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HK1201190B - Methods for attenuating release of inflammatory mediators and peptides useful therein - Google Patents

Methods for attenuating release of inflammatory mediators and peptides useful therein Download PDF

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HK1201190B
HK1201190B HK15101730.6A HK15101730A HK1201190B HK 1201190 B HK1201190 B HK 1201190B HK 15101730 A HK15101730 A HK 15101730A HK 1201190 B HK1201190 B HK 1201190B
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peptide
seq
amino acid
inflammatory
acetyl
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HK15101730.6A
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Chinese (zh)
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HK1201190A1 (en
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I．帕里克
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拜欧马克医药有限公司
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Publication of HK1201190B publication Critical patent/HK1201190B/en

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Description

Methods for reducing inflammatory mediator release and peptides useful therefor
The present invention is a divisional application of a patent application having a filing date of 26/7/2007 under application No. 200780035710.6 entitled "method of reducing inflammatory mediator release and peptides useful for the method".
Cross Reference to Related Applications
This application claims priority from U.S. patent application No. 60/833,239, filed on 26.7.2006, which is hereby incorporated by reference in its entirety.
Technical Field
The present invention relates to peptides or peptide compositions and methods of using them to reduce (or inhibit or reduce) the stimulated release of inflammatory mediators from inflammatory cells during inflammation. The invention also relates to the use of these peptides or peptide compositions for modulating intracellular signaling mechanisms that modulate the secretion of inflammatory mediators from inflammatory cells.
Background
Inflammatory leukocytes synthesize a variety of inflammatory mediators that are isolated intracellularly and stored in plasma membrane-bound particles. Examples of such media include, but are not limited to, neutrophil Myeloperoxidase (MPO) (see, for example, Borragard N, Cowland JB. granules of the human neutrophilic polymorphorocyte leukocyte. Blood 1997; 89:3503-3521), Eosinophil Peroxidase (EPO) and Major Basic Protein (MBP) (see, for example, Gleich GJ. mechanism of eosinophial-associated interferon in. J. interferon Immunol 2000; 105:651-663), lysozyme of monocyte/macrophage (see, for example, Saff T, SpencT, Emmenopter A., ppel-Structure M. Effect of leucocyte, Liposomal-peroxidase J. faecal et 35; see, for example, Borbecular peroxidase J. faecal et 52; see, Balbecular peroxidase J. faecal J. 35; see, Biocide J. faecal. 250; see, Biocide J. faecal. J. 52; see, Biocide J. faecal J. 5276; see, Biocide J. faecal. J. faecal J. 52; see, Biocide J. faecal. J. faecal J. 52; and B. faecal, goebel WS, Brahmiz.Stable transformed antisense gram B and perforin constraints inhibited human grain-mediated qualitative ability.cell immunological 1995; 164: 234-; gong JH., Maki G, Klingemann HG. characteristics of a human cell line (NK-92) with pharmaceutical and functional characteristics of activated natural killers. Leukemia 1994; 8: 652-; maki G, Klingemann HG, Martinson JA, Tam YK. Factorregelating the cytoxic activity of the human natural killer Cell line, NK-92.J Hematother Stem Cell Res 2001; 10: 369-383; and Takayama H, Trenn G, Sitkovsky MV.A novel cytoxic T lymphocyte activation assay.J Immunol Methods 1987; 104:183-190). Such mediators are released at the site of loss and promote inflammation and tissue repair, for example, in the lungs and elsewhere. Leukocytes are known to release these particles by the exocytosis mechanism (see, e.g., Burgoyne RD, Morgan A. Secreetolysis. physiol Rev 2003; 83: 581-.
Some exogenous stimuli may promote leukocyte degranulation involving a pathway through events of protein kinase C activation and subsequent phosphorylation (see, for example, Burgoyne RD, Morgan A. secretory genomic DNA Rev 2003; 83: 581-; Logan MR, Odemuywa SO, Moqbel R. Understand transcriptional endothelial in immune and biochemical cells: the molecular biology of molecular biology J Allergy Clin immune 2003; 111:923 932; Smolen JE, Sandborg RR. C2 + -induced molecular biology isolated human genes: the enzymes of Ca2+, protein kinase C. 1990. 12. molecular biology J. Lipase J. Bioparticle J. 1990; protein J. Bioparticle of molecular biology J. 198142; protein J. 12. molecular biology J. 12. molecular biology; protein J. 12. Bioparticle of molecular biology J. Biophycin J. 20. 12. Biophycin. 12. Biophytyl. 4. molecular biology. 4. molecular biology. 3. molecular biology. 4. molecular biology. 4. 3. molecular biology. molecular cloning. 3. molecular tissue. 4. molecular biology. 3. molecular biology. 4. molecular biology. 3. 4. molecular biology. J. molecular cloning. 3. molecular cloning. 4. molecular cloning. 4. 3. molecular cloning.
MARCKS proteins, wherein MARCKS is referred to herein as "Myristoylated Alanine-Rich Kinase C Substrate" (Myrstoylated Alanine-Rich C Kinase Substrate) are ubiquitous phosphorylation targets of Protein Kinase C (PKC) and are highly expressed in leukocytes (see, for example, Aderem AA, Albert KA, Keum MM, Wang JK, GreengardP, Cohn ZA. Stimulus-dependent mutagenesis of a major Substrate for protein Kinase C. Nature 1988; 332: 362-; Theren M, Rosen A, Nairn AC, Aderem A. Regulation by phosphorylation of transformation of a major Substrate for protein Kinase C. Nature 1988; 332: 362-; Theren M, Rosen A. Nature AC; Jarm A. Regulation by phosphorylation of transformation of a major Substrate with growth promoter and protein C. Nature et al., Japan K. 351. 35; incorporated protein, et al., Japan K. D. S. et al.: coding. S.320. Nature protein, Inc. 2. supplement A. Nature protein, protein C. Nature protein, et al., Nature protein, D. 1992. Ab. As). MARCKS proteins are mechanically involved in the exocytosis of mucins by goblet cells lining the respiratory tract (see, e.g., Li et al, J Biol Chem 2001; 276: 40982-. MARCKS are myristoylated at the alpha-amine position of the glycine (i.e., position 1) located at the N-terminus of the amino acid sequence through the amide bond of the N-terminal amino acid of the MARCKS protein amino acid sequence. In airway epithelial cells, the myristoylated N-terminal region of MARCKS appears to be essential for the secretion process. The N-terminus of the MARCKS protein refers to the MANS peptide, which has myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), both L-amino acids. Furthermore, peptide fragments of the MANS peptides disclosed herein also preferably consist of L-amino acids. The mechanism appears to involve MARCKS, a myristoylated protein, in association with the membrane of intracellular granules.
N-terminal myristoylated peptides from the N-terminus of MARCKS have been found to block mucin secretion and the binding of MARCKS to mucin granule membranes in goblet cells (see e.g., Singer et al, Nat Med 2004; 10: 193-196). The peptide contains 24 amino acids of MARCKS protein, starting from the N-terminal glycine of MARCKS protein that is myristoylated through an amide bond, called myristoylated α -N-terminal sequence (MANS); i.e., myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1). Furthermore, Vergeres et al, j. biochem.1998, 330; 5-11 also disclose that the N-terminal glycine residue of MARCKS proteins is myristoylated by a reaction catalyzed by myristoyl CoA, protein N-myristoyl transferase (NMT).
In inflammatory diseases such as asthma, COPD and chronic bronchitis; in genetic diseases such as cystic fibrosis; in allergic diseases (atopy, allergic inflammation); in bronchiectasis; and in a variety of acute infectious respiratory diseases such as pneumonia, rhinitis, influenza or the common cold, arthritis or autoimmune diseases, inflammatory cells are commonly found in or migrate to the damaged or infected areas associated with inflammatory disease states, particularly in the respiratory tract or airways of patients suffering from such diseases. These inflammatory cells can cause tissue damage through the inflammatory mediators they release, thereby playing a great role in the pathology of the disease. One example of such tissue damage or destruction by such chronic inflammation is observed in cystic fibrosis patients, where mediators released by neutrophils (e.g., Myeloperoxidase (MPO)) cause the shedding of airway epithelial tissue.
MARCKS is an approximately 82kD protein with 3 evolutionarily conserved regions (Aderem et al., Nature 1988; 332: 362-: the N-terminus, the phosphorylation site domain (or PSD), and the multiple homology 2(MH2) domain. Human MARCKS cDNA and proteins are known, see Harlan et al, J.biol.chem.1991,266:14399(GenBank Accession No. M68956) and Sakai et al, Genomics1992,14: 175. These sequences are also given in WO00/50062, which is hereby incorporated in its entirety by reference. The N-terminus is an alpha-amino acid sequence comprising 24 amino acid residues with a myristic acid moiety attached by an amide bond to the N-terminal glycine residue, which is involved in the binding of MARCKS to the intracellular membrane (Seykora et al, Jbiol Chem 1996; 271: 18797-. This 24 amino acid sequence is called MANS peptide.
Disclosure of Invention
Involvement of MARCKS proteins in the release of inflammatory mediators from the granules of infiltrating leukocytes is associated with inflammation in diseases of all tissues and organs, including pulmonary diseases characterized by airway inflammation, such as asthma, COPD and cystic fibrosis. However, inflammation of the airways and mucus secretion are two separate and independent processes (Li et al, J Biol Chem 2001; 276: 40982. sup. -. 40990; Singer et al, Nat Med 2004; 10: 193. sup. -. 196). While a variety of factors may promote the production and secretion of mucus, including mediators released by inflammatory cells, there is currently no direct link for excess mucus to cause inflammation.
In one aspect of the invention, the MANS peptide may act to reduce the rate and/or amount of inflammatory mediator particles or vesicles released by inflammatory leukocytes.
In another aspect, peptides derived from the N-terminus of MARCKS, in particular peptides derived from the N-terminal sequence of said 24 amino acids, i.e. active continuous peptide fragments within the sequence of amino acids 1 to 24N-terminus of MARCKS having a glycine at position 1, as well as N-terminal amides of such fragments, such as N-terminal acetic acid amide of such fragments, and/or C-terminal amides of such fragments, such as C-terminal amide formed with ammonia, may inhibit or reduce the rate and/or amount of release of inflammatory mediators from inflammatory leukocytes. Such inhibition or reduction of release includes inhibiting MARCKS-related inflammatory mediator release from inflammatory leukocytes.
In another aspect, peptides derived from the N-terminus of MARCKS, particularly peptides derived from the N-terminal sequence of amino acids 1 to 24, i.e., active contiguous peptide fragments within the sequence of amino acids 1 to 24N-terminus of MARCKS having glycine at position 1, as well as N-terminal amides of such fragments, e.g., N-terminal acetic acid amides of such fragments, and C-terminal amides of such fragments, e.g., C-terminal amides formed with ammonia, can inhibit the rate and/or amount of inflammatory mediator release, e.g., those identified herein, by inhibiting degranulation processes within inflammatory leukocytes.
In another aspect, the MANS peptides and active fragments thereof, as well as active amides of such fragments described herein, can compete with native MARCKS proteins for membrane binding in inflammatory cells to reduce (attenuate or reduce) MARCKS-related release of inflammatory mediators from granules or vesicles containing such inflammatory mediators in such inflammatory cells.
Leukocyte cell types and model cell types that secrete specific particulate components in response to phorbol ester-induced activation of PKC can be used to demonstrate the efficacy of the peptides of the invention and substituted peptides of the invention (e.g., α -N-amides, C-terminal amides and esters) in vitro.
Human leukocyte cell lines may be used to demonstrate that the compounds and compositions of the invention reduce the release of membrane-bound inflammatory mediators. For example, neutrophils isolated from human blood may be used to demonstrate a reduction or inhibition of Myeloperoxidase (MPO) release. Human promyelocytic cell line HL-60 clone 15 can be used to demonstrate that the compounds and compositions of the present invention reduce or inhibit the release or secretion of Eosinophil Peroxidase (EPO) (see, for example, Fischkoff SA. Graded secretion in promoter of eosinophilic differentiation of HL-60 prolycocytic peroxidase cells Leuk Res 1988; 12: 679-. Monocytic leukemia cell line U937 can be used to demonstrate that compounds and compositions of the invention reduce or inhibit lysozyme release or secretion (see, e.g., Hoff T, Spenker T, Emmenopter A., Goppelt-Struebe M.Effect of glucoportectics on the TPA-induced monoclonal differentiation. J LeucoC Biol 1992; 52: 173. sup. 182; BalboaM A, Saez Y, Balsinde J.calcium-independent phospholyase A2is obtained for lysozyme differentiation U937 prokaryotes. J Immunol 2003; 170: 5276. sup. 5280; and Sundstrom C, Nilsson K. Estasshzation of a lysozyme of intracellular lymphocyte J577; U.197577. gamma. 7. J.. Lymphocyte natural killer Cell line NK-92 may be used to demonstrate that the compounds and compositions of the invention reduce or inhibit granzyme release (see, for example, Gong JH., Maki G, Klingemann HG. characterization of a human Cell line (NK-92) with phenotypicals and functional characterization of activated natural killer cells.Leukemia 1994; 8: 652. 658; Maki G, Klingemann HG, Martinson JA, Tam YK. factor regulation of the cytotoxic activity of the human Cell line, NK-92.J Hemato Stem Cell Res 2001; 10: 369. 383; and Takayama H, Trenn G, SitkoskyMV. A. 183. molecular assay J. 183. strategy J. 198190. strategy 104). In an in vitro method of inhibiting or reducing the release of inflammatory mediators, such as those described herein, various cell types are pre-incubated with various concentration ranges of the peptide compounds or peptide compositions of the invention, followed by incubation of these cells with a stimulus for the release of inflammatory mediators, such as phorbol esters. Determining the percentage of inflammatory mediator release that is inhibited, e.g., in the form of a spectrophotometric reading of the concentration of the mediator released, as compared to the release of the mediator in the absence of the peptide compound or peptide composition.
To demonstrate the importance of relative amino acid sequence positions in the peptides of the invention, the relative ability of two classes of peptides, one identical to the 24 amino acid sequence of the N-terminal region of the MARCKS protein (i.e., MANS-myristoylated α -N-terminal sequence peptide) and the other containing the same 24 amino acid residues as MANS but with the order of these residues being random with respect to the sequence order in MANS (i.e., RNS peptides, alternatively referred to as "random N-terminal sequence peptides"), to inhibit or reduce the amount of inflammatory mediators released was compared. Among the various cell types tested, MANS peptide reduced the release of inflammatory mediators over a 0.5-3.0 hour period in a concentration-dependent manner, whereas RNS peptide did not. These results suggest that the relative amino acid sequence positions within the peptides of the invention that are identical to the order in the MARCKS protein, particularly in the N-terminal region thereof, and more particularly in the N-terminal region of 24 amino acid residues thereof, are involved in the treatment of at least one intracellular pathway that inhibits degranulation of leukocytes.
The present invention relates to a new use of said 24 amino acid peptide sequence and to said alpha-N-terminal acetylated peptide sequence, said myristoylated polypeptide, also called MANS peptide, and to an active fragment thereof, which may be selected from peptides having 4to 23 consecutive amino acid residues of the amino acid sequence of MANS peptide, and which fragments may be N-terminal myristoylated if not starting with the N-terminal glycine at position 1 of SEQ ID NO:1, or their N-terminal may be acylated with an acyl group from C2 to C12, including N-terminal acetylation, and/or their C-terminal amidation with NH 2.
The invention also relates to novel methods of blocking MARCKS-related cellular secretion processes, particularly those involving MARCKS-related inflammatory mediator release from inflammatory cells, whose stimulatory pathways involve the protein kinase c (pkc) substrate MARCKS protein and the release of components from intracellular vesicles or granules.
The present invention relates to a method of inhibiting exocytosis release of at least one inflammatory mediator from at least one inflammatory cell comprising contacting a cell comprising at least one inflammatory mediator located within a vesicle within the cell with an effective amount of at least one peptide selected from the group consisting of MANS peptide and active fragments thereof described herein, such that release of the inflammatory mediator from the inflammatory cell is reduced as compared to the release of the inflammatory mediator from the same type of inflammatory cell that would be possible in the absence of the at least one peptide.
The present invention further relates to a method of inhibiting the release of at least one inflammatory mediator from at least one inflammatory cell in a tissue or body fluid of a subject, comprising administering to the tissue and/or body fluid of the subject comprising at least one inflammatory cell comprising at least one inflammatory mediator located in a vesicle within the cell, a therapeutically effective amount of a pharmaceutical composition comprising a therapeutically effective amount of at least one peptide selected from the group consisting of MANS peptides and active fragments thereof, such that the release of the inflammatory mediator from at least one inflammatory cell is reduced compared to the release of the inflammatory mediator from at least one inflammatory cell of the same type that would be possible in the absence of the at least one peptide. More specifically, inhibiting the release of inflammatory mediators comprises blocking or reducing the release of inflammatory mediators from inflammatory cells.
More specifically, the present invention includes a method of reducing inflammation in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the MANS peptide (i.e., N-myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1)) or an active fragment thereof. The active fragment is at least 4 amino acids and preferably at least 6 amino acids in length. Herein, an "active fragment" of a MARCKS protein refers to a fragment that affects (inhibits or reduces) MARCKS protein-mediated release, such as MARCKS protein-mediated release of inflammatory mediators. The active fragment may be selected from GAQFSKTAAKGEAAAERPGEAAV (SEQ ID NO: 2); GAQFSKTAAKGEAAAERPGEAA (SEQ ID NO: 4); GAQFSKTAAKGEAAAERPGEA (SEQ ID NO: 7); GAQFSKTAAKGEAAAERPGE (SEQ ID NO: 11); GAQFSKTAAKGEAAAERPG (SEQ ID NO: 16); GAQFSKTAAKGEAAAERP (SEQ ID NO: 22); GAQFSKTAAKGEAAAER (SEQ ID NO: 29); GAQFSKTAAKGEAAAE (SEQ ID NO: 37); GAQFSKTAAKGEAAA (SEQ ID NO: 46); GAQFSKTAAKGEAA (SEQ ID NO: 56); GAQFSKTAAKGEA (SEQ ID NO: 67); GAQFSKTAAKGE (SEQ ID NO: 79); GAQFSKTAAKG (SEQ ID NO: 92); GAQFSKTAAK (SEQ ID NO: 106); GAQFSKTAA (SEQ ID NO: 121); GAQFSTA (SEQ ID NO: 137); GAQFSK (SEQ ID NO: 154); GAQFSK (SEQ ID NO: 172); GAQFS (SEQ ID NO:191) and GAQF (SEQ ID NO: 211). These peptides do not contain a myristoyl moiety at the N-terminal amino acid, but instead either do not contain any chemical moiety, or contain a non-myristoyl chemical moiety at the N-terminal amino acid and/or a chemical moiety at the C-terminal amino acid, such as the N-terminal acetyl and/or C-terminal amide groups described above. The presence of a hydrophobic N-terminal myristoyl moiety in MANS peptides and N-terminal myristoylated fragments thereof may enhance their compatibility with and possibly permeability to the plasma membrane and may allow uptake of the peptides by cells. Lipid bilayers of myristoyl hydrophobic insertion membranes can provide up to 104M-1The lipid partition coefficient or apparent association constant of (A) or a single Gibbs free Binding energy of about 8kcal/mol (see, e.g., Peitzsch, R.M., and McLaughlin, S.1993, Binding of acylated peptides and lipids to phosphopeptides: pertinence to myristoylated proteins. biochemistry.32:10436-10443), which is sufficient to (at least in part) allow the partitioning of MANS peptides and myristoylated MANS peptide fragments into the plasma membrane of cells, while the additional functional groups and their interaction inside the MANS peptide (which is myristoylated) and inside the acylated MANS peptide fragments enhance their relative membrane permeability. The fragments may each exhibit a partition coefficient and a membrane affinity that are representative of their respective structures. The fragments can be prepared by methods of peptide synthesis known in the art, e.g., by solid phase peptide synthesisMethods are well known (see, e.g., Chan, Weng C.and White, Peter D.eds., Fmoc Solid phase Peptide Synthesis: A Practical Approach, Oxford University Press, New York (2000); and Lloyd-Williams, P.et al.chemical applications to the Synthesis of Peptides and Proteins (1997)), and may be purified by methods known in the art, e.g., by high pressure liquid chromatography. The molecular weight of each peptide can be determined by mass spectrometry analysis, wherein each peptide shows a peak with the appropriate molecular mass. The efficacy of these individual peptides or various combinations of peptides (e.g., 2 combinations of the peptides, 3 combinations of the peptides, 4 combinations of the peptides) in the methods of the invention can be readily determined without undue experimentation using the methods disclosed in the examples herein. A preferred combination may comprise two of said peptides; the preferred molar ratio of the peptides may be from 50:50 (i.e., 1:1) to 99.99 to 0.01, which ratio can be readily determined using the methods disclosed in the examples herein.
Preferably, the MANS peptide or active fragment thereof is placed in a pharmaceutical composition for blocking inflammation. The invention also includes a method of inhibiting a cellular secretory process in a subject, the method comprising administering a therapeutically effective amount of a compound comprising a MANS peptide, or an active fragment thereof, that inhibits an inflammatory mediator in the subject. The administration is typically selected from topical administration, parenteral administration, rectal administration, pulmonary administration, inhalation, and nasal or oral administration, wherein pulmonary administration typically includes an aerosol, a dry powder spray inhaler, a metered dose spray inhaler, or a nebulizer.
