory mediator is selected from the group consisting of myeloperoxidase (MPO), eosinophil peroxidase (EPO), major basic protein (MBP), lysozyme, granzyme and a combination thereof. 
     
     
         20 . The method according to  claim 1 , wherein said effective inflammatory mediator release-reducing amount of said peptide comprises a degranulation-inhibiting amount of peptide that reduces the amount of an inflammatory mediator released from at least one inflammatory cell from about 1% to about 99% as compared to the amount released from at least one inflammatory cell in the absence of the peptide. 
     
     
         21 . The method according to  claim 1 , wherein said effective inflammatory mediator release-reducing amount of said peptide comprises a degranulation-inhibiting amount of peptide that reduces the amount of an inflammatory mediator released from at least one inflammatory cell from between about 5-50% to about 99% as compared to the amount released from at least one inflammatory cell in the absence of the peptide. 
     
     
         22 . The method according to  claim 1 , wherein said subject is afflicted by a respiratory disease. 
     
     
         23 . The method according to  claim 22 , wherein said respiratory disease is selected from the group consisting of asthma, chronic bronchitis, COPD and cystic fibrosis. 
     
     
         24 . The method according to  claim 1 , wherein said subject is a mammal. 
     
     
         25 . The method according to  claim 24 , wherein said mammal is selected from the group consisting of humans, canines, equines and felines. 
     
     
         26 . The method according to  claim 1 , wherein said administration is selected from the group consisting of topical administration, parenteral administration, rectal administration, pulmonary administration, nasal administration, and oral administration. 
     
     
         27 . The method according to  claim 26 , wherein said pulmonary administration comprises an aerosol. 
     
     
         28 . The method according to  claim 27 , wherein said aerosol is generated from a dry powder inhaler, a metered dose inhaler or nebulizer. 
     
     
         29 . The method according to  claim 1 , further comprising administration to said subject of a second molecule selected from the group consisting of an antibiotic, an antiviral compound, an antiparasitic compound, an anti-inflammatory compound, and an immunomodulator. 
     
     
         30 . The method according to  claim 1 , wherein said subject is suffering from a disease selected from the group consisting of a bowel disease, a skin disease, an autoimmune disease, a pain syndrome, and combinations thereof. 
     
     
         31 . The method according to  claim 30 , wherein said bowel disease is selected from the group consisting of ulcerative colitis, Crohn's disease and irritable bowel syndrome. 
     
     
         32 . The method according to  claim 30 , wherein said skin disease is selected from the group consisting of rosacea, eczema, psoriasis and severe acne. 
     
     
         33 . The method according to  claim 1 , wherein said subject is suffering from arthritis. 
     
     
         34 . The method according to  claim 1 , wherein the amino acid sequence of (c) substantially identical to the amino acid sequence of (a) is selected from the group consisting of SEQ ID NOS: 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 249, 250, 251 and 252. 
     
     
         35 . The method according to  claim 34 , wherein said peptide is acetylated at the alpha N-terminal amino acid. 
     
     
         36 . The method according to  claim 34 , wherein said peptide is myristoylated at the alpha N-terminal amino acid. 
     
     
         37 . The method according to  claim 34 , wherein said peptide is amidated with ammonia at the alpha C-terminal amino acid. 
     
     
         38 . An isolated peptide having an amino acid sequence selected from the group consisting of:
 (a) an amino acid sequence having from 4 to 23 contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1);   (b) an amino acid sequence having the sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1); and   (c) an amino acid sequence substantially identical to the sequence defined in (a),   wherein the C-terminal amino acid of the peptide is optionally independently chemically modified, and the N-terminal amino acid of the peptide is independently chemically modified by acylation with a carboxylic acid selected from the group consisting of a C2 to C13 saturated or unsaturated aliphatic carboxylic acid, a C14 saturated or unsaturated aliphatic carboxylic acid, a C15 to C24 saturated or unsaturated aliphatic carboxylic acid, and trifluoroacetic acid, or is not chemically modified, with the proviso that said peptide is modified by acylation when its amino acid sequence begins with the sequence GAQF of the reference sequence by acylation only with a carboxylic acid selected from the group consisting of a C2 to C13 saturated or unsaturated aliphatic carboxylic acid, a C14 unsaturated aliphatic carboxylic acid, a C15 to C24 saturated or unsaturated aliphatic carboxylic acid, and trifluoroacetic acid, or is not chemically modified, wherein said peptide, optionally combined with a pharmaceutically acceptable carrier, and in a therapeutically effective inflammatory mediator release-reducing amount to reduce the release of said inflammatory mediator from at least one inflammatory cell as compared to release of said inflammatory mediator from at least one of the same type of inflammatory cell that would occur in the absence of said at least one peptide.   
     
     
         39 . The peptide according to  claim 38 , wherein said peptide is acetylated at the alpha N-terminal amino acid. 
     
     
         40 . The peptide according to  claim 39 , wherein said peptide consists of at least ten contiguous amino acid residues. 
     
     
         41 . The peptide according to  claim 40 , wherein said peptide consists of acetyl-peptide 106 (SEQ ID NO: 106). 
     
     
         42 . The method according to  claim 38 , wherein said peptide consists of at least four contiguous amino acid residues. 
     
     
         43 . The peptide according to  claim 38 , wherein said peptide consists of at least six contiguous amino acid residues. 
     
     
         44 . The peptide according to  claim 38 , wherein said peptide is myristoylated at the alpha N-terminal amino acid. 
     
     
         45 . The peptide according to  claim 38 , wherein said peptide is amidated with ammonia at the alpha C-terminal amino acid. 
     
     
         46 . The peptide according to  claim 38 , wherein the peptide comprises an amino acid sequence of (a) wherein the N-terminal amino acid of the amino acid sequence of (a) is selected from amino acid position 2 to 21 of the reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1). 
     
     
         47 . The peptide according to  claim 46 , wherein said peptide is myristoylated at the alpha N-terminal amino acid. 
     
     
         48 . The peptide according to  claim 46 , wherein said peptide is amidated with ammonia at the alpha C-terminal amino acid. 
     
     
         49 . The peptide according to  claim 38 , wherein the amino acid sequence of (c) substantially identical to the amino acid sequence of (a) is selected from the group consisting of SEQ ID NOS: 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 249, 250, 251 and 252. 
     
     
         50 . The peptide according to  claim 49 , wherein said peptide is acetylated at the alpha N-terminal amino acid. 
     
     
         51 . The peptide according to  claim 49 , wherein said peptide is myristoylated at the alpha N-terminal amino acid. 
     
     
         52 . The peptide according to  claim 49 , wherein said peptide is amidated with ammonia at the alpha C-terminal amino acid. 
     
     
         53 . A composition comprising an isolated peptide according to any one of  claims 38 - 52  and an excipient. 
     
     
         54 . A pharmaceutical composition comprising an isolated peptide according to any one of  claims 38 - 52  and a pharmaceutically acceptable carrier. 
     
     
         55 . The pharmaceutical composition according to  claim 54 , wherein said composition is sterile, sterilizable or sterilized. 
     
     
         56 . A kit comprising at least one isolated peptide according to any one of  claims 38 - 52 . 
     
     
         57 . The method of any one of  claims 1 - 37 , wherein said administration of said peptide reduces MARCKS-related mucus hypersecretion from at least one mucus secreting cell or tissue in said subject, whereby mucus hypersecretion in the subject is reduced compared to that which would occur in the absence of said administration of said peptide.