Administration of a composition comprising a degranulation-inhibiting amount of MANS peptide or a degranulation-inhibiting amount of an active fragment of MANS peptide, e.g., a pharmaceutical composition of MANS peptide or an active fragment thereof, for use in a human or animal can provide MANS peptide or an active fragment thereof at least to a site in or on a tissue where inflammatory granulocytes are located or will invade, or into a liquid-containing layer in contact with a surface of the tissue, thereby contacting MANS peptide or an active fragment thereof with inflammatory granulocytes. In one aspect, the composition may be administered at the very beginning of the onset or at the very beginning of the detection of inflammation, at the very beginning of the perception of inflammation in a human or animal, or at the very beginning of a perceptible change in the level of inflammation in a human or animal, such that the amount of inflammation is lower than would be possible in the absence of the MANS peptide or active fragment thereof. In another aspect, administration may be performed during the progression of inflammation of a tissue of a human or animal to reduce the amount of additional inflammation that may occur in the absence of a MANS peptide or active fragment thereof. The dose and frequency of administration can be determined by clinical evaluation and is related to the source of the disease or inflammation and the extent of tissue involvement and the age and size of the patient, and it is contemplated that the pharmaceutical composition can be administered repeatedly 3 to 8 hours after the first administration of the pharmaceutical composition, preferably 6 to 8 hours.
The invention also includes a method of reducing inflammation in a subject comprising administering a therapeutically effective amount of a compound that inhibits the release of MARCKS-related inflammatory mediators, such that the release of at least one inflammatory mediator is reduced in the subject compared to what would occur in the absence of the treatment. Herein, "reducing" generally refers to a reduction in the effects of inflammation. Preferably, the release of inflammatory mediators is inhibited or blocked by the methods disclosed herein.
Another embodiment of the invention includes a method of reducing inflammation in a subject comprising administering a therapeutically effective amount of a compound that inhibits MARCKS-related inflammatory mediator release such that inflammation in the subject is reduced as compared to what would occur in the absence of the treatment. Also disclosed are methods of reducing or inhibiting inflammation in a subject comprising administering a therapeutically effective amount of a MANS peptide or an active fragment thereof effective to inhibit an inflammatory mediator at a site of inflammation. The term "inhibition" refers to a decrease in the amount of secretion of inflammatory mediators. The term "complete inhibition" refers to a reduction in the amount of inflammatory mediator secretion to zero. Also, as noted above, the active fragment is at least 4, and preferably at least 6 amino acids in length. The term "exocytosis process" refers to exocytosis, i.e., the process of cellular secretion or excretion, in which substances contained within vesicles located inside cells are expelled through fusion of the vesicle membranes of the vesicles with the outer cell membrane. "degranulation" refers to the release of cellular particulate components. The term "degranulation inhibition" refers to a reduction in the release of inflammatory mediators contained within inflammatory cell granules. Thus, a degranulation-inhibiting amount of MANS peptide and/or active fragments thereof means that the amount of these peptides is sufficient such that the release of inflammatory mediators contained within the particles is reduced as compared to the release in the absence of the same peptide.
In reference peptide GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), at the N-terminal position of the reference peptide, G is at position 1; adjacent to the 1-position G is a 2-position A; adjacent to a in position 2is Q in position 3; adjacent to Q at position 3 is F at position 4; adjacent to the 4-bit F is the 5-bit S; adjacent to S at position 5 is K at position 6; adjacent to K at position 6 is T at position 7; adjacent to T of 7 bits is a of 8 bits; adjacent to the 8-bit a is a 9-bit a; adjacent to the 9-bit a is a 10-bit K; adjacent to K at position 10 is G at position 11; adjacent to the 11-bit G is the 12-bit E; adjacent to the 11-bit G is a of 13 bits; adjacent to the 13-bit a is a of 14 bits; adjacent to 14-bit a is 15-bit a; adjacent to 15 bits of a is 16 bits of E; adjacent to E at position 16 is R at position 17; adjacent to R at position 17 is P at position 18; adjacent to P at position 18 is G at position 19; adjacent to the 19-bit G is the 20-bit E; adjacent to E of 20 bits is a of 21 bits; adjacent to the 21-bit a is a 22-bit a; adjacent to a of 22 bits is V of 23 bits; and adjacent to the V at position 23 is an A at position 24, wherein position 24 is the C-terminal position of the reference peptide.
A "variant" of a reference peptide or a variant of a 4-23 amino acid fragment of a reference peptide is a peptide having an amino acid sequence which differs from the amino acid sequence of the reference peptide or the amino acid sequence of a fragment of the reference peptide, respectively, at least one amino acid position in the amino acid sequence of the reference peptide or a reference peptide fragment, respectively, but which still retains mucin or mucus inhibiting activity which is typically 0.1-10 times, preferably 0.2-6 times, more preferably 0.3-5 times the activity of the reference peptide or fragment, respectively, of the reference peptide or fragment, respectively. A "variant" of a reference amino acid sequence or a variant of a 4-23 amino acid fragment of a reference amino acid sequence is an amino acid sequence which differs from the reference amino acid sequence or fragment of a reference amino acid sequence by at least one amino acid, but which has an amino acid sequence of a peptide which retains the mucin or mucus inhibiting activity of the peptide or fragment encoded by the reference amino acid sequence, which activity is typically 0.1-10 times the activity of the peptide or fragment of the reference sequence, respectively, preferably 0.2-6 times the activity of the peptide or fragment of the reference sequence, respectively, more preferably 0.3-5 times the activity of the peptide or fragment of the reference sequence, respectively. A substitution variant peptide or substitution variant amino acid sequence differs (i.e., differs) from a reference peptide or reference amino acid sequence by one or more amino acid substitutions in the reference amino acid sequence; a deletion variant peptide or deletion variant amino acid sequence differs (i.e., differs) from a reference peptide or reference amino acid sequence by the deletion of one or more amino acids in the reference amino acid sequence; and the addition of a variant peptide or addition of a variant amino acid sequence differs (i.e., differs) from the reference peptide or reference amino acid sequence by the addition of one or more amino acids in the reference amino acid sequence. A variant peptide or variant amino acid sequence can be the result of a substitution of one or more amino acids in the reference sequence (e.g., at least 1,2, 3, 4, 5, 6, 7, or 8 amino acid substitutions), or can be the result of a deletion of one or more amino acids in the reference sequence (e.g., at least 1,2, 3, 4, 5, 6, 7, or 8 amino acid deletions), or can be the result of an addition of one or more amino acids in the reference sequence (e.g., at least 1,2, 3, 4, 5, 6, 7, or 8 amino acid additions), or a combination thereof in any order. The peptide fragment of the substitution variant 4-23 amino acids or the fragment sequence of the substitution variant 4-23 amino acids may be different (i.e., different) from the peptide fragment of the reference 4-23 amino acids or the fragment sequence of the reference 4-23 amino acids by substitution of one or more amino acids in the reference amino acid fragment sequence; deletion variants 4-23 amino acid peptide fragments or 4-22 amino acid deletion variants amino acid fragment sequences can be different (i.e., different) from 5-23 amino acid reference peptide fragments or 5-23 amino acid reference amino acid fragment sequences by deletion of one or more amino acids in the reference amino acid fragment sequence; and the 4-23 amino acid addition variant peptide or the 4-23 amino acid addition variant amino acid sequence may differ (i.e., differ) from the 4-22 amino acid reference peptide sequence or the 4-22 amino acid reference amino acid sequence by the addition of one or more amino acids in the reference amino acid sequence. A 4-23 amino acid variant peptide or a 4-23 amino acid variant amino acid sequence can be the result of substitution of one or more amino acids (e.g., at least 1,2, 3, 4, 5, 6, 7, 8 amino acid substitutions) in a 4-23 amino acid fragment of the reference amino acid sequence, or can be the result of deletion of one or more amino acids in a relatively larger reference amino acid sequence (e.g., at least 1,2, 3, 4, 5, 6, 7, or 8 amino acid deletions), or can be the result of addition of one or more amino acids in a relatively smaller reference amino acid sequence (e.g., at least 1,2, 3, 4, 5, 6, 7, or 8 amino acid additions), or a combination thereof in any order. Preferably, the variant peptide or amino acid sequence differs from the reference peptide or fragment of the reference peptide or reference amino acid sequence or fragment of the reference amino acid sequence, respectively, by: less than 10 amino acid substitutions, deletions and/or additions; more preferably less than 8 amino acid substitutions, deletions and/or additions; more preferably less than 6 amino acid substitutions, deletions and/or additions; more preferably less than 5 amino acid substitutions, deletions and/or additions; more preferably less than 4 amino acid substitutions, deletions and/or additions. Most preferably the variant amino acid sequence differs from the reference peptide or fragment amino acid sequence by 1 or 2 or 3 amino acids.
By "sequence identity" is meant, with respect to the amino acid sequences of two peptides, the number of positions having the same amino acid divided by the number of amino acids in the shorter of the two sequences.
By "substantial identity" is meant that the amino acid sequence of the peptide or peptide fragment has at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and most preferably at least 95% sequence identity with respect to a comparison of the amino acid sequences of the two peptides or a comparison of the amino acid sequences of the two peptide fragments (e.g., a fragment of a reference peptide amino acid sequence).
The term "peptide" as used herein includes peptides and pharmaceutically acceptable salts thereof.
An "isolated" peptide herein means a naturally occurring peptide that has been separated or substantially separated from its naturally associated cellular components (e.g., nucleic acids and other peptides) by purification, recombination or chemical synthesis, and also includes non-naturally occurring recombinant or chemically synthesized peptides that have been purified or substantially purified from cellular components, biological substances, chemical precursors or other chemical substances.
The following three-letter and one-letter abbreviations for amino acids are used throughout: alanine: (Ala) A; arginine: (Arg) R; asparagine (b): (Asn) N; aspartic acid: (Asp) D; cysteine: (Cys) C; glutamine (b): (Gln) Q; glutamic acid: (Glu) E; glycine: (Gly) G; histidine: (His) H; isoleucine: (Ile) I; leucine: (Leu) L; lysine: (Lys) K; methionine: (Met) M; phenylalanine: (Phe) F; proline: (Pro) P; serine: (Ser) S; threonine: (Thr) T; tryptophan: (Trp) W; tyrosine: (Tyr) Y; valine: (Val) V. Other amino acid three letter symbols used herein include, in parentheses, (Hyp) for hydroxyproline, (Nle) for norleucine, (Orn) for ornithine, (Pyr) for pyroglutamic acid, and (Sar) for sarcosine. Typically, the amino terminus (or N-terminus) of a peptide occurs at the left end of the amino acid sequence of the indicated peptide, while the carboxy terminus (or C-terminus) occurs at the right end of the amino acid sequence. The amino acid sequence of a peptide can be written in one letter notation to represent the amino acids in the peptide covalently linked through a peptide amide bond.
Active fragments of MANS peptides are useful for preventing or reducing the amount of inflammation caused by inflammatory mediators in animal tissues. Active fragments of MANS peptides are also useful for preventing or reducing the amount of tissue loss produced or caused by inflammatory mediators in animals. An active fragment of MANS peptide consists of at least 4 contiguous amino acids and NO more than 23 contiguous amino acids of MANS peptide (SEQ ID NO: 1). The term "active fragment" is intended to encompass within the invention those fragments of MANS peptide which are capable of preventing or reducing the release of inflammatory mediators from inflammatory cells. The active fragment may reduce the release of inflammatory mediators by at least 5% to at least 99% compared to a reference peptide, such as MANS peptide.
Table 1 is a list of amino acid sequences in the single letter shorthand form along with the corresponding peptide numbering and SEQ ID NO. The reference peptide amino acid sequence (MANS peptide) is peptide 1. Peptides 2 to 231 are amino acid sequences of the peptides of the invention having amino acid sequences of 4to 23 consecutive amino acids of the reference amino acid sequence, and furthermore peptide 232 is an amino acid sequence comprising the random N-terminal sequence (RNS) of the amino acids of the MANS peptide. Representative variant amino acid sequences of the peptides of the invention described herein are peptides 233 to 245 and 247 to 251. The listing of such variant peptides is not intended as a limiting set of peptides, but is given merely as representative examples of variant peptides of the present invention. Also given are a representative reverse amino acid sequence (peptide 246) and a representative random amino acid sequence (peptide 232) for the peptides of the invention. The inverted and random amino acid sequences in the tables are not representative of the present invention.
Table 1 gives a list of peptides of the invention and their corresponding amino acid sequences and the corresponding SEQ ID NOs.
Table 1: peptide and amino acid sequences
Peptide numbering Sequence of Serial number
Peptide 1 GAQFSKTAAKGEAAAERPGEAAVA SEQ ID NO:1
Peptide 2 GAQFSKTAAKGEAAAERPGEAAV SEQ ID NO:2
Peptide 3 AQFSKTAAKGEAAAERPGEAAVA SEQ ID NO:3
Peptide 4 GAQFSKTAAKGEAAAERPGEAA SEQ ID NO:4
Peptide 5 AQFSKTAAKGEAAAERPGEAAV SEQ ID NO:5
Peptide 6 QFSKTAAKGEAAAERPGEAAVA SEQ ID NO:6
Peptide 7 GAQFSKTAAKGEAAAERPGEA SEQ ID NO:7
Peptide 8 AQFSKTAAKGEAAAERPGEAA SEQ ID NO:8
Peptide 9 QFSKTAAKGEAAAERPGEAAV SEQ ID NO:9
Peptide 10 FSKTAAKGEAAAERPGEAAVA SEQ ID NO:10
Peptide 11 GAQFSKTAAKGEAAAERPGE SEQ ID NO:11
Peptide 12 AQFSKTAAKGEAAAERPGEA SEQ ID NO:12
Peptide 13 QFSKTAAKGEAAAERPGEAA SEQ ID NO:13
Peptide 14 FSKTAAKGEAAAERPGEAAV SEQ ID NO:14
Peptide 15 SKTAAKGEAAAERPGEAAVA SEQ ID NO:15
Peptide 16 GAQFSKTAAKGEAAAERPG SEQ ID NO:16
Peptide 17 AQFSKTAAKGEAAAERPGE SEQ ID NO:17
Peptide 18 QFSKTAAKGEAAAERPGEA SEQ ID NO:18
Peptide 19 FSKTAAKGEAAAERPGEAA SEQ ID NO:19
Peptide 20 SKTAAKGEAAAERPGEAAV SEQ ID NO:20
Peptide 21 KTAAKGEAAAERPGEAAVA SEQ ID NO:21
Peptide 22 GAQFSKTAAKGEAAAERP SEQ ID NO:22
Peptide 23 AQFSKTAAKGEAAAERPG SEQ ID NO:23
Peptide 24 QFSKTAAKGEAAAERPGE SEQ ID NO:24
Peptide 25 FSKTAAKGEAAAERPGEA SEQ ID NO:25
Peptide 26 SKTAAKGEAAAERPGEAA SEQ ID NO:26
Peptide 27 KTAAKGEAAAERPGEAAV SEQ ID NO:27
Peptide 28 TAAKGEAAAERPGEAAVA SEQ ID NO:28
Peptide 29 GAQFSKTAAKGEAAAER SEQ ID NO:29
Peptide 30 AQFSKTAAKGEAAAERP SEQ ID NO:30
Peptide 31 QFSKTAAKGEAAAERPG SEQ ID NO:31
Peptide 32 FSKTAAKGEAAAERPGE SEQ ID NO:32
Peptide 33 SKTAAKGEAAAERPGEA SEQ ID NO:33
Peptide 34 KTAAKGEAAAERPGEAA SEQ ID NO:34
Peptide 35 TAAKGEAAAERPGEAAV SEQ ID NO:35
Peptide 36 AAKGEAAAERPGEAAVA SEQ ID NO:36
Peptide 37 GAQFSKTAAKGEAAAE SEQ ID NO:37
Peptide 38 AQFSKTAAKGEAAAER SEQ ID NO:38
Peptide 39 QFSKTAAKGEAAAERP SEQ ID NO:39
Peptide 40 FSKTAAKGEAAAERPG SEQ ID NO:40
Peptide 41 SKTAAKGEAAAERPGE SEQ ID NO:41
Peptide 42 KTAAKGEAAAERPGEA SEQ ID NO:42
Peptide 43 TAAKGEAAAERPGEAA SEQ ID NO:43
Peptide 44 AAKGEAAAERPGEAAV SEQ ID NO:44
Peptide 45 AKGEAAAERPGEAAVA SEQ ID NO:45
Peptide 46 GAQFSKTAAKGEAAA SEQ ID NO:46
Peptide 47 AQFSKTAAKGEAAAE SEQ ID NO:47
Peptide 48 QFSKTAAKGEAAAER SEQ ID NO:48
Peptide 49 FSKTAAKGEAAAERP SEQ ID NO:49
Peptide 50 SKTAAKGEAAAERPG SEQ ID NO:50
Peptide 51 KTAAKGEAAAERPGE SEQ ID NO:51
Peptide 52 TAAKGEAAAERPGEA SEQ ID NO:52
Peptide 53 AAKGEAAAERPGEAA SEQ ID NO:53
Peptide 54 AKGEAAAERPGEAAV SEQ ID NO:54
Peptide 55 KGEAAAERPGEAAVA SEQ ID NO:55
Peptide 56 GAQFSKTAAKGEAA SEQ ID NO:56
Peptide 57 AQFSKTAAKGEAAA SEQ ID NO:57
Peptide 58 QFSKTAAKGEAAAE SEQ ID NO:58
Peptide 59 FSKTAAKGEAAAER SEQ ID NO:59
Peptide 60 SKTAAKGEAAAERP SEQ ID NO:60
Peptide 61 KTAAKGEAAAERPG SEQ ID NO:61
Peptide 62 TAAKGEAAAERPGE SEQ ID NO:62
Peptide 63 AAKGEAAAERPGEA SEQ ID NO:63
Peptide 64 AKGEAAAERPGEAA SEQ ID NO:64
Peptide 65 KGEAAAERPGEAAV SEQ ID NO:65
Peptide 66 GEAAAERPGEAAVA SEQ ID NO:66
Peptide 67 GAQFSKTAAKGEA SEQ ID NO:67
Peptide 68 AQFSKTAAKGEAA SEQ ID NO:68
Peptide 69 QFSKTAAKGEAAA SEQ ID NO:69
Peptide 70 FSKTAAKGEAAAE SEQ ID NO:70
Peptide 71 SKTAAKGEAAAER SEQ ID NO:71
Peptide 72 KTAAKGEAAAERP SEQ ID NO:72
Peptide 73 TAAKGEAAAERPG SEQ ID NO:73
Peptide 74 AAKGEAAAERPGE SEQ ID NO:74
Peptide 75 AKGEAAAERPGEA SEQ ID NO:75
Peptide 76 KGEAAAERPGEAA SEQ ID NO:76
Peptide 77 GEAAAERPGEAAV SEQ ID NO:77
Peptide 78 EAAAERPGEAAVA SEQ ID NO:78
Peptide 79 GAQFSKTAAKGE SEQ ID NO:79
Peptide 80 AQFSKTAAKGEA SEQ ID NO:80
Peptide 81 QFSKTAAKGEAA SEQ ID NO:81
Peptide 82 FSKTAAKGEAAA SEQ ID NO:82
Peptide 83 SKTAAKGEAAAE SEQ ID NO:83
Peptide 84 KTAAKGEAAAER SEQ ID NO:84
Peptide 85 TAAKGEAAAERP SEQ ID NO:85
Peptide 86 AAKGEAAAERPG SEQ ID NO:86
Peptide 87 AKGEAAAERPGE SEQ ID NO:87
Peptide 88 KGEAAAERPGEA SEQ ID NO:88
Peptide 89 GEAAAERPGEAA SEQ ID NO:89
Peptide 90 EAAAERPGEAAV SEQ ID NO:90
Peptide 91 AAAERPGEAAVA SEQ ID NO:91
Peptide 92 GAQFSKTAAKG SEQ ID NO:92
Peptide 93 AQFSKTAAKGE SEQ ID NO:93
Peptide 94 QFSKTAAKGEA SEQ ID NO:94
Peptide 95 FSKTAAKGEAA SEQ ID NO:95
Peptide 96 SKTAAKGEAAA SEQ ID NO:96
Peptide 97 KTAAKGEAAAE SEQ ID NO:97
Peptide 98 TAAKGEAAAER SEQ ID NO:98
Peptide 99 AAKGEAAAERP SEQ ID NO:99
Peptide 100 AKGEAAAERPG SEQ ID NO:100
Peptide 101 KGEAAAERPGE SEQ ID NO:101
Peptide 102 GEAAAERPGEA SEQ ID NO:102
Peptide 103 EAAAERPGEAA SEQ ID NO:103
Peptide 104 AAAERPGEAAV SEQ ID NO:104
Peptide 105 AAERPGEAAVA SEQ ID NO:105
Peptide 106 GAQFSKTAAK SEQ ID NO:106
Peptide 107 AQFSKTAAKG SEQ ID NO:107
Peptide 108 QFSKTAAKGE SEQ ID NO:108
Peptide 109 FSKTAAKGEA SEQ ID NO:109
Peptide 110 SKTAAKGEAA SEQ ID NO:110
Peptide 111 KTAAKGEAAA SEQ ID NO:111
Peptide 112 TAAKGEAAAE SEQ ID NO:112
Peptide 113 AAKGEAAAER SEQ ID NO:113
Peptide 114 AKGEAAAERP SEQ ID NO:114
Peptide 115 KGEAAAERPG SEQ ID NO:115
Peptide 116 GEAAAERPGE SEQ ID NO:116
Peptide 117 EAAAERPGEA SEQ ID NO:117
Peptide 118 AAAERPGEAA SEQ ID NO:118
Peptide 119 AAERPGEAAV SEQ ID NO:119
Peptide 120 AERPGEAAVA SEQ ID NO:120
Peptide 121 GAQFSKTAA SEQ ID NO:121
Peptide 122 AQFSKTAAK SEQ ID NO:122
Peptide 123 QFSKTAAKG SEQ ID NO:123
Peptide 124 FSKTAAKGE SEQ ID NO:124
Peptide 125 SKTAAKGEA SEQ ID NO:125
Peptide 126 KTAAKGEAA SEQ ID NO:126
Peptide 127 TAAKGEAAA SEQ ID NO:127
Peptide 128 AAKGEAAAE SEQ ID NO:128
Peptide 129 AKGEAAAER SEQ ID NO:129
Peptide 130 KGEAAAERP SEQ ID NO:130
Peptide 131 GEAAAERPG SEQ ID NO:131
Peptide 132 EAAAERPGE SEQ ID NO:132
Peptide 133 AAAERPGEA SEQ ID NO:133
Peptide 134 AAERPGEAA SEQ ID NO:134
Peptide 135 AERPGEAAV SEQ ID NO:135
Peptide 136 ERPGEAAVA SEQ ID NO:136
Peptide 137 GAQFSKTA SEQ ID NO:137
Peptide 138 AQFSKTAA SEQ ID NO:138
Peptide 139 QFSKTAAK SEQ ID NO:139
Peptide 140 FSKTAAKG SEQ ID NO:140
Peptide 141 SKTAAKGE SEQ ID NO:141
Peptide 142 KTAAKGEA SEQ ID NO:142
Peptide 143 TAAKGEAA SEQ ID NO:143
Peptide 144 AAKGEAAA SEQ ID NO:144
Peptide 145 AKGEAAAE SEQ ID NO:145
Peptide 146 KGEAAAER SEQ ID NO:146
Peptide 147 GEAAAERP SEQ ID NO:147
Peptide 148 EAAAERPG SEQ ID NO:148
Peptide 149 AAAERPGE SEQ ID NO:149
Peptide 150 AAERPGEA SEQ ID NO:150
Peptide 151 AERPGEAA SEQ ID NO:151
Peptide 152 ERPGEAAV SEQ ID NO:152
Peptide 153 RPGEAAVA SEQ ID NO:153
Peptide 154 GAQFSKT SEQ ID NO:154
Peptide 155 AQFSKTA SEQ ID NO:155
Peptide 156 QFSKTAA SEQ ID NO:156
Peptide 157 FSKTAAK SEQ ID NO:157
Peptide 158 SKTAAKG SEQ ID NO:158
Peptide 159 KTAAKGE SEQ ID NO:159
Peptide 160 TAAKGEA SEQ ID NO:160
Peptide 161 AAKGEAA SEQ ID NO:161
Peptide 162 AKGEAAA SEQ ID NO:162
Peptide 163 KGEAAAE SEQ ID NO:163
Peptide 164 GEAAAER SEQ ID NO:164
Peptide 165 EAAAERP SEQ ID NO:165
Peptide 166 AAAERPG SEQ ID NO:166
Peptide 167 AAERPGE SEQ ID NO:167
Peptide 168 AERPGEA SEQ ID NO:168
Peptide 169 ERPGEAA SEQ ID NO:169
Peptide 170 RPGEAAV SEQ ID NO:170
Peptide 171 PGEAAVA SEQ ID NO:171
Peptide 172 GAQFSK SEQ ID NO:172
Peptide 173 AQFSKT SEQ ID NO:173
Peptide 174 QFSKTA SEQ ID NO:174
Peptide 175 FSKTAA SEQ ID NO:175
Peptide 176 SKTAAK SEQ ID NO:176
Peptide 177 KTAAKG SEQ ID NO:177
Peptide 178 TAAKGE SEQ ID NO:178
Peptide 179 AAKGEA SEQ ID NO:179
Peptide 180 AKGEAA SEQ ID NO:180
Peptide 181 KGEAAA SEQ ID NO:181
Peptide 182 GEAAAE SEQ ID NO:182
Peptide 183 EAAAER SEQ ID NO:183
Peptide 184 AAAERP SEQ ID NO:184
Peptide 185 AAERPG SEQ ID NO:185
Peptide 186 AERPGE SEQ ID NO:186
Peptide 187 ERPGEA SEQ ID NO:187
Peptide 188 RPGEAA SEQ ID NO:188
Peptide 189 PGEAAV SEQ ID NO:189
Peptide 190 GEAAVA SEQ ID NO:190
Peptide 191 GAQFS SEQ ID NO:191
Peptide 192 AQFSK SEQ ID NO:192
Peptide 193 QFSKT SEQ ID NO:193
Peptide 194 FSKTA SEQ ID NO:194
Peptide 195 SKTAA SEQ ID NO:195
Peptide 196 KTAAK SEQ ID NO:196
Peptide 197 TAAKG SEQ ID NO:197
Peptide 198 AAKGE SEQ ID NO:198
Peptide 199 AKGEA SEQ ID NO:199
Peptide 200 KGEAA SEQ ID NO:200
Peptide 201 GEAAA SEQ ID NO:201
Peptide 202 EAAAE SEQ ID NO:202
Peptide 203 AAAER SEQ ID NO:203
Peptide 204 AAERP SEQ ID NO:204
Peptide 205 AERPG SEQ ID NO:205
Peptide 206 ERPGE SEQ ID NO:206
Peptide 207 RPGEA SEQ ID NO:207
Peptide 208 PGEAA SEQ ID NO:208
Peptide 209 GEAAV SEQ ID NO:209
Peptide 210 EAAVA SEQ ID NO:210
Peptide 211 GAQF SEQ ID NO:211
Peptide 212 AQFS SEQ ID NO:212
Peptide 213 QFSK SEQ ID NO:213
Peptide 214 FSKT SEQ ID NO:214
Peptide 215 SKTA SEQ ID NO:215
Peptide 216 KTAA SEQ ID NO:216
Peptide 217 TAAK SEQ ID NO:217
Peptide 218 AAKG SEQ ID NO:218
Peptide 219 AKGE SEQ ID NO:219
Peptide 220 KGEA SEQ ID NO:220
Peptide 221 GEAA SEQ ID NO:221
Peptide 222 EAAA SEQ ID NO:222
Peptide 223 AAAE SEQ ID NO:223
Peptide 224 AAER SEQ ID NO:224
Peptide 225 AERP SEQ ID NO:225
Peptide 226 ERPG SEQ ID NO:226
Peptide 227 RPGE SEQ ID NO:227
Peptide 228 PGEA SEQ ID NO:228
Peptide 229 GEAA SEQ ID NO:229
Peptide 230 EAAV SEQ ID NO:230
Peptide 231 AAVA SEQ ID NO:231
Peptide 232 GTAPAAEGAGAEVKRASAEAKQAF SEQ ID NO:232
Peptide 233 GKQFSKTAAKGE SEQ ID NO:233
Peptide 234 GAQFSKTKAKGE SEQ ID NO:234
Peptide 235 GKQFSKTKAKGE SEQ ID NO:235
Peptide 236 GAQASKTAAK SEQ ID NO:236
Peptide 237 GAQASKTAAKGE SEQ ID NO:237
Peptide 238 GAEFSKTAAKGE SEQ ID NO:238
Peptide 239 GAQFSKTAAAGE SEQ ID NO:239
Peptide 240 GAQFSKTAAKAE SEQ ID NO:240
Peptide 241 GAQFSKTAAKGA SEQ ID NO:241
Peptide 242 AAQFSKTAAK SEQ ID NO:242
Peptide 243 GAAFSKTAAK SEQ ID NO:243
Peptide 244 GAQFAKTAAK SEQ ID NO:244
Peptide 245 GAQFSATAAK SEQ ID NO:245
Peptide 246 KAATKSFQAG SEQ ID NO:246
Peptide 247 GAQFSKAAAK SEQ ID NO:247
Peptide 248 GAQFSKTAAA SEQ ID NO:248
Peptide 249 GAQFSATAAA SEQ ID NO:249
Peptide 250 GAQASKTA SEQ ID NO:250
Peptide 251 AAGE SEQ ID NO:251
Peptide 252 GKASQFAKTA SEQ ID NO:252
The amino acid sequences of the peptides listed in table 1 may be chemically modified. For example, if the amino acid sequence of the peptides listed in Table 1 is chemically modified at the N-terminal amine to form an amide compound with a carboxylic acid, the resulting peptide is sometimes referred to in the present invention as a combination in which the identifier of the carboxylic acid is prefixed by a hyphen to the peptide number. For example, in the case of peptide 79, the N-terminally myristoylated peptide 79 may sometimes be referred to herein as "myristoylated peptide 79" or "myr-peptide 79"; the N-terminal acetylated peptide 79 may sometimes be referred to herein as an "acetyl-peptide 79" or "Ac-peptide 79". The cyclized form of peptide 79 may be referred to as "ring-peptide 79" or "cyc-peptide 79". Furthermore, for example if the amino acid sequence of the peptides listed in table 1 is chemically modified at the C-terminal carboxyl group, for example by an amine such as ammonia to form a C-terminal amide, the resulting peptide is sometimes indicated herein by a combination of hyphenated and peptide-numbered suffixes with an identifier of the amine residue. Thus, for example, the C-terminal amide of peptide 79 may sometimes be referred to as "peptide-NH 2". If the N-terminal amine of a peptide (e.g., peptide 79) is chemically modified by, for example, myristoyl and the C-terminal carboxyl group is chemically modified by, for example, an amino group to form the amide described above, the resulting peptide may sometimes be referred to as "myr-peptide 79-NH 2" using the prefix and suffix specifications.
The present invention relates to peptides having an amino acid sequence comprising less than 24 amino acids, wherein the amino acid sequence is related to the amino acid sequence of MANS peptide (i.e., MANS peptide is myristoyl-peptide 1and the reference 24 amino acid sequence of MANS peptide is peptide 1). The peptides of the invention consist of an amino acid sequence containing less than 24 amino acids, and may consist of 8-14, 10-12, 9-14, 9-13, 10-14, at least 9, at least 10, etc. amino acids. The peptides are typically linear, but may also be cyclic. Further, the peptide may be an isolated peptide.
For peptide 1(SEQ ID NO:1), a 24 amino acid reference sequence, a fragment of 23 contiguous amino acids of the reference amino acid sequence is sometimes referred to herein as a 23-mer. Similarly, a fragment of 22 contiguous amino acids of a reference sequence is sometimes referred to herein as a 22-mer; a sequence of 21 amino acids referred to as a 21-mer; a sequence of 20 amino acids referred to as a 20-mer; a19 amino acid sequence referred to as the 19-mer; a sequence of 18 amino acids referred to as 18-mer; a sequence of 17 amino acids referred to as a 17-mer; a sequence of 16 amino acids is called a 16-mer; a sequence of 15 amino acids is called a 15-mer; a sequence of 14 amino acids referred to as 14-mer; a sequence of 13 amino acids referred to as 13-mer; a sequence of 12 amino acids referred to as a 12-mer; a sequence of 11 amino acids is called 11-mer; a sequence of 10 amino acids is called a 10-mer; a sequence of 9 amino acids is called a 9-mer; a sequence of 8 amino acids is called an 8-mer; a 7 amino acid sequence is referred to as a 7-mer; a sequence of 6 amino acids is called a 6-mer; a sequence of 5 amino acids is referred to as a 5-mer; while a sequence of 4 amino acids is called a 4-mer. In one aspect, the amino acid sequence of these "4-to 23-mers" is itself a peptide (sometimes denoted herein as H)2N-peptide-COOH), any of which may independently be chemically modified, for example by chemical modification, wherein the chemical modification may be selected from the group consisting of: (i) at the N-terminal amino group (H)2N-peptide-) to form amides, such as with, for example, C1Or preferably with C2(acetic acid) to C22A carboxylic acid; (ii) formation of amides at the C-terminal carboxyl group (-peptide-COOH), such as with, for example, ammonia or with C1To C22Primary or secondary amines of (a); and (iii) combinations thereof.
The peptide has an amino acid sequence selected from the group consisting of: (a) 4to 23 consecutive with the reference sequence (peptide 1)An amino acid sequence of amino acids; (b) a sequence substantially similar to the amino acid sequence defined in (a); and (c) a variant of the amino acid sequence defined in (a) selected from the group consisting of: substitution variants, deletion variants, addition variants, and combinations thereof. In some embodiments, the peptide has an amino acid sequence selected from the group consisting of: (a) an amino acid sequence of 8 to 14 contiguous amino acids having the reference sequence (peptide 1); (b) an amino acid sequence substantially identical to the sequence defined in (a); and (c) a variant of the amino acid sequence defined in (a) selected from the group consisting of a substitution variant, a deletion variant, an addition variant, and combinations thereof. In other embodiments, the peptide has an amino acid sequence selected from the group consisting of: (a) an amino acid sequence of 10 to 12 contiguous amino acids having the reference sequence (peptide 1); (b) an amino acid sequence substantially identical to the sequence defined in (a); and (c) a variant of the amino acid sequence defined in (a) selected from the group consisting of a substitution variant, a deletion variant, an addition variant, and combinations thereof. In further embodiments, the peptide has an amino acid sequence having at least 9, at least 10, 9-14, 9-13, 10-14, etc. consecutive amino acids of the reference sequence (peptide 1); an amino acid sequence substantially identical thereto; or a variant thereof selected from the group consisting of: substitution variants, deletion variants, addition variants, and combinations thereof. As further explained below, one or more amino acids (e.g., the N-terminal and/or C-terminal amino acids) of the peptide optionally can be independently chemically modified; in some embodiments, one or more amino acids of a peptide may be chemically modified, while in other embodiments none of the amino acids of the peptide are modified. In one aspect, preferred modifications may occur at the amine group (H) of the N-terminal amino acid of the peptide or peptide fragment2N-) (the amine group is assumed to form a peptide amide bond if it is located inside the peptide sequence rather than at the N-terminal position). In another aspect, preferred modifications may occur at the carboxyl (-COOH) group of the C-terminal amino acid of the peptide or peptide fragment (this carboxyl group would form a peptide amide bond if located internally within the peptide sequence rather than at the C-terminal position). In another aspect, preferred modifications may occur simultaneously at the N-terminal amine group (H)2N-) and a C-terminal carboxyl group (-COOH).
In some embodiments, the amino acid sequence of the peptide begins with the N-terminal amino acid of reference sequence peptide 1. For example, the peptide may have an amino acid sequence selected from the group consisting of: (a) an amino acid sequence having 4to 23 consecutive amino acids of reference sequence peptide 1, wherein the amino acid sequence is initiated from the N-terminal amino acid of the reference sequence (i.e., peptide 2, peptide 4, peptide 7, peptide 11, peptide 16, peptide 22, peptide 29, peptide 37, peptide 46, peptide 56, peptide 67, peptide 79, peptide 92, peptide 106, peptide 121, peptide 137, peptide 154, peptide 172, peptide 191, or peptide 211); (b) a sequence substantially similar to the amino acid sequence defined in (a); and (c) a variant of the amino acid sequence defined in (a). These peptides do not contain a chemical moiety or the chemical moiety on their N-terminal glycine is not myristoyl. Preferably, the chemical moiety is an acyl group in the form of an amide bond, such as acetyl, or an alkyl group.
In other embodiments, the amino acid sequence of the peptide terminates at the C-terminal amino acid of reference sequence peptide 1. For example, a peptide can have an amino acid sequence selected from the group consisting of (a) an amino acid sequence of 4to 23 consecutive amino acids of reference sequence peptide 1, wherein the amino acid sequence terminates at the C-terminal amino acid of the reference sequence (i.e., peptide 3, peptide 6, peptide 10, peptide 15, peptide 21, peptide 28, peptide 36, peptide 45, peptide 55, peptide 66, peptide 78, peptide 91, peptide 105, peptide 120, peptide 136, peptide 153, peptide 171, peptide 190, peptide 210, or peptide 231); (b) a sequence substantially similar to the amino acid sequence defined in (a); and (c) a variant of the amino acid sequence defined in (a).
In other embodiments, the amino acid sequence of the peptide is not the N-terminal amino acid from the reference sequence peptide 1(SEQ ID NO:1), but from the amino acid at position 2 to the amino acid at position 21 of the reference sequence peptide 1. For example, the peptide may have an amino acid sequence selected from the group consisting of (a) an amino acid sequence of 4to 23 consecutive amino acids having the reference sequence peptide 1, wherein the amino acid sequence starts from any amino acid between position 2 to position 21 of the reference sequence. These peptides may be between 4 and 23 consecutive amino acids in length and may represent peptides in the middle of the reference sequence peptide 1; (b) a sequence substantially similar to the amino acid sequence defined in (a); and (c) a variant of the amino acid sequence defined in (a). These peptides are disclosed in table 1 or 2. These peptides may contain NO covalently bound chemical moiety, or a chemical moiety at the N-terminal amino acid which is not the N-terminal glycine from or equivalent to the amino acid sequence of SEQ ID NO. 1. Preferably, the chemical moiety is an acyl group, such as acetyl or myristoyl, in the form of an amide bond, or an alkyl group.
Peptide amino acid sequences useful in the present invention to inhibit mucin hypersecretion in a mammal, and to reduce mucin hypersecretion in a mammal, in methods of inhibiting mucin hypersecretion, and in methods of reducing mucin hypersecretion, the peptide amino acid sequence comprising the amino acid sequence of an isolated peptide of the present invention and optionally the amino acid sequence of a peptide containing an N-terminal and/or C-terminal chemically modified group, the peptide amino acid sequence being selected from the group consisting of: 23-mer (i.e. a peptide having a sequence of 23 amino acids): a peptide 2; and peptide 3; 22-mer (i.e. a peptide having a sequence of 22 amino acids): peptide 4; peptide 5; and peptide 6; 21-mer (i.e. a peptide having a sequence of 21 amino acids): peptide 7; a peptide 8; peptide 9; and a peptide 10; 20-mer (i.e. a peptide having a sequence of 20 amino acids): a peptide 11; a peptide 12; a peptide 13; a peptide 14; and peptide 15; 19-mer (i.e. a peptide having a sequence of 19 amino acids): a peptide 16; peptide 17; peptide 18; peptide 19; a peptide 20; and peptide 21; 18-mer (i.e. a peptide having a sequence of 18 amino acids): a peptide 22; peptide 23; a peptide 25; a peptide 26; a peptide 27; and peptide 28; 17-mer (i.e. a peptide having a sequence of 17 amino acids): a peptide 29; a peptide 30; a peptide 31; a peptide 32; a peptide 33; a peptide 34; a peptide 35; and peptide 36; 16-mer (i.e. a peptide having a sequence of 16 amino acids): peptide 37; a peptide 38; a peptide 39; a peptide 40; peptide 41; a peptide 42; a peptide 43; a peptide 44; and peptide 45; 15-mer (i.e. a peptide having a sequence of 15 amino acids): a peptide 46; peptide 47; peptide 48; peptide 49; a peptide 50; a peptide 51; a peptide 52; a peptide 53; a peptide 54; and peptide 55; 14-mer (i.e. a peptide having a sequence of 14 amino acids): peptide 56; a peptide 57; a peptide 58; peptide 59; a peptide 60; a peptide 61; a peptide 62; a peptide 63; peptide 64; peptide 65; and a peptide 66; 13-mer (i.e. a peptide having a sequence of 13 amino acids): peptide 67; peptide 68; a peptide 69; a peptide 70; peptide 71; a peptide 72; peptide 73; peptide 74; a peptide 75; peptide 76; peptide 77; and peptide 78; 12-mer (i.e. a peptide having a sequence of 12 amino acids): a peptide 79; a peptide 80; a peptide 81; a peptide 82; a peptide 83; a peptide 84; a peptide 85; a peptide 86; a peptide 87; a peptide 88; peptide 89; a peptide 90; and peptide 91; 11-mer (i.e. a peptide having a sequence of 11 amino acids): a peptide 92; a peptide 93; peptide 94; a peptide 95; a peptide 96; peptide 97; a peptide 98; a peptide 99; a peptide 100; a peptide 101; a peptide 102; a peptide 103; a peptide 104; and a peptide 105; 10-mer (i.e. peptide having a sequence of 10 amino acids): a peptide 106; a peptide 107; a peptide 108; a peptide 109; a peptide 110; a peptide 111; a peptide 112; a peptide 113; a peptide 114; a peptide 115; a peptide 116; peptide 117; a peptide 118; peptide 119; and a peptide 120; 9-mer (i.e. a peptide having a sequence of 9 amino acids): a peptide 121; a peptide 122; peptide 123; a peptide 124; a peptide 125; a peptide 126; a peptide 127; a peptide 128; a peptide 129; a peptide 130; a peptide 131; a peptide 132; a peptide 133; a peptide 134; a peptide 135; and peptide 136; 8-mer (i.e. a peptide having a sequence of 8 amino acids): peptide 137; a peptide 138; a peptide 139; a peptide 140; peptide 141; a peptide 142; a peptide 143; a peptide 144; a peptide 145; peptide 146; a peptide 147; a peptide 148; peptide 149; a peptide 150; a peptide 151; a peptide 152; and peptide 153; 7-mer (i.e. a peptide having a sequence of 7 amino acids): peptide 154; a peptide 155; a peptide 156; a peptide 157; a peptide 158; a peptide 159; a peptide 160; a peptide 161; a peptide 162; a peptide 163; a peptide 164; a peptide 165; a peptide 166; a peptide 167; peptide 168; a peptide 169; a peptide 170; and peptide 171; 6-mer (i.e. a peptide having a sequence of 6 amino acids): a peptide 172; peptide 173; a peptide 174; a peptide 175; a peptide 176; (ii) peptide 177; a peptide 178; 179 a peptide; a peptide 180; a peptide 181; a peptide 182; a peptide 183; a peptide 184; a peptide 185; a peptide 186; a peptide 187; a peptide 188; a peptide 189; and peptide 190; 5-mer (i.e. peptide having a sequence of 5 amino acids): a peptide 191; a peptide 192; a peptide 193; a peptide 194; a peptide 195; peptide 196; a peptide 197; a peptide 198; peptide 199; a peptide 200; a peptide 201; a peptide 202; a peptide 203; a peptide 204; a peptide 205; a peptide 206; a peptide 207; a peptide 208; peptide 209; and peptide 210; and 4-mers (i.e., peptides having a sequence of 4 amino acids): a peptide 211; a peptide 212; a peptide 213; a peptide 214; a peptide 215; a peptide 216; a peptide 217; a peptide 218; a peptide 219; a peptide 220; a peptide 221; a peptide 222; a peptide 223; a peptide 224; a peptide 225; a peptide 226; peptide 227; a peptide 228; peptide 229; a peptide 230; and a peptide 231.
Preferred amino acid sequences of the isolated peptides and N-terminally-and/or C-terminally-chemically modified peptides of the invention are selected from the group consisting of: the 23-mer: a peptide 2; and peptide 3; 22-mer: peptide 4; peptide 5; and peptide 6; 21-mer: peptide 7; a peptide 8; peptide 9; and a peptide 10; 20-mer: a peptide 11; a peptide 12; a peptide 13; a peptide 14; and peptide 15; 19-mer: a peptide 16; peptide 17; peptide 18; peptide 19; a peptide 20; and peptide 21; 18-mer: a peptide 22; peptide 23; a peptide 24; a peptide 25; a peptide 26; a peptide 27; and peptide 28; 17-mer: a peptide 29; a peptide 30; a peptide 31; a peptide 32; a peptide 33; a peptide 34; a peptide 35; and peptide 36; 16-mer: peptide 37; a peptide 38; a peptide 39; a peptide 40; peptide 41; a peptide 42; a peptide 43; a peptide 44; and peptide 45; 15-mer: a peptide 46; peptide 47; peptide 48; peptide 49; a peptide 50; a peptide 51; a peptide 52; a peptide 53; and peptide 54; 14-mer: peptide 56; a peptide 57; a peptide 58; peptide 59; a peptide 60; a peptide 61; a peptide 62; a peptide 63; and peptide 64; 13-mer: peptide 67; peptide 68; a peptide 69; a peptide 70; peptide 71; a peptide 72; peptide 73; peptide 74; and peptide 75; 12-mer: a peptide 79; a peptide 80; a peptide 81; a peptide 82; a peptide 83; a peptide 84; a peptide 85; a peptide 86; and peptide 87; 11-mer: a peptide 92; a peptide 93; peptide 94; a peptide 95; a peptide 96; peptide 97; a peptide 98; a peptide 99; and peptide 100; 10-mer: a peptide 106; a peptide 107; a peptide 108; a peptide 109; a peptide 110; a peptide 111; a peptide 112; a peptide 113; and peptide 114; 9-mer: a peptide 122; peptide 123; a peptide 124; a peptide 125; a peptide 126; a peptide 127; a peptide 128; and peptide 129; 8-mer: a peptide 139; a peptide 140; peptide 141; a peptide 142; a peptide 143; a peptide 144; and peptide 145; a 7-mer: a peptide 157; a peptide 158; a peptide 159; a peptide 160; a peptide 161; and peptide 162; 6-mer: a peptide 176; (ii) peptide 177; a peptide 178; 179 a peptide; and a peptide 180; 5-mer: peptide 196; a peptide 197; a peptide 198; and peptide 199; and 4-mers: a peptide 217; and peptide 219.
More preferred amino acid sequences of the isolated peptides and the N-terminally and/or C-terminally chemically modified peptides of the invention are selected from the group consisting of: the 23-mer: a peptide 2; and peptide 3; 22-mer: peptide 4; peptide 5; and peptide 6; 21-mer: peptide 7; a peptide 8; peptide 9; and a peptide 10; 20-mer: a peptide 11; a peptide 12; a peptide 13; a peptide 14; and peptide 15; 19-mer: a peptide 16; peptide 17; peptide 18; peptide 19; a peptide 20; and peptide 21; 18-mer: a peptide 22; peptide 23; a peptide 24; a peptide 25; a peptide 26; a peptide 27; and peptide 28; 17-mer: a peptide 29; a peptide 30; a peptide 31; a peptide 32; a peptide 33; a peptide 34; a peptide 35; and peptide 36; 16-mer: peptide 37; a peptide 38; a peptide 39; a peptide 40; peptide 41; a peptide 42; a peptide 43; a peptide 44; and peptide 45; 15-mer: a peptide 46; peptide 47; peptide 48; peptide 49; a peptide 50; a peptide 51; a peptide 52; a peptide 53; and peptide 54; 14-mer: peptide 56; a peptide 57; a peptide 58; peptide 59; a peptide 60; a peptide 61; a peptide 62; a peptide 63; and peptide 64; 13-mer: peptide 67; peptide 68; a peptide 69; a peptide 70; peptide 71; a peptide 72; peptide 73; peptide 74; a peptide 80; a peptide 81; a peptide 82; a peptide 83; a peptide 84; a peptide 85; a peptide 86; and peptide 87; 11-mer: a peptide 92; a peptide 93; peptide 94; a peptide 95; a peptide 96; peptide 97; a peptide 98; a peptide 99; and peptide 100; 10-mer: a peptide 106; a peptide 108; a peptide 109; a peptide 110; a peptide 111; a peptide 112; a peptide 113; and peptide 114; 9-mer: a peptide 124; a peptide 125; a peptide 126; a peptide 127; a peptide 128; and peptide 129; 8-mer: peptide 141; a peptide 142; a peptide 143; a peptide 144; and peptide 145; a 7-mer: a peptide 159; a peptide 160; a peptide 161; and peptide 162; 6-mer: a peptide 178; 179 a peptide; and a peptide 180; 5-mer: a peptide 198; and peptide 199; and 4-mers: peptide 219.
In other embodiments, the amino acid sequence of the peptide includes contiguous residues A, K, G, E, as in peptide 219 of reference sequence peptide 1. For example, the peptide may have an amino acid sequence selected from the group consisting of: (a) an amino acid sequence having 4to 23 contiguous amino acids of reference sequence peptide 1, wherein the amino acid sequence of the peptide includes contiguous residues A, K, G, and E as in peptide 219 of reference peptide 1 (e.g., peptide 219, peptide 45, peptide 79, peptide 67, peptide 80, etc.); (b) a sequence substantially similar to the amino acid sequence defined in (a); and (c) a variant of the amino acid sequence defined in (a).
Examples of peptide fragments comprising the amino acid sequence AKGE of the reference peptide amino acid sequence (peptide 1) include: (a) the 23-mer: a peptide 2; and peptide 3; 22-mer: peptide 4; peptide 5; and peptide 6; 11-mer: peptide 7; a peptide 8; peptide 9; and a peptide 10; 20-mer: a peptide 11; a peptide 12; a peptide 13; a peptide 14; and peptide 15; 19-mer: a peptide 16; peptide 17; peptide 18; peptide 19; a peptide 20; and peptide 21; 18-mer: a peptide 22; peptide 23; a peptide 24; a peptide 25; a peptide 26; a peptide 27; and peptide 28; 17-mer: a peptide 29; a peptide 30; a peptide 31; a peptide 32; a peptide 33; a peptide 34; a peptide 35; and peptide 36; 16-mer: peptide 37; a peptide 38; a peptide 39; a peptide 40; peptide 41; a peptide 42; a peptide 43; a peptide 44; and peptide 45; 15-mer: a peptide 46; peptide 47; peptide 48; peptide 49; a peptide 50; a peptide 51; a peptide 52; a peptide 53; and peptide 54; 14-mer: peptide 56; a peptide 57; a peptide 58; peptide 59; a peptide 60; a peptide 61; a peptide 62; a peptide 63; and peptide 64; 13-mer: peptide 67; peptide 68; a peptide 69; a peptide 70; peptide 71; a peptide 72; peptide 73; peptide 74; and peptide 75; 12-mer: a peptide 79; a peptide 80; a peptide 81; a peptide 82; a peptide 83; a peptide 84; a peptide 85; a peptide 86; and peptide 87; 11-mer: a peptide 93; peptide 94; a peptide 95; a peptide 96; peptide 97; a peptide 98; a peptide 99; and peptide 100; 10-mer: a peptide 108; a peptide 109; a peptide 110; a peptide 111; a peptide 112; a peptide 113; and peptide 114; 9-mer: a peptide 124; a peptide 125; a peptide 126; a peptide 127; a peptide 128; and peptide 129; 8-mer: peptide 141; a peptide 142; a peptide 143; a peptide 144; and peptide 145; a 7-mer: a peptide 159; a peptide 160; a peptide 161; and peptide 162; 6-mer: a peptide 178; 179 a peptide; and a peptide 180; 5-mer: a peptide 198; and peptide 199; and 4-mers: a peptide 219, (b) a sequence substantially similar to the amino acid sequence defined in (a); and (c) a variant of the amino acid sequence defined in (a) selected from the group consisting of a substitution variant, a deletion variant, an addition variant, and combinations thereof, wherein the fragment comprises or consists of 4to 23 consecutive amino acids.
In another embodiment, a preferred peptide sequence has an amino acid sequence selected from the group consisting of: (a) an amino acid sequence of 10 to 23 consecutive amino acids having the reference sequence (peptide 1); (b) a sequence substantially similar to the amino acid sequence defined in (a); and (c) a variant of the amino acid sequence defined in (a) selected from the group consisting of a substitution variant, a deletion variant, an addition variant, and combinations thereof, wherein a preferred amino acid sequence comprises the 23-mer: a peptide 2; 22-mer: peptide 4; 21-mer: peptide 7; 20-mer: a peptide 11; 19-mer: a peptide 16; 18-mer: a peptide 22; 17-mer: a peptide 29; 16-mer: peptide 37; 15-mer: a peptide 46; 14-mer: peptide 56; 13-mer: peptide 67; 12-mer: a peptide 79; 11-mer: a peptide 92; and 10-mers: peptide 106.
In further embodiments, the amino acid sequence of the peptide begins at the N-terminal amino acid of reference sequence peptide 1and includes contiguous residues A, K, G, and E as with peptide 219 of reference sequence peptide 1, while in other embodiments, the amino acid sequence of the peptide terminates at the C-terminal amino acid of reference sequence peptide 1and includes contiguous residues A, K, G, and E as with peptide 219 of reference sequence peptide 1.
The peptide may comprise one or more amino acid deletions, substitutions and/or additions relative to a reference amino acid sequence. Preferably, the substitution may be a conservative amino acid substitution, or the substitution may be a non-conservative amino acid substitution. In some embodiments, the peptides, including peptides having an amino acid sequence that is substantially identical to or a variant of a reference amino acid sequence, do not have deletions or additions as compared to the corresponding contiguous amino acids of the reference amino acid sequence, but may have conservative or non-conservative substitutions. Amino acid substitutions that can be made to a reference amino acid sequence in a peptide of the invention include, but are not limited to, the following substitutions: alanine (a) may be substituted with lysine (K), valine (V), leucine (L), or isoleucine (I); glutamic acid (E) may be substituted with aspartic acid (D); glycine (G) may be substituted with proline (P); lysine (K) may be substituted with arginine (R), glutamine (Q), or asparagine (N); phenylalanine (F) may be substituted with leucine (L), valine (V), isoleucine (I), or alanine (a); proline (P) may be substituted for glycine (G); glutamine (Q) may be substituted with glutamic acid (E) or asparagine (N); arginine (R) may be substituted with lysine (K), glutamine (Q), or asparagine (N); serine (S) may be substituted with threonine; threonine (T) may be substituted with serine (S); and valine (V) may be substituted for leucine (L), isoleucine (I), methionine (M), phenylalanine (F), alanine (a), or norleucine (Nle). For example, substitutions that may be made in a peptide of the invention to a reference amino acid sequence include: a substitution of phenylalanine (F) with alanine (a) (e.g., at amino acid position 4 of the reference amino acid sequence), a substitution of glutamine (Q) with glutamic acid (E) (e.g., at amino acid position 3 of the reference amino acid sequence), a substitution of alanine (a) with lysine (K) (e.g., at amino acid positions 2 and/or 8 of the reference amino acid sequence), and/or a substitution of threonine (T) with serine (S) (e.g., at amino acid position 7 of the reference amino acid sequence).
If substitutions are introduced in the amino acid sequence of a peptide of the invention (which peptide comprises unmodified peptide as well as chemically modified peptides, e.g. modified by N-terminal and/or C-terminal modification such as amide formation) relative to a reference amino acid sequence, it is preferred that the amino acid sequence of the peptide has at least 80% sequence identity to the reference amino acid sequence. Peptides having 5 to 23 amino acids and including one amino acid substitution relative to a reference amino acid sequence have about 80% to about 96% (i.e., -95.7%) sequence identity to the reference amino acid sequence. Peptides having 10 to 23 amino acids and including one amino acid substitution relative to a reference amino acid sequence have about 90% to about 96% (i.e., -95.7%) sequence identity to the reference amino acid sequence. Peptides having 20 to 23 amino acids and including one amino acid substitution relative to a reference amino acid sequence have about 95% to about 96% (i.e., -95.7%) sequence identity to the reference amino acid sequence. Peptides having 10 to 23 amino acids and including 2 amino acid substitutions relative to a reference amino acid sequence have about 80% to about 92% (i.e., -91.3%) sequence identity to the reference amino acid sequence. Peptides having 16 to 23 amino acids and including 2 amino acid substitutions relative to a reference amino acid sequence have about 87.5% to about 92% (i.e., -91.3%) sequence identity to the reference amino acid sequence. Peptides having 20 to 23 amino acids and including 2 amino acid substitutions relative to a reference amino acid sequence have about 90% to about 92% (i.e., -91.3%) sequence identity to the reference amino acid sequence. A peptide having 15 to 23 amino acids and comprising 3 amino acid substitutions relative to a reference amino acid sequence has about 80% to about 87% sequence identity to the reference amino acid sequence. A peptide having 20 to 23 amino acids and comprising 3 amino acid substitutions relative to a reference amino acid sequence has about 85% to about 87% sequence identity to the reference amino acid sequence. Peptides having 20 to 23 amino acids and including 4 amino acid substitutions relative to a reference amino acid sequence have about 80% to about 83% (i.e., -82.6%) sequence identity to the reference amino acid sequence.
In the peptide of the present invention, regarding the contiguous amino acid sequence of the reference peptide (which is a 24-mer), in a sequence of contiguous 23 amino acids (23-mer) selected from the reference sequence of 24 amino acids, substitution of one amino acid provides an amino acid sequence of the peptide having 95.65% (or 96%) sequence identity with the amino acid fragment having identity with the 23-mer in the reference peptide. Similarly, substitutions of 2,3, 4, and 5 amino acids in the 23-mer provide peptide amino acid sequences having 91.30% (or-91%), 86.96% (or-87%), 82.61% (or-83%), and 78.27% (or-78%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitutions of 1,2, 3, 4, and 5 amino acids in the 22-mer provide peptide amino acid sequences having 95.45% (or 95%), 90.91% (or 91%), 86.36% (or 86%), 81.82% (or 82%), and 77.27% (or 77%), respectively, sequence identity to the reference peptide amino acid sequence. Similarly, substitutions of 1,2, 3, 4, and 5 amino acids in the 21-mer provide amino acid sequences of peptides having 95.24% (-95%), 90.48 (-91%), 85.71% (-86%), 80.95 (-81%), and 76.19% (-76%), respectively, sequence identity to the reference peptide amino acid sequence. Similarly, substitutions of 1,2, 3, 4, and 5 amino acids in the 20-mer provide peptide amino acid sequences having 95.00% (95%), 90.00% (90%), 85.00% (85%), 80.00% (80%), and 75.00% (75%), respectively, sequence identity to the reference peptide amino acid sequence. Similarly, substitutions of 1,2, 3, and 4 amino acids in the 19-mer provided the amino acid sequence of the peptide with 94.74% (-95%), 89.47% (-89%), 84.21% (-84%), and 78.95% (-79%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitutions of 1,2, 3, and 4 amino acids in the 18-mer provide amino acid sequences of peptides having 94.44% (-94%), 88.89% (-89%), 83.33% (-83%), and 77.78% (-78%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitutions of 1,2, 3, and 4 amino acids in the 17-mer provided the amino acid sequence of the peptide with 94.12% (-94%), 88.23% (-88%), 82.35% (-82%), and 76.47% (-76%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitutions of 1,2, 3, and 4 amino acids in the 16-mer provide amino acid sequences of peptides having 93.75% (-94%), 87.50% (-88%), 81.25% (-81%), and 75.00% (75%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitutions of 1,2, and3 amino acids in the 15-mer provide peptide amino acid sequences having 93.33% (-93%), 86.67% (-87%), and 80.00% (80%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitutions of 1,2, and3 amino acids in the 14-mer provided the amino acid sequence of the peptide with 92.86% (-93%), 85.71% (-86%), and 78.57% (79%), respectively, sequence identity to the reference peptide amino acid sequence. Similarly, substitutions of 1,2, and3 amino acids in the 13-mer provide peptide amino acid sequences having 92.31% (-92%), 84.62% (-85%), and 76.92% (-77%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitutions of 1,2, and3 amino acids in the 12-mer provide peptide amino acid sequences having 91.67% (-92%), 83.33% (-83%), and 75.00% (75%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitutions of 1and 2 amino acids in the 11-mer provide peptide amino acid sequences having 90.91% (-91%) and 81.82% (-82%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitutions of 1and 2 amino acids in the 10-mer provide peptide amino acid sequences having 90.00% (90%) and 80.00% (80%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitutions of 1and 2 amino acids in the 9-mer provide peptide amino acid sequences having 88.89% (-89%) and 77.78% (-78%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitutions of 1and 2 amino acids in the 8-mer provide peptide amino acid sequences with 87.50% (-88%) and 75.00% (75%) sequence identity to the reference peptide amino acid sequence, respectively. Similarly, substitution of 1 amino acid in the 7-mer, 6-mer, 5-mer, and 4-mer provides a peptide amino acid sequence having 85.71% (-86%), 83.33% (-83.3%), 80.00% (80%), and 75.00% (75%), respectively, sequence identity to the reference peptide amino acid sequence. Preferred amino acid sequences of the invention have more than 80% sequence identity with an amino acid sequence in a reference sequence, more preferably 81% to 96% sequence identity with an amino acid sequence in a reference sequence, and more preferably 80% to 96% sequence identity with an amino acid sequence in a reference sequence. Preferred amino acid sequences optionally can be N-terminally chemically bonded at the terminal peptide amino group to a C2 to C22 linear aliphatic carboxylic acid moiety, more preferably a C2 to C16 linear aliphatic carboxylic acid moiety, more preferably a C2 or C16 linear aliphatic carboxylic acid moiety, and optionally can be C-terminally chemically bonded at the terminal peptide carboxyl group to an amine, such as ammonia or a primary or secondary amine, such as a C1 to C16 linear aliphatic primary amine, through an amide bond.
Peptide 79 is a 12-mer, examples of substituted variants thereof include, for example, peptide 238, wherein Q at position 3 in peptide 79 is substituted with E in sequence 238; peptide 233, wherein a at position 2 in peptide 79 is substituted with K in peptide 233; peptide 234, wherein a at position 8 in peptide 79 is substituted with K in peptide 234; peptide 235, wherein a at positions 2 and 8 in peptide 79 are substituted by K in peptide 235; peptide 237, wherein F at position 4 of peptide 79 is substituted with a in peptide 237; peptide 239 in which K at position 10 in peptide 79 is substituted with a in peptide 239; peptide 240, wherein the G at position 11 in peptide 79 is substituted with the a in peptide 240; and peptide 241, wherein E at position 12 in peptide 79 is substituted with a in peptide 241.
Peptide 106 is a 10-mer, examples of substituted variants thereof include, for example, peptide 236, wherein F at position 4 in peptide 106 is substituted with a in peptide 236; peptide 242, wherein the G at position 1 in peptide 106 is substituted with the a in peptide 242; peptide 243, where Q at position 3 in peptide 106 is substituted with a in peptide 243; peptide 244, wherein S at position 5 in peptide 106 is substituted with a in peptide 244; peptide 245, where the K at position 6 in peptide 106 is substituted with a in peptide 245; peptide 247, where T at position 7 in peptide 106 was replaced with a in peptide 247; peptide 248, wherein K at position 10 in peptide 106 is substituted with a in peptide 248; and peptide 249, wherein both K at positions 16 and 10 of peptide 106 are substituted with A in peptide 249.
Examples of substituted variants of 8-mer peptide 137 include, for example, peptide 250, wherein the F at position 4 in peptide 137 is substituted with an a in peptide 250.
Examples of substituted variants of 4-mer peptide 219 include, for example, peptide 251, wherein the K at position 2 in peptide 219 is substituted with a in peptide 251.
The substituted variant peptides described herein can be in the form of isolated peptides or chemically modified peptides, such as N-terminal amides, e.g., myristoyl amides, acetyl amides, and the like, as described herein, and, e.g., C-terminal amides, e.g., amides with ammonia, and, e.g., both N-terminal amides and C-terminal amides.
If a deletion is introduced in the amino acid sequence of a peptide of the invention relative to a reference amino acid sequence, it is preferred that the amino acid sequence of the peptide has at least 80% sequence identity to the reference amino acid sequence. Peptides having 5 to 23 amino acids and including one amino acid deletion relative to a reference peptide have 80% to about 96% (i.e., -95.7%) sequence identity to the reference amino acid sequence. Peptides having 10 to 23 amino acids and including one amino acid deletion relative to a reference peptide have about 90% to about 96% (i.e., -95.7%) sequence identity to the reference amino acid sequence. Peptides having 20 to 23 amino acids and including one amino acid deletion relative to a reference peptide have 95% to about 96% (i.e., -95.7%) sequence identity to the reference amino acid sequence. Peptides having 10 to 23 amino acids and including 2 amino acid deletions relative to a reference peptide have about 80% to about 92% (i.e., -91.3%) sequence identity to the reference amino acid sequence. Peptides having 16 to 23 amino acids and including 2 amino acid deletions relative to a reference peptide have about 87.5% to about 92% (i.e., -91.3%) sequence identity to the reference amino acid sequence. Peptides having 20 to 23 amino acids and including 2 amino acid deletions relative to a reference peptide have about 90% to about 92% (i.e., -91.3%) sequence identity to the reference amino acid sequence. A peptide having 15 to 23 amino acids and comprising a deletion of 3 amino acids relative to a reference peptide has about 80% to about 87% sequence identity to the reference amino acid sequence. Peptides having 20 to 23 amino acids and including 3 amino acid deletions relative to a reference peptide have about 85% to about 87% sequence identity to the reference amino acid sequence. Peptides having 20 to 23 amino acids and including 4 amino acid deletions relative to a reference peptide have about 80% to about 83% (i.e., -82.6%) sequence identity to the reference amino acid sequence.
As mentioned above, one or more amino acids of the peptide may also be chemically modified. The amino acid sequence of the peptide may be subjected to any amino acid modification known in the art using any method known in the art.
In some embodiments, the N-terminal and/or C-terminal amino acids may be modified. For example, the N-terminal alpha-amino acid of the peptide may be alkylated, amidated, or acylated at the alpha-N-terminal (N-terminal) amino group (. alpha. -H2N-), and for example, the C-terminal amino acid of the peptide may be amidated or esterified at the C-terminal carboxyl group (-COOH). For example, the N-terminal amino group may be modified by acylation to introduce any acyl or fatty acyl group to form an amide, including acetyl (i.e., CH 3-C (═ O) -or myristoyl, both of which are presently preferred groups). In some embodiments, the N-terminal amino group can be modified to introduce an acyl group of the formula-c (o) R, wherein R is a linear or branched alkyl group having 1 to 15 carbon atoms, or can be modified to introduce an acyl group of the formula-c (o) R1, wherein R1 is a linear alkyl group having 1 to 15 carbon atoms. The N-amide may also be a carboxamide (R ═ H). The C-terminal amino acid of the peptide may also be chemically modified. For example, the C-terminal carboxyl group of the C-terminal amino acid may be chemically modified (i.e., amidated) by conversion to a carboxamide (carboxamide group) in place of the carboxyl group. In some embodiments, the N-terminal and/or C-terminal amino acids are not chemically modified. In some embodiments, the N-terminal group is modified and the C-terminal group is not modified. In some embodiments, both the N-terminal and C-terminal groups are modified.
The peptide may be acylated at the amino group of the N-terminal amino acid to form an N-terminal amide with an acid selected from the group consisting of:
(i-a) C2 (acetyl) to C13 aliphatic (saturated or optionally unsaturated) carboxylic acids (e.g., N-terminal amides with acetic acid (which is the preferred group), propionic acid, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid), which may be linear, branched (greater than C3), or contain rings (greater than C3);
(i-b) a saturated C14 aliphatic carboxylic acid, which may be linear, branched, or contain rings;
(i-C) an unsaturated C14 aliphatic carboxylic acid which may be linear, branched, or contain a ring;
(i-d) C15 to C24 aliphatic (saturated or optionally unsaturated) carboxylic acids, which may be linear, branched, or contain rings (e.g., tetradecanoic acid (myristic acid, which is a preferred group), hexadecanoic acid, 9-hexadecenoic acid, octadecanoic acid, 9-octadecenoic acid, 11-octadecenoic acid, 9, 12-octadecadienoic acid, 9,12, 15-octadecatrienoic acid, 6,9, 12-octadecatrienoic acid, eicosanoic acid, 9-eicosenoic acid, 5,8,11, 14-eicosatetraenoic acid, 5,8,11,14, 17-eicosapentaenoic acid, docosanoic acid, 13-docosenoic acid, 4,7,10,13,16, 19-docosahexenoic acid, tetracosanoic acid, and the like);
(ii) trifluoroacetic acid;
(iii) benzoic acid; and
(iv-a) a C1 to C12 aliphatic alkylsulfonic acid which forms an aliphatic alkylsulfonamide in which the C1 to C12 aliphatic alkyl carbon chain of the sulfonic acid has a structure similar to the aliphatic alkyl carboxylic acid chain of the aliphatic alkyl carboxylic acid described above. For example, the peptide may be acylated with a carboxylic acid group denoted as (C1-C11) -alkyl-C (O) OH to form an amide denoted as (C1-C11) -alkyl-C (O) -NH-peptide by activating the carboxylic acid group for anhydro coupling. Similarly, sulfonamides can be formed by reacting a sulfonic acid species, represented as (C1-C12) -alkyl-S (O2) -X, for example, where X is halogen or OCH3 or other compatible leaving group, with the N-terminal amino group to form a sulfonamide represented by the (C1-C12) -alkyl-S (O2) -NH-peptide.
(iv-b) a C14 to C24 aliphatic alkylsulfonic acid which forms an aliphatic alkylsulfonamide in which the C14 to C24 aliphatic alkyl carbon chain of the sulfonic acid has a structure similar to the aliphatic alkyl carboxylic acid chain of the aliphatic alkyl carboxylic acid described above. For example, the peptide may be acylated with a carboxylic acid group denoted as (C13-C23) -alkyl-C (O) OH to form an amide denoted as (C13-C23) -alkyl-C (O) -NH-peptide by activating the carboxylic acid group for anhydro coupling. Similarly, sulfonamides can be formed by reacting a sulfonic acid species, represented as (C14-C24) -alkyl-S (O2) -X, for example, where X is halogen or OCH3 or other compatible leaving group, with the N-terminal amino group to form a sulfonamide represented by the (C14-C24) -alkyl-S (O2) -NH-peptide.
As another example, the N-terminal amino group of the N-terminal amino acid may be alkylated with a C1 to C12 aliphatic alkyl group, the structure of which is described above. Alkylation can be achieved, for example, by using aliphatic alkyl halides or aliphatic alkyl sulfonates (methanesulfonate, toluenesulfonate, etc.), preferably primary alkyl halides or primary alkyl sulfonates. The N-terminal amino acid may be modified at the terminal amino group to introduce any acyl or aliphatic acyl fatty acyl group as an amide, including acetyl (i.e., -C (O) CH3, which is a preferred group), myristoyl (which is a preferred group), butyryl, hexanoyl, octanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, 9-hexadecanoyl, octadecanoyl, 9-octadecenoyl, 11-octadecenoyl, 9, 12-octadecadienoyl, 9,12, 15-octadecatrienoyl, 6,9, 12-octadecatrienoyl, eicosanoyl, 9-eicosenoyl, 5,8,11, 14-eicosatetraenoyl, 5,8,11,14, 17-eicosapentaenoyl, docosanoyl, 13-docosahexenoyl, 4,7,10,13,16, 19-docosahexenoyl, tetracosanyl, covalently linked to the terminal amino group on the peptide by amide bonds.
The C-terminal carboxylic acid group of the C-terminal amino acid of the peptide of the invention may also be chemically modified. The C-terminal amino acid may be modified, for example, by reacting the C-terminal carboxylic acid group of the peptide with an amine to form an amide group, e.g., an amide of ammonia (which is the preferred group); amides of C1 to C12 aliphatic alkylamines, preferably linear aliphatic alkylamines; amides of hydroxy-substituted C2 to C12 aliphatic alkylamines; amides of linear 2- (C1 to C12 aliphatic alkyl) oxyethylamino; and amides of omega-methoxy-poly (ethyleneoxy) n-ethylamino (also known as omega-methoxy-PEG-alpha-amino or omega-methoxy- (polyethylene glycol) amino), where n is 0-10. The C-terminal carboxylic acid of the C-terminal amino acid of the peptide may be in the form of an ester selected from the group consisting of: esters of C1 to C12 aliphatic alkyl alcohols, esters of 2- (omega-methoxy-poly (ethyleneoxy) n) -ethanol groups, wherein n is 0-10. In one aspect, preferably the polyethylene glycol component, such as in PEG esters, MPEG esters, PEG amides, MPEG amides, and the like, has a molecular weight of about 500 to 40,000 daltons, more preferably 1000 to 25,000 daltons, and most preferably about 1000 to about 10,000 daltons.
The C-terminal carboxylic acid group of the peptide may be represented by the formula peptide-C (o) OH, which may also be amidated by conversion to an active form such as a carboxylic acid halide, carboxylic acid anhydride, N-hydroxysuccinimide ester, pentafluorophenyl (OPfp) ester, 3-hydroxy-2, 3-dihydro-4-oxo-benzo-triazinone (ODhbt) ester, or the like, to facilitate reaction with ammonia or a primary or secondary amine, preferably ammonia or a primary amine, and preferably any other active group in the peptide is also protected by synthetic chemically compatible protecting groups known in the art of peptide synthesis, especially synthetic chemically compatible protecting groups for solid phase synthesis of peptides such as benzyl ester, tert-butyl ester, phenyl ester, or the like. The peptide amide produced may be represented by the formula peptide-C (o) -NR3R4 (amine at the C-terminus of the peptide), wherein R3 and R4 are independently selected from: hydrogen; c1 to C12 alkyl groups such as methyl, ethyl, butyl, isobutyl, cyclopropylmethyl, hexyl, dodecyl; and optionally higher alkyl groups such as C14 to C24 alkyl groups, such as tetradecyl and the like as described above.
The C-terminal carboxylic acid of the C-terminal amino acid can also be converted to an amide of a hydroxy-substituted C2 to C12 aliphatic alkylamine (the hydroxy group being attached to a carbon atom of the amine rather than to the nitrogen atom of the amine), such as 2-hydroxyethylamine, 4-hydroxybutylamine, 12-hydroxydodecylamine, and the like.
The C-terminal carboxylic acids may also be converted to hydroxy-substituted amides of C2 to C12 aliphatic alkylamines, wherein the hydroxy group may be acylated to form esters with the C2 to C12 aliphatic carboxylic acids described above. Preferably, in the peptide, the C-terminal amide of the peptide is represented by the formula peptide-C (o) NR5R6, R5 is hydrogen, and R6 is selected from hydrogen, C1 to C12 alkyl, and hydroxy-substituted C1 to C12 alkyl.
The C-terminal carboxylic acid of the C-terminal amino acid can be converted to an amide of a linear 2- (C1 to C12 aliphatic alkyl) oxyethylamine. The amides can be prepared, for example, by reacting a linear C1 to C12 aliphatic alcohol with a hydride of potassium in diglyme containing 2-chloroethanol to provide a linear C1 to C12 aliphatic alkyl ethanol that can be converted to an amine by oxidation to an aldehyde followed by reductive amination to an amine (e.g., with ammonia), or by conversion to an alkyl halide (e.g., with thionyl chloride) followed by treatment with an amine such as ammonia.
The C-terminal carboxylic acid of the C-terminal amino acid may also be converted to an amide of a linear PEG-amine (e.g., omega-hydroxy-PEG-alpha-amine; omega- (C1 to C12) -PEG-alpha-amine, e.g., omega-methoxy-PEG-alpha-amine, i.e., MPEG-amine). In one aspect, preferably, the polyethylene glycol or PEG component has a molecular weight of about 500 to 40,000 daltons, more preferably 1000 to 25,000 daltons, and most preferably about 1000 to about 10,000 daltons.
The C-terminal carboxylic acid of the C-terminal amino acid may also be converted to the amide of omega-methoxy-poly (ethyleneoxy) n-ethylamine, where n is 0-10, which may be prepared from the corresponding omega-methoxy-poly (ethyleneoxy) n-ethanol, for example by converting the alcohol to an amine as described above.
In another embodiment, the C-terminal carboxyl group can be converted to an amide of the formula-C (O) -NR7R8, where R7 is hydrogen and R8 is a linear 2- (C1 to C12 aliphatic alkyl) oxyethyl group, where the C1 to C12 aliphatic alkyl moieties are as described above and include groups such as methoxyethyl (i.e., CH3O-CH2CH2-), 2-dodecyloxyethyl, and the like; or R7 is hydrogen, R8 is ω -methoxy-poly (ethylene oxide) n-ethyl, wherein n of the poly (ethylene oxide) moiety is 0-10, such as 2-methoxyethyl (i.e., CH3O-CH2CH2-), ω -methoxyethoxyethyl (i.e., CH3O-CH 2O-CH2CH2-) through CH3O- (CH 2O)10-CH2CH 2-.
The C-terminal carboxylic acid group of the C-terminal amino acid of the peptide may also be in the form of an ester of a C1 to C12 aliphatic alkyl alcohol, the aliphatic alkyl portion of the alcohol being as described above. The C-terminal carboxylic acid group of the C-terminal amino acid of the peptide may also be in the form of an ester of a 2- (ω -methoxy-poly (ethylene oxide) n) -ethanol group, where n is 0-10, which may be prepared from 2-methoxy alcohol as sodium2-methoxy dehydroacetate (sodium 2-methoxythonate) reacted with a stoichiometric amount (stoichiometric ammonium) of ethylene oxide, where the stoichiometric amount depends on the size of n.
The side chains of amino acids in peptides may also be chemically modified. For example, the phenyl group in phenylalanine or tyrosine may be replaced by a substituent selected from the group consisting of:
c1 to C24 aliphatic alkyl groups (i.e., linear or branched, and/or saturated or unsaturated, and/or containing cyclic groups), such as methyl (preferred), ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, 2-methylcyclopropyl, cyclohexyl, octyl, decyl, dodecyl, hexadecyl, octadecyl, eicosyl, docosyl, tetracosyl, 9-hexadecenyl, 9-octadecenyl, 11-octadecenyl, 9, 12-octadecadienyl, 9,12, 15-octadecatrienoyl, 6,9, 12-octadecatrienoyl, 9-eicosenyl, 5,8,11, 14-eicosatetraenyl, 5,8,11,14, 17-eicosapentaenyl, 13-docosadienyl, and 4,7,10,13,16, 19-docosahexaenyl;
a C1 to C12 aliphatic alkyl group substituted on at least one carbon atom other than the unsaturated position with a hydroxyl group, examples of the hydroxyalkyl group include hydroxymethyl, hydroxyethyl, hydroxydodecyl and the like;
a C1 to C12 alkyl substituted with a hydroxyl group esterified with a C2 to C25 aliphatic carboxyl group of an acid such as: acetic acid, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, 9-hexadecenoic acid, stearic acid, 9-octadecenoic acid, 11-octadecenoic acid, 9, 12-octadecadienoic acid, 9,12, 15-octadecatrienoic acid, 6,9, 12-octadecatrienoic acid, eicosanoic acid, 9-eicosenoic acid, 5,8,11, 14-eicosatetraenoic acid, 5,8,11,14, 17-eicosapentaenoic acid, docosanoic acid, 13-docosenoic acid, 4,7,10,13,16, 19-docosahexaenoic acid, tetracosanoic acid, etc.; dicarboxylic acids such as succinic acid; or hydroxy acids such as lactic acid, wherein the total number of carbon atoms of the ester substituents is from 3 to 25;
halogen such as fluoro-, chloro-, bromo-, and iodo-; nitro-;
amino-such as NH2, methylamino, dimethylamino; trifluoromethyl-;
a carboxyl group (-COOH);
c1 to C24 alkoxy (which may be formed, for example, by alkylation of tyrosine), for example methoxy, ethoxy, propoxy, isopropyloxy, butoxy, isobutyloxy, cyclopropoxy, 2-methoxycyclopropoxy, cyclohexyloxy, octyloxy, decyloxy, dodecyloxy, hexadecyloxy, octadecyloxy, eicosyloxy, docosyloxy, tetracosyloxy, 9-hexadecenyloxy, 9-octadecenyloxy, 11-octadecenyloxy, 9, 12-octadecadienyloxy, 9,12, 15-octadecatrienoyloxy, 6,9, 12-octadecatrienoyloxy, 9-eicosenyloxy, 5,8,11, 14-eicosatetraenyloxy, 5,8,11,14, 17-eicosapentaenyloxy, 13-docosahexenyloxy, and 4,7,10,13,16, 19-docosahexenyloxy;
c2 to C12 hydroxyalkoxy groups, such as 2-hydroxyethyloxy and its esters with the carboxylic acids or trifluoroacetic acid mentioned above.
The serine hydroxyl group may be esterified with a substituent selected from the group consisting of:
c2 to C12 aliphatic carboxylic acid groups as described above;
a trifluoroacetate group; and
a benzoic acid group.
The amino group in lysine can be modified by chemical means, for example by amide formation with: c2 to C12 aliphatic carboxylic acid groups as described above (e.g., by reaction of an amine with a chemically active form of a carboxylic acid such as an acid chloride, anhydride, N hydroxysuccinimide ester, pentafluorophenyl (OPfp) ester, 3-hydroxy-2, 3-dihydro-4-oxo-benzo-triazinone (ODhbt) ester, etc.), or a benzoic acid group, or an amino acid group. In addition, the amino group in lysine can be chemically modified by alkylation with one or two C1 to C4 aliphatic alkyls.
The carboxylic acid group in glutamic acid can be modified by amide formation with amines such as: ammonia; c1 to C12 primary aliphatic alkylamines (alkyl portions of which are described above), including methylamines; or an amino group of an amino acid.
The carboxylic acid group in glutamic acid can be modified by forming an ester with a C1 to C12 aliphatic hydroxyalkyl group as described above, preferably an ester with a C1 to C12 aliphatic alkyl primary alcohol, e.g., methanol, ethanol, 1-propanol, n-dodecanol, etc., as described above.
In a preferred embodiment, the present invention comprises a method of inhibiting the release of at least one inflammatory mediator from a particle within at least one inflammatory cell in a tissue and/or a body fluid of a subject, said method comprising administering to said tissue and/or body 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 of 4to 23 consecutive amino acids having the 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 or not by acylation with a carboxylic acid selected from the group consisting of: c2 to C13 saturated or unsaturated aliphatic carboxylic acid, C14 saturated (myristic acid) or unsaturated aliphatic carboxylic acid, C15 to C24 saturated or unsaturated aliphatic carboxylic acid, and trifluoroacetic acid, with the proviso that when the amino acid sequence of the peptide starts with the sequence of the reference sequence GAQF, the peptide is acylated modified or not chemically modified by acylation with only a carboxylic acid selected from the group consisting of: c2 to C13 saturated or unsaturated aliphatic carboxylic acid, C14 unsaturated aliphatic carboxylic acid, C15 to C24 saturated or unsaturated aliphatic carboxylic acid, and trifluoroacetic acid, wherein said peptide, optionally in combination with a pharmaceutically acceptable carrier, is present in a therapeutically effective amount to reduce the release of inflammatory mediators such that the release of said inflammatory mediators from at least one inflammatory cell is reduced as compared to the release of said inflammatory mediators from at least one same type of inflammatory cell in the absence of said at least one peptide.
The method is preferably performed using a peptide which can be acetylated at the N-terminal alpha amino acid. The peptide may consist of at least 10 consecutive amino acid residues and is preferably acetyl-peptide 106(SEQ ID NO: 106).
The method may also use peptides consisting of at least 4 consecutive amino acid residues and more preferably at least 6 consecutive amino acid residues. In addition, the N-terminal alpha amino acid of the peptide may be myristoylated. The method may also use peptides that can be amidated with ammonia at the C-terminal alpha amino acid.
In a further embodiment, the method uses a peptide comprising the amino acid sequence: (a) an amino acid sequence of 4to 23 consecutive amino acids having the reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), wherein the N-terminal amino acid of the amino acid sequence of (a) is selected from the amino acids 2 to 21 of the reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1). In addition, the N-terminal alpha amino acids of these peptides may be myristoylated, and the C-terminal alpha amino acids may also be amidated with ammonia.
The administration method of the present invention defines reducing the release of inflammatory mediators as blocking or inhibiting the mechanism of release of inflammatory mediators from inflammatory cells within the subject.
The method of administration includes combining or mixing the peptide with a pharmaceutically acceptable carrier to form a pharmaceutical composition.
The administration method of the present invention inhibits the release of at least one inflammatory mediator in a release-inhibiting amount such that the release of the inflammatory mediator from at least one inflammatory cell is reduced as compared to the release of the inflammatory mediator from at least one inflammatory cell of the same type in the absence of the at least one peptide. The inflammatory cell of the subject may be a leukocyte, granulocyte, basophil, eosinophil, monocyte, macrophage or a combination thereof.
The inflammatory mediators released from the at least one particle of the at least one inflammatory cell are selected from the group consisting of Myeloperoxidase (MPO), Eosinophil Peroxidase (EPO), Major Basic Protein (MBP), lysozyme, granzyme, histamine, proteoglycan, protease, chemokine, cytokine, arachidonic acid metabolite, defensin, bactericidal permeability-increasing protein (BPI), elastase, cathepsin G, cathepsin B, cathepsin D, β -D-glucuronidase, α -mannosidase, phospholipase A24-chondroitin sulphate, protease 3, lactoferrin, collagenase, complement activator, complement receptor, N-formylmethionyl-leucyl-phenylalanine (FMLP) receptor, laminin receptor, cytochrome b558Preferably the inflammatory mediator is selected from the group consisting of Myeloperoxidase (MPO), Eosinophil Peroxidase (EPO), Major Basic Protein (MBP), lysozyme, granzyme and combinations thereof.
The method of claim 13, 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 by about 1% to about 99%, or preferably by about 5-50% to about 99%, as compared to the amount released from at least one inflammatory cell in the absence of said peptide.
The methods of the invention are useful for treating a subject having a respiratory disease. The respiratory disease may be asthma, chronic bronchitis, Chronic Obstructive Pulmonary Disease (COPD) and cystic fibrosis. The subject treatable by the present invention is preferably a mammal such as a human, dog, horse and feline.
The method of administration of the peptide of the present invention may be topical administration, parenteral administration, rectal administration, pulmonary administration, nasal administration and oral administration. More preferably, pulmonary administration comprises an aerosol, which may be produced by a dry powder inhaler, a metered dose inhaler or a nebulizer. In addition, administering to the subject may further comprise administering a second molecule selected from the group consisting of: antibiotics, antiviral compounds, antiparasitic compounds, anti-inflammatory compounds, and immunomodulators.
The method may also be used to treat a subject having a disease selected from the group consisting of: bowel disorders, skin disorders, autoimmune disorders, pain syndromes, and combinations thereof. More specifically, the bowel disease is selected from ulcerative colitis, crohn's disease, and irritable bowel syndrome. Skin disorders treatable by the present method include rosacea, eczema, psoriasis and severe acne. The invention may also treat subjects suffering from arthritis.
One embodiment of the invention encompasses the administration of a peptide comprising an amino acid sequence that is substantially identical to amino acid sequence (a) having 4to 23 consecutive amino acids of reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1). Preferably, these peptides are selected from the group consisting of: 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 249, 250, 251 and 252. The peptides may also be acetylated at the N-terminal alpha amino acid or myristoylated at the N-terminal alpha amino acid, and optionally amidated at the C-terminal alpha amino acid with ammonia.
By administering the peptide of the invention as described above, the method of the invention may also be used to reduce mucus hypersecretion in a subject, and may be used to reduce MARCKS-related mucus hypersecretion in mucus secreting cells or tissues of a subject, whereby mucus hypersecretion in a subject is reduced compared to what may occur in the absence of administration of the peptide.
The present invention relates to an isolated peptide having an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence of 4to 23 consecutive amino acids having the 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 or not by acylation with a carboxylic acid selected from the group consisting of: c2 to C13 saturated or unsaturated aliphatic carboxylic acid, C14 saturated or unsaturated aliphatic carboxylic acid, C15 to C24 saturated or unsaturated aliphatic carboxylic acid, and trifluoroacetic acid, with the proviso that when the amino acid sequence of the peptide starts with the sequence of the reference sequence GAQF, the peptide is acylated modified or not chemically modified by acylation with only a carboxylic acid selected from the group consisting of: c2 to C13 saturated or unsaturated aliphatic carboxylic acid, C14 unsaturated aliphatic carboxylic acid, C15 to C24 saturated or unsaturated aliphatic carboxylic acid, and trifluoroacetic acid, wherein said peptide, optionally in combination with a pharmaceutically acceptable carrier, is present in a therapeutically effective amount to reduce the release of inflammatory mediators such that the release of said inflammatory mediators from at least one inflammatory cell is reduced as compared to the release of said inflammatory mediators from at least one same type of inflammatory cell in the absence of said at least one peptide.
The N-terminal alpha amino acid of the isolated peptide can be acetylated. The isolated peptide consists of at least 10 contiguous amino acid residues, and is preferably an isolated peptide consisting of acetyl-peptide 106(SEQ ID NO: 106).
In another embodiment, the peptide consists of at least 4 consecutive amino acid residues or the peptide consists of at least 6 consecutive amino acid residues.
The N-terminal alpha amino acid of the peptide may be myristoylated and/or the C-terminal alpha amino acid of the peptide may be amidated with ammonia.
The isolated peptide may further comprise the amino acid sequence of (a) above, (a) an amino acid sequence of 4to 23 consecutive amino acids having the reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1); wherein the N-terminal amino acid of the amino acid sequence of (a) is selected from amino acids 2 to 21 of reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1). The N-terminal alpha amino acid of the peptide may also be myristoylated or acetylated, or the C-terminal alpha amino acid may optionally be amidated with ammonia.
In another embodiment, the amino acid sequence of the isolated peptide is substantially identical to the amino acid sequence of (a) having 4to 23 contiguous amino acids of reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1). These peptides are preferably selected from the group consisting of: 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 249, 250, 251 and 252. These peptides may be further acetylated at the N-terminal alpha amino acid or myristoylated at the N-terminal alpha amino acid, and optionally amidated with ammonia. (c) Is substantially identical to the amino acid sequence of (a) 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.
The invention also encompasses compositions comprising the above paragraphs as well as the isolated peptides and excipients disclosed herein. The invention also encompasses pharmaceutical compositions of the foregoing paragraphs as well as the isolated peptides disclosed herein and a pharmaceutically acceptable carrier. The pharmaceutical composition further preferably may be sterile, sterilizable or sterilizable. These peptides may be placed in a kit with reagents for administration.
Drawings
FIGS. 1A-1B show that PKC-dependent phosphorylation releases MARCKS from the plasma membrane to the cytoplasm.
FIGS. 2A-2C show that PKG induces MARCKS dephosphorylation by activating PP 2A.
The bar graph presented in FIG. 3 confirms that PP2A is an essential component of the mucin secretory pathway.
The gel of fig. 4 shows MARCKS binding to actin and myosin in the cytoplasm.
FIG. 5 depicts the signaling mechanism controlling MPO secretion in neutrophils.
FIG. 6 is a bar graph depicting that MANS peptide is capable of blocking myeloperoxidase secretion by isolated canine neutrophils.
FIG. 7 is a bar graph depicting that MANS peptide is capable of blocking myeloperoxidase secretion by isolated human neutrophils.
FIG. 8 is a bar graph showing that PMA stimulation increases the secretion of MPO from LPS-stimulated human neutrophils in small amounts, and that co-stimulation with 8-Br-cGMP leads to an enhancement in a concentration-dependent manner.
The bar graph of FIG. 9 shows that 8-Br-cGMP stimulation had little effect on LPS-stimulated human neutrophil secretion of MPO until co-stimulated with PMA in a concentration-dependent manner.
FIG. 10 is a bar graph showing that PMA stimulation increases the secretion of MPO from LPS-stimulated canine neutrophils in small amounts, and that co-stimulation with 8-Br-cGMP leads to an enhancement in a concentration-dependent manner.
FIG. 11 is a bar graph showing that 8-Br-cGMP stimulation had little effect on LPS-stimulated canine neutrophil secretion of MPO until co-stimulated with PMA in a concentration-dependent manner.
FIG. 12 is a bar graph showing that co-stimulation of PMA +8-Br-cGMP is necessary to maximize MPO secretion from LPS-stimulated canine neutrophils.
Detailed Description
The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The use of the terms a or an herein in describing any aspect of the invention is to be construed to refer to one or more.
The present invention relates to a method of inhibiting exocytosis release of at least one inflammatory mediator from at least one inflammatory cell comprising at least one inflammatory mediator located within a vesicle within said cell, comprising contacting said at least one inflammatory cell with an effective amount of at least one peptide selected from the group consisting of MANS peptide and active fragments thereof as described herein, such that release of said inflammatory mediator from said inflammatory cell is reduced as compared to said inflammatory mediator that would be released from the same type of inflammatory cell in the absence of said at least one peptide.
The present invention further relates to a method of inhibiting the release of at least one inflammatory mediator from at least one inflammatory cell in a tissue or body fluid of a subject, comprising administering to the tissue and/or body fluid of the subject comprising at least one inflammatory cell comprising at least one inflammatory mediator located in a vesicle within the cell, a therapeutically effective amount of a pharmaceutical composition comprising a therapeutically effective amount of at least one peptide selected from the group consisting of MANS peptides and active fragments thereof, such that the release of the inflammatory mediator from at least one inflammatory cell is reduced compared to the release of the inflammatory mediator from at least one inflammatory cell of the same type that would be possible in the absence of the at least one peptide. More specifically, reducing the release of inflammatory mediators comprises blocking or inhibiting the mechanism of inflammatory mediator release from inflammatory cells.
The present invention relates to contacting and/or administering the aforementioned and throughout the specification peptides to any known inflammatory cell that may be located within a tissue or body fluid of a subject, said cell containing at least one inflammatory mediator located within a vesicle within said cell. The inflammatory cells are preferably leukocytes, more preferably granulocytes, which can be further classified as neutrophils, basophils, eosinophils or a combination thereof. The inflammatory cells contacted in the methods of the invention may also be monocytes/macrophages.
The present invention is directed to reducing the release of inflammatory mediators contained in vesicles of inflammatory cells, the inflammatory mediators being selected from the group consisting of Myeloperoxidase (MPO), Eosinophil Peroxidase (EPO), Major Basic Protein (MBP), lysozyme, granzyme, histamine, proteoglycan, protease, chemokine, cytokine, arachidonic acid metabolite, defensin, bactericidal permeability-increasing protein (BPI), elastase, cathepsin G, cathepsin B, cathepsin D, β -D-glucuronidase, α -mannosidase, phospholipase A24-chondroitin sulphate, protease 3, lactoferrin, collagenase, complement activator, complement receptor, N-formylmethionyl-leucyl-phenylalanine (FMLP) receptor, laminin receptor, cytochrome b558Monocyte-chemotaxisPreferably, these inflammatory mediators are selected from the group consisting of Myeloperoxidase (MPO), Eosinophil Peroxidase (EPO), Major Basic Protein (MBP), lysozyme, granzyme and combinations thereof.
The present invention contacts an effective amount of the peptide with an inflammatory cell, wherein the effective amount is defined as a degranulation-inhibiting amount of MANS peptide or an active fragment thereof that reduces the amount of an inflammatory mediator released from at least one inflammatory cell by about 1% to about 99% as compared to the amount released from at least one inflammatory cell in the absence of MANS peptide or an active fragment thereof. This amount is also referred to as an effective reduction in inflammatory mediator release. More preferably, the effective amount of the peptide for contacting comprises a degranulation-inhibiting amount of MANS peptide or an active fragment thereof that reduces the amount of an inflammatory mediator released from at least one inflammatory cell by about 5-50% to about 99% as compared to the amount released from the at least one inflammatory cell in the absence of MANS peptide or an active fragment thereof.
In one embodiment, the present invention is directed to administering a therapeutically effective amount of at least one peptide comprising MANS peptide and active fragments thereof into a tissue or body fluid of a subject, wherein the subject has a respiratory disease, preferably asthma, chronic bronchitis, or COPD. In another embodiment, the subject has a bowel disease, a skin disease, an autoimmune disease, a pain syndrome, and combinations thereof. The bowel disease may be ulcerative colitis, crohn's disease, or irritable bowel syndrome. The subject may be suffering from a skin disorder, such as rosacea, eczema, psoriasis or severe acne. The subject may also be suffering from arthritis, such as rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus. Subjects with cystic fibrosis can also be treated with the methods and peptides of the invention. The methods of the invention are preferably used to treat subjects, such as mammals, preferably humans, dogs, horses and felines.
Methods of treatment of the invention by administering to a subject one or more peptides comprising a MANS peptide or active fragment described herein include topical administration, parenteral administration, rectal administration, pulmonary administration, nasal administration, or oral administration. More specifically, pulmonary administration is selected from the group consisting of aerosols, dry powder spray inhalers, metered dose spray inhalers, and nebulizers. In addition, the method of the present invention may further comprise administering to the subject a second molecule selected from the group consisting of: antibiotics, antiviral compounds, antiparasitic compounds, anti-inflammatory compounds, and immunomodulators.
In one aspect, the invention relates to a method of administering a pharmaceutical composition. The pharmaceutical compositions comprise a therapeutically effective amount of a known compound and a pharmaceutically acceptable carrier. A "therapeutically effective" amount is herein an amount of a compound sufficient to alleviate a symptom exhibited by a subject. The therapeutically effective amount may vary with the age and physical condition of the patient, the basic severity of the patient being treated, the duration of the treatment, the nature of any concurrent therapy, the pharmaceutically acceptable carrier employed, and other similar factors known to those skilled in the art. The pharmaceutically acceptable carrier is preferably a solid dosage form such as tablets and capsules. Liquid preparations for oral use can also be used, which can be prepared in the form of syrups and suspensions, for example, solutions containing the active ingredient, sugar and a mixture of ethanol, water, glycerol and propylene glycol. If desired, such liquid formulations may include one or more of the following components: coloring agents, flavoring agents and saccharin. In addition, thickeners such as carboxymethyl cellulose and other acceptable carriers may also be used, the skilled artisan knows how to select them.
As indicated above, the present invention relates to methods for modulating cellular secretory processes, and in particular to methods for modulating cellular secretory processes in which inflammatory mediators are released from inflammatory cells. As used herein, the term "modulate" refers to blocking, inhibiting, decreasing, increasing, enhancing, or stimulating. Some cellular secretion processes involve the release of components from membrane-bound vesicles or particles within the cell. Membrane-bound vesicles or particles are defined as intracellular particles that are predominantly vesicular (or intracellular vesicles) and contain a depot substance that can be secreted. And the domestic discovery that some components of these vesicles, such as those contained in inflammatory cells, contribute to a variety of pathologies in a large number of mammalian tissues. Some of the effects of these secretions appear to include damage to previously healthy tissue during inflammation. The present invention provides means to block secretion from membrane-bound vesicles, including those in inflammatory cells, by targeting specific important molecules in the intracellular endocrine pathway with synthetic peptides. The method has therapeutic significance for treating various hypersecretion and inflammatory diseases of human and animals.
More specifically, the invention targets inflammatory cells that contain inflammatory mediators within one or more granules or vesicles within the cytoplasm. The cells are contacted with one or more peptides selected from the group consisting of MANS peptides or active fragments thereof, as described in detail in the specification. Preferably, the peptides are contacted with inflammatory cells by administering the peptides to a subject having a disease in which these inflammatory cells are present in a particular tissue or a bodily fluid of a tissue. Upon administration or upon contact of the peptide with a cell, the peptide competitively competes for and competitively inhibits binding of the native MARCKS protein to the membrane of an intracellular particle or vesicle containing inflammatory mediators. Thereby blocking the binding of MARCKS proteins to vesicles within inflammatory cells so that these vesicles within these cells do not migrate to the plasma membrane of the cell as they would normally do if stimulated to release their inflammatory mediator components extracellularly to exocytosis. Thus, the methods of the invention inhibit the migration of vesicles to the plasma membrane of a cell, thereby reducing the release of inflammatory mediators by inflammatory cells. Since both the rate and amount of mediator released from inflammatory cells depend on the concentration of peptide administered and in contact with inflammatory cells, the amount of inflammatory mediators released from the cells over time is reduced.
One advantage of the present invention is that it can combine a treatment comprising direct blocking of mucus secretion with a unique anti-inflammatory treatment. One advantage of the present invention over current anti-inflammatory therapies that affect a broad suppression of the immune system is that the peptides are believed to block secretion of only intracellular components secreted from inflammatory cells. Thus, although inflammatory mediators are suppressed, many aspects of the immune system will still play a role.
The compounds of the invention modulate, i.e., block, the release of inflammatory mediators from cells. Inhibition of inflammatory mediator release is an attractive approach for the prevention and treatment of a variety of diseases, e.g., diseases and pathological conditions involving inflammation. Thus, the compounds of the present invention are useful for treating such disorders. These diseases include airway diseases and chronic inflammatory diseases including, but not limited to, osteoarthritis, multiple sclerosis, Guillain-Barre syndrome, Crohn's disease, ulcerative colitis, psoriasis, graft-versus-host disease, and systemic lupus erythematosus. The compounds of the invention are also useful in the treatment of other diseases associated with elevated levels of proinflammatory mediators and enzyme activity, such as response to various infectious agents, as well as a variety of autoimmune diseases such as rheumatoid arthritis, toxic shock syndrome, diabetes and inflammatory bowel disease.
Uses of the peptides and methods of the invention include treatments to combat inflammation and treatments that combine the anti-inflammatory activity of the peptides with their ability to block mucus secretion. Diseases that can be treated by the ability of the peptides to simultaneously block inflammation and mucus secretion include, but are not limited to, inflammatory bowel disease, digestive system diseases (i.e., cholecystitis, Menetier's disease), and inflammatory airway diseases.
Other pro-inflammatory mediators have been found to be associated with a variety of disease states associated with the invasion of inflammation or injury sites by neutrophils. Blocking antibodies have been shown to be useful in the treatment of neutrophil-associated tissue injury in acute inflammation (Harada et al, 1996, Molecular Medicine today2, 482). Other cells other than neutrophils that release inflammatory mediators include other leukocytes such as basophils, eosinophils, monocytes and lymphocytes, and can be treated for secretion from these cells. Neutrophils, eosinophils and basophils are all types of granulocytes, i.e. they are leukocytes with granules in the cytoplasm. Leukocytes synthesize a variety of inflammatory mediators, which are packaged and stored within granules of the cytoplasm. Such media are, for example, Myeloperoxidase (MPO) in neutrophils (Borregaard N, Cowland JB. Granules of the neutrophilic polymorphhonuclear peroxidase. Bloodshab 1997; 89: 3503-mediated 3521), Eosinophilic Peroxidase (EPO) and Major Basic Protein (MBP) (Gleich G J. Mechanisms of eosiniophil-assisted kinase. J. Allergy Clin 2000; 105:651-663), lysozyme in monocytes/macrophages (HoffT, Spencker T, Emmenopter A. Goppelst-Structure M. Effects glucose complexes. It. J. interferon-. 52; Goppelsberg M. interferon-induced nuclear peroxidase J. lysate J. 11. kappa. J. Biozyme J. kappa. J. Biozyme.; J. kappa. J. lysate 52; Batheromyc. kappa. M. Effe. 11. kappa. M. 11; Goldenlysiphylvanine J. kappa. J. lysate J. 11. kappa. lysate J. kappa. J. IV. kappa. and B5276; natural peroxidase J. lysate J. 12. lysate J. kappa. lysate J. 11. kappa. IV. lysate J. kappa. 11. kappa. lysate J. kappa. 11. kappa. IV. kappa. expressing peroxidase and B2003. lysate J. 11. kappa Maki G, Klingemann HG. Characterisation of ahuman cell line (NK-92) with phenotypicals and functional characteristics of activated natural killer cells. Leukemia 1994; 8: 652-; maki G, Klingemann HG, Martinson JA, Tam YK. fans regulating the cytoxic activity of the humannatural killer Cell line, NK-92.J Hematother Stem Cell Res 2001; 10: 369-383; and Takayama H, Trenn G, Sitkovsky MV.A novel cytoxic T lymphocyte activity.J Immunol Methods 1987; 104:183-1907-10). As a result of the infiltration of these cells into the tissue site of injury or disease, these mediators are released at the site of injury and can contribute to inflammation and repair, for example, in the lung and other sites. Leukocytes release these particles by the exocytosis mechanism (Burgoyne RD, Morgan A. Secreassociated granulocytosis. physiol Rev 2003; 83: 581-.
Mast cells, which are not normally circulating in the bloodstream, and basophils contain secretory cytoplasmic granules that store and (when the cells are activated) release inflammatory (allergic) mediators, such as histamine; proteoglycans such as heparin and chondroitin sulfate; proteases such as tryptase, chymotrypsin, carboxypeptidase, and cathepsin G-like proteases; chemokines, cytokines and arachidonic acid metabolites that act on blood vessels, smooth muscle, connective tissue, mucous glands and inflammatory cells.
Neutrophils, also known as polymorphonuclear leukocytes (PMNs), account for 50 to 60% of the total number of circulating leukocytes. Neutrophils act against infectious agents, such as bacteria, fungi, protozoa, viruses, virus-infected cells, and tumor cells, that penetrate the physical barriers of the body at the site of infection or injury. The maturation of neutrophils goes through 6 morphological stages: myeloblasts, promyelocytes, mesogranulocytes, metagranulocytes, nonphyllous nucleus (rod nucleus) neutrophils, and phyllular nucleus (functionally active) neutrophils.
Among the various inflammatory mediators, the primary (azurophil) particle contains Myeloperoxidase (MPO), lysozyme, defensin, bactericidal permeability-increasing protein (BPI), elastase, cathepsin G, cathepsin B, cathepsin D, β -D-glucuronidase, α -mannosidase, phospholipase A24-chondroitin sulfate, and protease 3 (see, e.g., Hartwig JH, Thelen M, Rosen A, Janmey PA, Nairn AC, Aderem A. MARCKS isactan membrane cross protein regulated by protein kinase C and calcium-calmodulin. Nature 1992; 356: 618-; the secondary (specific) particles comprise lysozyme, lactoferrin, collagenase, complement activator, phospholipase A2Complement receptors such as CR3, CR4, N-formylmethionyl-leucyl-phenylalanine (FMLP) receptor, laminin receptor, cytochrome b558Monocyte-chemotactic factor, histaminase, and vitamin B12 binding protein, and small storage particles comprising gelatinase, plasminogen activator, cathepsin B, cathepsin D, β -D-glucoseUronic acid glycosidase, α -mannosidase and cytochrome b558。
The neutrophil granule contains an antimicrobial or cytotoxic substance, a neutral protease, an acid hydrolase and a number of cytoplasmic membrane receptors. In the azurophilic granule component, Myeloperoxidase (MPO) is a key enzyme to convert hydrogen peroxide to hypochlorous acid. Which, together with hydrogen peroxide and halide cofactors, form the myeloperoxidase system, a potent microbicidal and cytotoxic mechanism of leukocytes.
Defensins constitute 30 to 50% of the protein of azurophil granules, they are small (molecular weight <4000) strong antimicrobial peptides, cytotoxic to a large number of bacteria, fungi and certain viruses. The toxicity may consist in membrane permeation of the target cells, similar to other channel-forming proteins (perforin).
Bacterial Permeability Increasing (BPI) proteins are perforin members. It is highly toxic to gram-negative bacteria but not to gram-positive bacteria or fungi, and it also neutralizes endotoxins, the toxic lipopolysaccharide component of the cell envelope of gram-negative bacteria.
Lactoferrin chelates free iron, thereby preventing the growth of ingested microorganisms that are not killed and increasing the permeability of bacteria to lysozyme.
Serine proteases such as elastase and cathepsin G hydrolyze proteins in bacterial cell envelopes. Substrates for granulocyte elastase include collagen crosslinks and proteoglycans, as well as the elastin component of blood vessels, ligaments, and cartilage. Cathepsin D cleaves cartilage proteoglycans, while granulocyte collagenase actively cleaves type I collagen from bone, cartilage and tendons and to a lesser extent type III collagen. The collagen breakdown products have chemotactic activity on neutrophils, monocytes and fibroblasts.
Protease inhibitors such as alpha-2-macroglobulin and alpha-1-antiproteases mediate the regulation of the tissue destruction capacity of lysosomal proteases. These anti-proteases are present in serum and synovial fluid. They act by binding to and covering the active site of the protease. Protease-antipain imbalances play an important role in the pathogenesis of emphysema.
Azurophil granules act primarily in the intracellular environment (within phagolysosomal vacuoles), where they participate in killing and degrading microorganisms. Neutrophil-specific particles readily release their components outside the cell and play an important role in the initiation of inflammation. Specific particles represent various plasma membrane components of intracellular stores, including cytochrome b (a component of NADPH oxidase, which is responsible for the production of peroxides), receptors for the complement fragment iC3b (CR3, CR4), receptors for laminin, and formylmethionyl-peptide chemokines. Among others, histamine enzymes associated with histamine degradation, vitamin binding proteins, and plasminogen activator responsible for the formation of plasmin and cleavage of C5a from C5.
The importance of neutrophil granules in inflammation is clearly seen in the study of some patients with congenital abnormalities of these granules. Patients with the Ch diak-Higashi syndrome have severe abnormalities in the rate at which inflammatory responses are established and have abnormally large lysosomal particles. This congenital specific particle deficiency syndrome is a very rare disease characterized by a reduced inflammatory response and severe bacterial infection of the skin and deep tissues.
Although only partially understood are the mechanisms regulating exocytic secretion of these particles, several key molecules in this process have been identified, including intracellular Ca2+ transients (Ca2+ transitions) (Richter et al Proc Natl Acad Sci USA 1990; 87: 9472-9476; Blackwood et al, Biochem J1990; 266:195-200), G-protein, tyrosine and protein kinase (PK, especially PKC) (Smolen et al, Biochim Biophys Acta 1990; 1052: 133-142; Niessen et al, Biophys. Acta 1994; 1223: 267-273; Naucler et al, Petteren et al, Chest 2002; 142-150), Rac2 (Absel-Latif et al, 2004; 104: 832; Thercy. 358; 2003-J-70; Australin et al; SNmp 79; Australin et al: 2679; Australin et al; 26: 76, J. 76, 2003; Australin et al; 26; Australin et al; 26; Australin et al; Aust.
SNARE (soluble N-ethylmaleimide attachment protein receptor) proteins are a family of membrane-associated proteins characterized by an alpha-helical coiled-coil domain called the SNARE motif (Li et al, cell. mol. Life Sci.60:942-960 (2003)). These proteins are classified into the v-SNARE class and the t-SNARE class based on their position on the vesicle and the target membrane; another classification scheme based on conserved arginine or glutamine residues at the center of the SNARE motif classifies them into the R-SNARE class and the Q-SNARE class. SNARE is located in separate membranous compartments of secretory and endocytic trafficking pathways and contributes to the specificity of intracellular membrane fusion processes. the t-SNARE domain consists of 4 helical bundles with coiled-coil twists. The SNARE motif contributes to the fusion of the two membranes. SNARE motifs are classified into 4 classes: syntaxin 1a (t-SNARE) homolog, VAMP-2(v-SNARE), and the N-and C-terminal SNARE motifs of SNAP-25. One member from each class may interact to form a SNARE complex. SNARE motifs are found in the N-terminal domain of certain syntaxin family members such as syntaxin 1a, which are essential for neurotransmitter release (Lerman et al, Biochemistry39:8470-8479(2000)), andsyntaxin6, which is found in endosomol transport vehicles (Misura et al, Proc. Natl. Acad. Sci. U.S.A.99:9184-9189 (2002)).
The SNAP-25 (synaptosome associated protein 25kDa) protein is a component of the SNARE complex, which contributes to the specificity of membrane fusion and directly effects fusion by forming a tight complex (SNARE complex or core complex) that brings the synaptic vesicle and plasma membrane together. SNARE constitutes a large family of proteins characterized by a sequence of 60 residues called SNARE motif, which is highly prone to form coiled coils and is usually located before the carboxy-terminal transmembrane region. The synaptic axial complex is formed by 4 SNARE motifs (2 from SNAP-25, one from each of synaptophysin and synaptoxin 1) which are loose in isolation but form parallel 4-helix bundles upon assembly. The crystal structure of the axial complexes reveals that the helix bundle is highly twisted and contains several salt bridges on the surface, with an internal hydrophobic residual layer. The polar layer in the center of the complex is formed by 3 glutamines (2 from SNAP-25 and one from synaptotagmin 1) and one arginine (from synaptophysin) (Rizo et al, Nat Rev Neurosci3:641-653 (2002)). Membranes of the SNAP-25 family contain a cluster of cysteine residues that can be hexadecanoylated for membrane attachment (Risinger et al, J.biol.chem.268:24408-24414 (1993)).
The primary role of neutrophils is to phagocytose and destroy infectious agents. They may also limit the growth of certain microorganisms before an adaptive (specific) immune response occurs. Although neutrophils are essential for host defense, they are also thought to be involved in the pathology of a variety of chronic inflammatory diseases and ischemia reperfusion injury. Hydrolytic enzymes and oxidatively inactivated protease inhibitors from neutrophils can be detected in fluid isolated from the site of inflammation. Normally, neutrophils can migrate to the site of infection without destroying host tissues. However, sometimes deleterious host tissue damage can occur. This damage can occur by a number of independent mechanisms. These include premature activation during migration, release of toxic products out of the cell during killing of certain microorganisms, removal of infected or damaged host cells and debris as a first step in tissue reconstruction, or failure to terminate acute inflammatory responses. Ischemia reperfusion injury is associated with the entry of neutrophils into the affected tissue and subsequent activation. This may be triggered by substances released by the disrupted host cell or by peroxides produced by xanthine oxidase.
Under normal conditions, blood may contain normal, primary activated (primary), activated and depleted (patient) neutrophils. At the site of inflammation, predominantly activated and depleted neutrophils. Activated neutrophils flourish to produce reactive oxygen species intermediates (ROIs). A subpopulation of neutrophils with a vigorous respiratory burst was detected in the blood of patients with acute bacterial infections and patients with Adult Respiratory Distress Syndrome (ARDS). This is an example of a neutrophil contradiction. Neutrophils are thought to be involved in the pathology of this condition because of the massive entry of these cells into the lung and the associated tissue destruction caused by the oxidative and hydrolytic enzymes released by activated neutrophils. The impairment of neutrophil microbicidal activity induced locally by inflammatory products may be a protective response in the host when ARDS is exacerbated.
The acute phase of thermal injury is also associated with neutrophil activation, followed by extensive impairment of various neutrophil functions. Activation of neutrophils by immune complexes in synovial fluid contributes to the pathology of rheumatoid arthritis. Chronic activation of neutrophils can also trigger tumorigenesis because some ROIs produced by neutrophils destroy DNA, while proteases promote tumor cell migration. In patients with severe burns, a correlation was found between the occurrence of bacterial infection and a reduction in the proportion and absolute number of antibodies and complement receptor positive neutrophils. It has also been found that oxidants from neutrophils oxidize Low Density Lipoproteins (LDL), making the latter more efficiently bound to the plasma membrane of macrophages via specific scavenger receptors. These oxidized LDL may cause arteriosclerosis after being taken up by macrophages. Furthermore, the presence of primarily activated neutrophils is found in patients with essential hypertension, hodgkin's disease, inflammatory bowel disease, psoriasis, sarcoidosis and septicaemia, where the primary activation is associated with high concentrations of circulating TNF-alpha (cachectin).
After the protective action of antioxidants and antiproteases is lost, hydrolytic destruction of host tissues and chronic inflammatory diseases can occur. Protease resistance deficiency is thought to contribute to the pathology of emphysema. Various antiproteases are members of the SERPIN family. Although the blood circulation is rich in antiproteases, these macromolecular proteins may be selectively excluded from the site of inflammation due to the adhesion of neutrophils to their targets. Oxidative stress can initiate tissue destruction by reducing the extracellular anti-protease concentration below the level required to inhibit the released protease. Chlorine-containing oxidants and hydrogen peroxide inactivate anti-proteases such as alpha 1-protease inhibitors and alpha 2-macroglobulin, which are endogenous inhibitors of elastase, but at the same time activate latent metalloproteinases such as collagenase and gelatinase, which in turn cause further inactivation of the anti-protease.
The cytoplasmic component of neutrophils may also be a cause of the production of specific anti-neutrophil cytoplasmic antibodies (ANCA), which are closely associated with the development of systemic vasculitis and glomerulonephritis. ANCA are antibodies directed against enzymes located predominantly inside the azurophil granules or primary granules of neutrophils. There are three types of ANCAs that can be distinguished by the pattern they produce when detected by indirect immunofluorescence on conventional ethanol-fixed neutrophils. Diffuse fine granular cytoplasmic fluorescence (cANCA) is typically seen in wegener granulomatosis, some microscopic polyarteritis and churg strauss syndrome cases, and some cases of crescentic and segmental dying glomerulonephritis. The target antigen is typically protease 3. Pericyclic fluorescence (pANCA) is seen in many cases of polyarteritis and glomerulonephritis under the microscope. These antibodies are typically directed against myeloperoxidase, but other targets include elastase, cathepsin G, lactoferrin, lysozyme and β -D-glucuronidase. The third group, termed "atypical" ANCAs, includes neutrophil nuclear fluorescence and some rare cytoplasmic patterns, and although a few target antigens are identical to pANCA, others have not been identified. One third of crohn's disease also presents with pANCA. Reports on the positive rates of ANCA in rheumatoid arthritis and SLE vary greatly, but the patterns are mainly pANCA and atypical ANCA.
Eosinophils are terminally differentiated, terminally-stage leukocytes that are predominantly located in submucosal tissues and recruited to sites of specific immune responses, including allergic disease. The cytoplasm of eosinophils contains large ellipsoidal particles with electron dense crystalline nuclei and partially permeable stroma. In addition to these large primary crystalline particles, there is another type of particle that is smaller (small particles) and lacks a crystalline core. Large specific particles of eosinophils contain at least four different cationic proteins: major Basic Protein (MBP), Eosinophil Cationic Protein (ECP), eosinophil-derived neurotoxin (EDN), and Eosinophil Peroxidase (EPO), which have a range of biological effects on host cell and microbial targets. Basophils contain a major basic protein relative to one fourth of eosinophils, and also contain detectable amounts of EDN, ECP and EPO. Neutrophiles also have small amounts of EDN and ECP (Gleich G J. mechanisms of eosinophia-associated inflammation. J. Allergy Clin Immunol 2000; 105: 651-663). MBP appears to be a highly cationic polypeptide lacking enzymatic activity, which exerts its toxic activity by interacting with the lipid membrane to perturb it. Both MBP and EPO act as selective allosteric inhibitors of agonists that bind M2 muscarinic receptors. These proteins can cause dysfunction of the M2 receptor and exacerbate vagaries-mediated bronchoconstriction in asthma. EDN can specifically destroy the myelin sheath of neurons. Large specific granules of eosinophils also possess histaminase and a variety of hydrolysosomal enzymes. Enzymes within the eosinophil granule include arylsulfatase, acid phosphatase, and a 92kDa metalloprotease, gelatinase. Eosinophils can produce cytokines, including those with potential autocrine growth factor activity on eosinophils as well as those with potential roles in acute and chronic inflammatory responses. Three cytokines have growth factor activity on eosinophils: granulocyte-macrophage colony stimulating factor (GM-CSF), IL-3, and IL-5. Other cytokines produced by human eosinophils that may be active in acute and chronic inflammatory responses include: IL-1-alpha, IL-6, IL-8, TNF-alpha and two transforming growth factors TGF-alpha and TGF-beta.
Eosinophils contain crystalline particles that contain MBP, eosinophil cationic protein, EPO, and eosinophil-derived neurotoxin (Gleich, J Allergy Clin Immunol 2000; 105: 651-. The human promyelocytic cell line HL-60 clone 15 was used to detect EPO secretion. The cell line was established by growing HL-60 at high pH for 2 months (Fischkoff, Leuk Res 1988; 12: 679-.
Eosinophils can participate in hypersensitivity reactions, particularly through two lipid inflammatory mediators, leukotriene C4(LTC4) And Platelet Activating Factor (PAF). Both mediators constrict airway smooth muscle, promote mucus secretion, alter vascular permeability and cause eosinophil and neutrophil infiltration. In addition to the direct activity of these eosinophil-derived mediators, MBP stimulates histamine release from basophils and mast cells, which in turn stimulate EPO release from mast cells. Eosinophils can act as a local source of specific lipid mediators and induce the release of mediators from mast cells and basophils. Eosinophil granule components can be released upon stimulation by stimuli similar to neutrophil granules, for example during phagocytosis of opsonized granules or by stimulation by chemokines. Neutrophil lysosomal enzymes act primarily on substances engulfed in phagolysosomes, while eosinophil granule components act primarily on extracellular target structures such as parasites and inflammatory mediators.
Monocytes and macrophages develop in the bone marrow and undergo the following stages: stem cells; directing the stem cells; a primary monocyte; a baby monocyte; monocytes in the bone marrow; monocytes in peripheral blood; and macrophages in the tissue. Monocytes differentiated rapidly in the bone marrow (1.5 to 3 days). During differentiation, granules are formed in the cytoplasm of monocytes, which can be classified into at least two classes, as can neutrophils. However, they are less than and smaller than their neutrophil counterparts (azurophil and specific particles). Their enzymatic components are similar.
The monocyte/macrophage particle-bound enzymes include lysozyme, acid phosphatase and β -glucuronidase. As an in vivo study model, lysozyme secretion from U937 cells was used. This cell line is derived from human tissue cell-type lymphomas and has been used as a monocyte cell line that can be activated by a variety of agonists, such as PMA (Hoff et al, JLEUkoc Biol 1992; 52: 173-.
Natural Killer (NK) cells and cytotoxic lymphocytes contain potent cytotoxic particles, including perforin (a pore-forming protein) and granzymes (lymphocyte-specific serine proteases). For example, the NK-92 cell line is an IL-2-dependent human cell line established from a rapidly progressing non-Hodgkin lymphoma patient (Gong JH., Maki G, Klingemann HG. characteristics of a human cell line (NK-92) with a phenotypal and functional characteristics of activated natural killer cells Leukemia 1994; 8: 652-. NK-92 cells express high levels of molecules involved in the perforin-granzyme cytotoxic pathway against a variety of malignant cells (Gong et al, vide infra, and Maki G, Klingemann HG, Martinson JA, Tam YK. Factorizing the cytoxic activity of the human natural killer Cell line, NK-92.J Hematother Stem Cell 2001; 10: 369-.
Granzymes are exogenous serine proteases released by cytoplasmic granules within cytotoxic T cells and natural killer cells. Granzymes can induce apoptosis in cells infected with the virus, thereby destroying them.
The extracellular release of inflammatory mediators (inflammatory mediators) by granulocytes (or leukocytes) and the extracellular release of more than one inflammatory mediator (inflammatory mediator) by granulocytes (or leukocytes) is sometimes referred to herein as degranulation. In a preferred embodiment, releasing the inflammatory mediators comprises releasing the mediators from particles located inside granulocytes or leukocytes. Releasing inflammatory mediators is preferably the release of inflammatory mediators from these particles.
Upon primary stimulation with proinflammatory factors (inflammatory stimuli) such as TNF α, neutrophils and macrophages dramatically increase their MARCKS protein synthesis: up to 90% of the novel proteins synthesized by neutrophils in response to TNF α or Lipopolysaccharide (LPS) are MARCKS (Thelen M, Rosen A, Nairn AC, Aderema. tumor across alpha. modifications, synthesis-dependent responses in human therapeutics by induction and methylation of a specific protein kinase, C Natl Acad Sci USA 1990; 5603-. Thus, MARCKS may play an important role in the subsequent inflammatory mediator release when granulosa-containing cells (e.g., neutrophils and macrophages) are stimulated by agonists, particularly those that act by activating PKC (Burgoyne et al, PhysiolRev 2003; 83: 581-632; Logan et al J Allergy Clin Immunol 2003; 111: 923-932; Smolene et al, Biochim Biophys Acta; 1052: 133-142; Niessen et al, Biochim. Biophys.acta 1994; 1223: 267-273; Naucler ukbiol 2002; Leoc Biol 2002; 71: 701-710).
In one aspect of the invention, a degranulation-inhibiting amount of MANS peptide or an active fragment thereof as described herein is administered to a site of inflammation in a subject (the site of inflammation being a result of a disease, disorder, trauma, invasion by foreign objects, and combinations thereof at the site of inflammation in the subject), the administration reducing the amount of inflammatory mediators released by inflammatory site-infiltrating leukocytes, wherein the leukocytes are preferably granulocytes. Administration of MANS peptide and/or at least one active fragment thereof may reduce the amount of inflammatory mediators released by leukocytes, such as granulocytes, infiltrating into the site of inflammation. A degranulation-inhibiting amount of MANS peptide or a degranulation-inhibiting amount of an active fragment thereof is sufficient to reduce or inhibit exocytosis release of inflammatory mediators from granules contained within inflammatory cells infiltrating said site. The degranulation inhibition efficacy is determined at a time after administration of the MANS peptide or active fragment thereof, which can be determined by comparing the percent inhibition (i.e., percent reduction) of the level or amount or concentration of inflammatory mediators released by the cells (leukocytes or granulocytes or other inflammatory cells) relative to the level or amount or concentration of the inflammatory mediators released or produced at about the same time in the absence of the MANS peptide and/or in the absence of the active fragment thereof. In addition, one skilled in the art can determine whether inflammation has been reduced at the tissue site by measuring symptoms or inflammatory parameters known to be indicative of the disease, in order to determine whether a sufficient or therapeutically effective amount of MANS peptide and/or active fragments thereof has been administered. A sufficient degranulation-inhibiting amount means that the degranulation-inhibiting amount reduces the percentage of inflammatory mediators released from granulocytes at the site of inflammation by from about 1% to about 99%, preferably from 5% to about 99%, more preferably from about 10% to about 99%, even more preferably from about 25% to 99%, and even more preferably from about 50% to about 99%, as compared to the amount of said inflammatory mediators released from said granulocytes as determined under the same conditions in the absence of said MANS peptide or active fragment thereof.
In one aspect of the invention, a degranulation-inhibiting amount of MANS peptide is administered to an inflammatory stimulation site in an animal generated by administering an inflammatory stimulating amount of an inflammatory stimulus to the site, the administration being such that the amount of inflammatory mediators released by granulocytes stimulated by the inflammatory stimulus at the site of inflammatory stimulation is reduced by about 1% to about 99%, preferably 5% to about 99%, more preferably about 10% to about 99%, even more preferably about 25% to 99%, and even more preferably about 50% to about 99%, as compared to the amount of inflammatory mediators released from granulocytes in the presence of the same amount of the inflammatory stimulus but in the absence of MANS peptide.
In another aspect of the invention, a degranulation-inhibiting amount of MANS peptide is administered to an inflammatory stimulation site in an animal (the inflammatory stimulation site being generated by administering an inflammatory stimulating amount of an inflammatory stimulus to the site) such that the amount of inflammatory mediators released from granulocytes at the inflammatory stimulation site that are stimulated by the inflammatory stimulus is reduced by 100% as compared to the amount of inflammatory mediators released from the granulocytes in the presence of the same inflammatory stimulating amount of the inflammatory stimulus but in the absence of the MANS peptide.
An example of an inflammatory stimulant used in the in vitro examples herein is phorbol (12-) myristate (-13-) acetate (PMA). Monocyte chemoattractant protein (MCP-1) is almost as effective as C5a in that it releases histamine from basophils degranulation, and is much stronger than IL-8. Stimulation with chemokines (i.e., chemoattractant cytokines), RANTES, and MIP-1 can cause histamine release.
In preferred embodiments, the degranulation-inhibiting amount of the MANS peptide administered at the site of inflammatory stimulation in an animal is from about 1-fold to about 1,000,000-fold greater than the concentration of the MARCKS peptide at the site of inflammatory stimulation, preferably from about 1-fold to about 100,000-fold greater than the concentration of the MARCKS peptide at the site of inflammatory stimulation, more preferably from about 1-fold to about 10,000-fold greater than the concentration of the MARCKS peptide at the site of inflammatory stimulation, even more preferably from about 1-fold to about 1,000-fold greater than the concentration of the MARCKS peptide at the site of inflammatory stimulation, even more preferably from about 1-fold to about 100-fold greater than the concentration of the MARCKS peptide at the site of inflammatory stimulation, and even more preferably from about 1-fold to about 10-fold greater than the concentration of the MARCKS peptide at the site of inflammatory stimulation.
In a preferred embodiment, the granulocytes are located on or within the airways of an animal, preferably a human, and the MANS peptide is administered by inhalation, e.g. by inhalation of a pharmaceutical composition comprising the MANS peptide, e.g. a pharmaceutical composition comprising the MANS peptide and an aqueous solution, said composition being administered in the form of an aerosol, or a pharmaceutical composition comprising the MANS peptide in dry powder form, said composition being administered by a dry powder spray inhaler. Other methods and devices known in the art for administering solutions or dry powders by inhalation may also be used, such as droplets, sprays, and nebulizers.
In some embodiments, it is possible that the peptides of the invention can block secretory processes of physiological importance, including basal secretory functions. Without intending to be limited by any particular theory of the invention, it is believed that the mechanism of modulating basal secretion is different from the mechanism of modulating stimulated secretion. Alternatively, the basal secretion mechanism may require less MARCKS protein than stimulated secretion. Basal secretion is retained since all treatments that block MARCKS-mediated secretion do not result in complete loss of MARCKS function.
As used herein, the term "MARCKS nucleotide sequence" refers to any nucleotide sequence derived from a gene encoding a MARCKS protein, including, for example, DNA or RNA sequences, DNA sequences of the gene, any transcribed RNA sequences, RNA sequences of a precursor mRNA or mRNA transcript, and DNA or RNA bound to a protein.
Accurate delivery of MARCKS-blocking peptides can also overcome any potential limitation of blocking important secretion processes. Such drugs can be readily delivered to the respiratory tract using inhalation-type formulations. Since these drugs are useful for the treatment of inflammatory bowel disease, it is contemplated that these blockers are delivered to the rectum/colon/small intestine by enema or suppository. Intra-articular injection or transdermal delivery into inflamed joints can lead to remission in arthritic or autoimmune patients by reducing local inflammatory cell secretion. Injection into the area around the nerve endings can inhibit the secretion of certain types of neurotransmitters, blocking the transmission of severe pain or intractable muscle spasms. The peptides can be readily delivered for the treatment of inflammatory skin conditions using various topical formulations known in the art.
MARCKS is thought to interact with actin and myosin within the cytoplasm and therefore it is possible to bind (teter) the particles to the cell contraction machinery, thereby mediating subsequent particle movement and exocytosis. The inflammation-mediated secretion of MPO from neutrophils may be maximized by activating PKC and PKG. MARCKS has the potential to act as a junction of these two protein kinases that control secretion from the membrane-bound compartment of inflammatory cells (i.e., secretion of MPO from neutrophils).
The present invention demonstrates that simultaneous activation of PKC and PKG results in enhanced secretion of the canine or human neutrophil inflammatory mediator, MPO, whereas activation of either kinase alone is insufficient to induce maximal secretory responses. The literature reports that PMA alone enhances the secretory response in NHBE cells as well as in neutrophils as demonstrated herein, but the magnitude of the response is much lower than that observed by others in the rat goblet cell line. See Abdullah et al, supra. Furthermore, although cGMP analogues have previously been reported to induce significant mucin secretion by cultured guinea pig tracheal epithelial cells (Fischer et al, supra), it was noted that this response did not reach significant levels until 8 hours of exposure. A secretory response with such a long lag phase is unlikely to be a direct consequence and is likely to involve de novo protein synthesis rather than the release of previously synthesized and stored cytoplasmic granules.
As noted above, the present invention may be used in pharmaceutical formulations. In a particular embodiment, the pharmaceutical product is present in a solid pharmaceutical composition suitable for oral administration. The solid substance composition of the present invention may be formed and mixed with and/or diluted with excipients. The solid composition may also be placed in a carrier, which may be, for example, a capsule, sachet, tablet, paper or other container. If the excipient serves as a diluent, it may be a solid, semi-solid, or liquid material that serves as a vehicle, carrier, or matrix for the composition of matter.
Various suitable excipients are known to those skilled in the art and may be found in National Formulary,19: 2404-. Examples of suitable excipients include, but are not limited to, starch, gum arabic, calcium silicate, microcrystalline cellulose, methacrylate, shellac, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The pharmaceutical product formulations may additionally contain lubricating agents such as talc, magnesium stearate and mineral oil; a humectant; emulsifying and suspending agents; preservatives such as methyl paraben and propyl paraben; a sweetener; or a flavoring agent. Polyols, buffers and inert fillers may also be used. Examples of polyols include, but are not limited to, mannitol, sorbitol, xylitol, sucrose, maltose, glucose, lactose, dextrose, and the like. Suitable buffers include, but are not limited to, phosphate, citrate, tartrate, succinate, and the like. Other inert fillers that may be used include those known in the art and useful in the preparation of various dosage forms. The solid formulation may include other ingredients such as fillers and/or granulating agents and the like, if desired. The pharmaceutical products of the present invention may be formulated so as to enable rapid, sustained or controlled release of the active ingredient after administration to a patient by methods known in the art.
To form an oral tablet, the composition of matter of the present invention can be prepared by direct compression. In this method, the active pharmaceutical ingredient may be mixed with: solid powdered carriers such as lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin, and combinations thereof; and antiwear agents such as magnesium stearate, calcium stearate, and polyethylene glycol waxes. The mixture may then be compressed into tablets using a machine with suitable punches and dies to obtain the desired tablet size. The person skilled in the art may select the operating parameters of the machine. Alternatively, oral tablets may be formed by a wet granulation process. The active pharmaceutical ingredient may be mixed with excipients and/or diluents. The solid material is ground or sieved to obtain the desired particle size. The binder is added to the drug a binder. The binder may be suspended and homogenized in a suitable solvent. The active ingredient and adjuvants may also be mixed with the binder solution. The resulting dry mixture was uniformly humidified with a solution. Wetting typically results in a slight aggregation of the particles, and the resulting material is then passed through a stainless steel screen having the desired pore size. The mixture is then dried in a controlled drying unit for a predetermined length of time to achieve the desired particle size and consistency. The granules of the dry mixture were sieved to remove the powder. A disintegrant, an antiwear agent, and/or an antiadherent agent may be added to the mixture. Finally, the mixture is compressed into tablets using a machine with suitable punches and dies to obtain the desired tablet size. The person skilled in the art may select the operating parameters of the machine.
If a coated tablet is desired, the core prepared as described above may be coated using concentrated solutions of sugar or cellulose polymer, which may contain gum arabic, gelatin, talc, titanium dioxide, or using a lacquer dissolved in a volatile organic solvent or solvent mixture. For this coating, various dyes can be added in order to distinguish tablets with different active compounds or with different amounts of active compound. In one embodiment, the active ingredient is present in the core of the tablet which is coated with one or more layers, including an enteric coating layer.
Soft gelatin capsules can be prepared containing a mixture of the active ingredient and vegetable oil. Hard gelatin capsules may contain granules of the active ingredient together with solid, powdered carriers, for example lactose, sucrose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives, and/or gelatin.
Oral liquid preparations may be prepared in the form of syrups or suspensions, for example, solutions containing the active ingredient, sugar, and a mixture of ethanol, water, glycerol, and propylene glycol. Such liquid formulations may, if desired, include one or more of the following ingredients: coloring agents, flavoring agents, and saccharin. Thickeners such as carboxymethyl cellulose may also be used.
If the above drugs are intended for parenteral administration, the formulation may comprise sterile aqueous injection solutions, non-aqueous injection solutions, or both, containing the composition of matter of the invention. If an aqueous injection solution is prepared, the composition of matter may be in the form of a water-soluble pharmaceutically acceptable salt. Parenteral formulations may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the subject. Aqueous and non-aqueous sterile suspensions may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials. Various sterile powders, granules and tablets of the kind described above may be used to prepare injectables and suspensions for use in the field.
The composition of matter may also be formulated to be suitable for topical application (e.g., a skin cream). These formulations may contain various excipients known to those skilled in the art. Suitable excipients may include, but are not limited to, cetyl esters wax, cetyl alcohol, white wax, glyceryl monostearate, propylene glycol, monostearate, methyl stearate, benzyl alcohol, sodium lauryl sulfate, glycerol, mineral oil, water, carbomer, ethanol, acrylate binders, polyisocyanate binders, and silicone-based binders.
In a preferred embodiment, the peptide fragments are disclosed in Table 2, are at least 4to 23 amino acid residues in length and have an amino acid sequence identical to the amino acid sequence of the MANS peptide, wherein the N-terminal amino acid of the peptide is selected from positions 2 to 21 of the MANS peptide sequence (SEQ ID NO: 1). More preferred peptide fragments are at least 6 amino acids to 23 amino acids in length. Preferably, the N-terminal alpha amino acids of these peptides are acylated, more preferably, the alpha-N-terminal amino acid positions of these peptides are myristoylated.
TABLE 2
Referring to fig. 5, MARCKS is phosphorylated by PKC and then translocated from the membrane to the cytoplasm. Here, PKG appears to induce MARCKS dephosphorylation (fig. 2A, lane 4, and fig. 2B). This dephosphorylation is inhibited by PKG inhibitor Rp-8-Br-PET-cGMPThe reversal (FIG. 2A, lane 5) suggests that dephosphorylation is PKG-dependent specifically. Referring to FIG. 2, using [ [ 2 ] ]32P]Orthophosphate-labeled NHBE cells were then exposed to the indicated reagents. Phosphorylation of MARCKS in response to the above treatments was analyzed by immunoprecipitation assay. Referring to FIG. 2A, 8-Br-cGMP reverses PMA-induced MARCKS phosphorylation, and this effect of 8-Br-cGMP can be reversed by Rp-8-Br-PET-cGMP (PKG inhibitor) or granite acid (PP1/2A inhibitor). Referring to FIG. 2B, PMA-induced MARCKS phosphorylation was reversed by subsequent exposure of cells to 8-Br-cGMP. Lane 1, medium alone for 8 min; lane 2, 100nM PMA treatment for 3 min; lane 3, 100nM PMA treatment for 3 min followed by 1 μ M8-Br-cGMP treatment for 5 min; lane 4, 100nM PMA treatment for 8 min; lane 5, medium alone for 3 min followed by 100nM PMA + 1. mu.M 8-Br-cGMP for 5 min. Referring to FIG. 2C, forstericin (fosstricin) attenuated 8-Br-cGMP-induced MARCKS dephosphorylation in a concentration-dependent manner.
PKG is thought to dephosphorylate MARCKS by activating protein phosphatases. Referring to fig. 2A (lane 6), 500nM of roughage acid (which inhibits both PP 1and PP2A) blocked PKG-induced phosphorylation of MARCKS, suggesting that PKG causes dephosphorylation by activating PP1 and/or PP 2A. Further studies using forsterin and direct determination of phosphatase activity indicated that only PP2A was activated by PKG and caused the removal of the phosphate group of MARCKS (fig. 2C). It is likely that either Gonostoc acid or forskocin (at concentrations that inhibit PKG-induced MARCKS dephosphorylation) attenuated PMA +8-Br-cGMP or UTP-induced mucin secretion (FIG. 3). FIG. 3 helps to demonstrate that PP2A is an essential component of the mucin secretory pathway. NHBE cells were preincubated with either forskocin, okadaic acid (500nM) or medium only at the indicated concentrations for 15 min followed by stimulation with PMA (100nM) +8-Br-cGMP (1. mu.M) for 15 min or UTP (100. mu.M) for 2 h. The secreted mucins were measured by ELISA. Data are expressed as mean values (+/-. s.e. (each point n-6), where represents a significant difference from the media control (p)<0.05)；Representing a marked distinction from PMA +8-Br-cGMP stimulation (p)<0.05); whileRepresentation is significantly different from UTP stimulation (p)<0.05). Thus, dephosphorylation of MARCKS by PKG-activated PP2A appears to be an essential component of the signaling pathway leading to exocytosis of mucin granules.
In order to reveal which molecular events MARCKS links kinase activation to mucin secretion, extensive studies were conducted on MARCKS phosphorylation in response to PKC/PKG activation. Referring to FIG. 1A, PMA (100nM) caused a significant increase (3 to 4 fold) in MARCKS phosphorylation in NHBE cells, which was attenuated by the PKC inhibitor, prohibitin C (500 nM). Once phosphorylated, MARCKS was transferred from the plasma membrane to the cytoplasm (fig. 1B). More specifically, fig. 1A shows that activation of PKC leads to MARCKS phosphorylation in NHBE cells. Use 232P]Orthophosphate-labeled cells 2h were then exposed to stimulatory and/or inhibitory reagents the phosphorylation of MARCKS in response to treatment was analyzed by immunoprecipitation as described above, lane 1, media control, lane 2, vehicle, 0.1% Me.sub.2SO, lane 3, 100nM4 α -PMA, lane 4, 100nM PMA, lane 5, 100 nPMA +500nM prohibitin C, lane 6, 500nM prohibitin C, FIG. 1B confirming the translocation of phosphorylated MARCKS from the plasma membrane to the cytoplasm.32P-labeled cells were exposed to PMA (100nM) or medium only for 5 min, followed by separation of membrane and cytoplasmic fractions. Activation of PKG by 8-Br-cGMP (1 μ M), another kinase activation event required to promote mucin secretion, did not result in MARCKS phosphorylation, but the opposite effect was in fact observed: PMA-induced MARCKS phosphorylation was reversed by 8-Br-cGMP (FIG. 2A). This effect of 8-Br-cGMP was not due to inhibition of PKC activity, since PMA-induced phosphorylation could be reversed by 8-Br-cGMP which was subsequently added to the cells (FIG. 2B). Thus, PKG activation is likely to lead to MARCKS dephosphorylation.
Further studies demonstrated that PKG-induced MARCKS dephosphorylation was blocked by 500nM granite acid, a protein phosphatase (type 1 and/or 2A (PP1/2A)) inhibitor (FIG. 2A, lane 6). Thus, it appears that this dephosphorylation is mediated by PP1 and/or PP 2A. To ensureIn addition to phosphorylation studies, a new, more specific PP2A inhibitor, forskocin (IC), was used to determine the subtype of protein phosphatase involved503.2 nM). Referring to fig. 2C, forskocin inhibited PKG-induced MARCKS dephosphorylation in a concentration-dependent manner (1-500nM), suggesting that PKG induces dephosphorylation by activating PP 2A. To further confirm that PKG activates PP2A in NHBE cells, cells were exposed to 8-Br-cGMP, after which cytosolic PP 1and PP2A activities were determined. At concentrations as low as 0.1. mu.M of 8-Br-cGMP, PP2A activity was still increased by nearly 3-fold (from 0.1 to 0.3nmol/min/mg protein, p)<0.01) while the activity of PP1 remained unchanged. These data indicate that PP2A can be activated by PKG and cause dephosphorylation of MARCKS. Thus, this PP2A activity appears to be crucial for mucin secretion; when PKG-induced MARCKS dephosphorylation was blocked by either taokamic acid or forskocin, secretion in response to PKC/PKG activation or UTP stimulation was improved (fig. 3).
MARCKS binds to actin and myosin in the cytoplasm
FIG. 4 shows a radiolabeled immunoprecipitation assay, which reveals that MARCKS can bind to two other proteins (about 200 and about 40kDa) in the cytoplasm. Referring to FIG. 4, the gene for NHBE cells3H]Leucine and [ alpha ], [ alpha ]3H]Proline was labelled overnight and membrane and cytoplasmic fractions were prepared as described in "experimental methods". The isolated fractions were pre-cleared (preclear) with a non-immune control antibody (6F 6). The cytoplasm was aliquoted in two aliquots for immunoprecipitation in the presence of 10 μ M cytochalasin D (Biomol, Plymout Meeting, Pa.), using anti-MARCKS antibody 2F12 (lane 2) and non-immune control antibody 6F6 (lane 3), respectively. MARCKS protein within the membrane fraction was also assessed by immunoprecipitation using antibody 2F12 (lane 1). The precipitated protein complexes were separated by 8% SDS-polyacrylamide gel electrophoresis and visualized by enhanced autoradiography. MARCKS appears to bind to two cytoplasmic proteins with molecular weights of about 200kDa and about 40kDa, respectively. These two MARCKS-binding proteins were excised from the gel and ionized/time-of-flight mass spectrometry/internal sequencing by matrix-assisted laser desorption (ma)A three-associated laser desorption/time of flight mass spectrometry/internal sequencing (Protein/DNA Technology Center of Rockffer University, N.Y.) was analyzed. Using the peptide masses and sequence data obtained, protein databases were searched by Internet programs supplied and MS-Fit. The results indicated that they were myosin (heavy chain, non-muscle type a) and actin, respectively. Matrix-assisted laser desorption ionization/time of flight mass spectrometry/internal sequence analysis (Matrix-assisted laser desorption/time of flight mass spectrometry/internal sequence analysis) indicated that the two MARCKS-binding proteins were myosin (heavy chain, non-myotype a) and actin, respectively.
These studies suggest that this is a new paradigm for the signaling mechanism controlling exocytosis of airway mucin granules, and this also gives the first direct evidence that MARCKS is believed to have specific biological functions in physiological processes. MARCKS acts as a key mediator molecule that regulates the release of mucin particles within human airway epithelial cells. It is believed that dual activation and synergy of PKC and PKG is required for the initiation of airway mucin secretion. Activated PKC phosphorylates MARCKS, resulting in the translocation of MARCKS from the medial side of the plasma membrane into the cytoplasm. PKG activation in turn activates PP2A, which dephosphorylates MARCKS within the cytoplasm. Since MARCKS' membrane binding capacity is dependent on its phosphorylation state, the dephosphorylated antibody restores its membrane binding capacity to MARCKS and may promote MARCKS attachment to the membrane of cytoplasmic mucin granules. In addition, MARCKS was able to subsequently bind the granule to the cell contraction machinery, mediating granule movement to the cell periphery and subsequent exocytosis release, by interacting with actin and myosin within the cytoplasm (fig. 4). MARCKS is widely distributed, suggesting that this or similar mechanisms may modulate the release of membrane-bound particles in various cell types under normal or pathological conditions.
Referring to fig. 5, MARCKS can function as a molecular linker by interacting with the particle membrane with its N-terminal domain and binding to actin filaments at its PSD site, thereby binding the particle to the contractile scaffold responsible for motility and exocytosis. Figure 5 presents one possible mechanism that depicts the interaction of the mucin secretagogue (secretogogue) with airway epithelial (goblet) cells and the activation of two different protein kinases, PKC and PKG. Activated PKC phosphorylates MARCKS, causing MARCKS to translocate from the plasma membrane to the cytoplasm, while PKG, activated through the Nitric Oxide (NO) → GC-S → cGMP → PKG pathway, in turn activates cytoplasmic PP2A, which dephosphorylates MARCKS. This dephosphorylation stabilizes MARCKS attachment to the particle membrane. In addition, MARCKS also interacts with actin and myosin, thereby linking the particles to the cell contractile machinery for subsequent movement and exocytosis to release inflammatory mediators, such as MPO. After MARCKS is released into the cytoplasm, it can also be directed by specific targeting proteins or some other form of protein-protein interaction in which the N-terminal domain of MARCKS is involved, allowing MARCKS to attach to the granule. In any case, the MANS peptide, or an active fragment thereof comprising at least 4 amino acids, competitively inhibits MARCKS targeting of mucin granule membranes, thereby inhibiting secretion.
The invention also relates to novel methods for blocking exocytosis processes of any cell, particularly those releasing inflammatory mediators from granules contained within inflammatory cells, wherein the stimulatory pathway involves the Protein Kinase C (PKC) substrate MARCKS protein and the release of components from membrane-bound vesicles. In particular, the inventors have demonstrated that MANS peptide can block the stimulated release of inflammatory mediators myeloperoxidase from human (fig. 6) or canine (fig. 7) neutrophils in a concentration-dependent manner. Specifically, FIG. 6 shows that isolated neutrophils secrete Myeloperoxidase (MPO) following stimulation with 100nM PMA and 10. mu.M 8-Br-cGMP. 100 μ M MANS peptide reduced the secretion of MPO to control levels (═ p < 0.05). 10 μ M MANS resulted in a slight decrease in MPO secretion. Control peptide (RNS) at 10 or 100. mu.M did not affect MPO secretion. Referring to FIG. 7, isolated neutrophils secrete Myeloperoxidase (MPO) following stimulation with 100nM PMA and 10. mu.M 8-Br-cGMP. 100 μ M MANS peptide reduced the secretion of MPO to control levels (═ p < 0.05). 10 μ M MANS resulted in a slight decrease in MPO secretion. Control peptide (RNS) at 10 or 100. mu.M did not affect MPO secretion. Thus, the peptides can be used therapeutically to block the release of secreted inflammatory mediators by inflammatory cells infiltrating any tissue. Many of these mediators released are mediators that cause extensive tissue damage observed in various chronic inflammatory diseases (i.e., respiratory diseases such as asthma, chronic bronchitis, and COPD; inflammatory bowel diseases including ulcerative colitis and crohn's disease; autoimmune diseases; skin diseases such as rosacea, eczema; and severe acne, arthritis, and pain syndromes such as rheumatoid arthritis and fibromyalgia). The invention is useful in the treatment of diseases such as arthritis, chronic bronchitis, COPD and cystic fibrosis. Thus, the present invention is useful for treating human and animal diseases, particularly those involving horses, dogs, felines, and other domestic pets.
FIGS. 8-12 show MPO secretion in humans and dogs. In all these experiments, LPS (at a concentration of 1X 10) was used-6M) stimulation of isolated neutrophils for 10 min, 37 ℃, then assume stimulus as shown in the figure. LPS initially activates these cells, so they are able to respond to secretagogues.
In one embodiment, the present invention discloses a method of modulating inflammation in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a MANS peptide or an active fragment thereof. In one aspect of this embodiment, said active fragment of MANS protein comprises at least 4 and preferably 6 amino acids. In another aspect, the cause of the inflammation is a respiratory disease, an intestinal disease, a skin disease, an autoimmune disease, and a pain syndrome. In another aspect, the respiratory disease is selected from asthma, chronic bronchitis, and COPD. In another aspect, the intestinal disease is selected from ulcerative colitis, crohn's disease, and irritable bowel syndrome. In another aspect, the skin disorder is selected from the group consisting of rosacea, eczema, psoriasis, and severe acne. In another aspect, the inflammation is caused by arthritis or cystic fibrosis. In another aspect, the subject is a mammal. In yet another aspect, the mammal is selected from the group consisting of humans, dogs, horses, and felines. In another aspect, the administering step is selected from the group consisting of topical administration, parenteral administration, rectal administration, pulmonary administration, nasal administration, inhalation, and oral administration. In another aspect, the pulmonary administration is selected from the group consisting of an aerosol, a dry powder spray inhaler, a metered dose spray inhaler, and a nebulizer.
In another embodiment, the present invention discloses a method for modulating a cellular secretory process in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition comprising at least one compound comprising a MANS peptide, or an active fragment thereof, which modulates an inflammatory mediator in the subject. In one aspect of this embodiment, said active fragment of MANS protein comprises at least 4, and preferably 6 amino acids. In another aspect, the modulating the secretory process of the cell is blocking or reducing the secretory process of the cell. In another aspect, the cause of the inflammation is a respiratory disease, an intestinal disease, a skin disease, an autoimmune disease, and a pain syndrome. In another aspect, the respiratory disease is selected from asthma, chronic bronchitis, and COPD. In another aspect, the intestinal disease is selected from ulcerative colitis, crohn's disease, and irritable bowel syndrome. In another aspect, the skin disorder is selected from the group consisting of rosacea, eczema, psoriasis, and severe acne. In another aspect, the inflammation is caused by arthritis or cystic fibrosis. In another aspect, the subject is a mammal. In another aspect, the mammal is selected from the group consisting of humans, dogs, horses, and felines. In another aspect, the administering step is selected from the group consisting of topical administration, parenteral administration, rectal administration, pulmonary administration, nasal administration, inhalation, and oral administration. In another aspect, the pulmonary administration is selected from the group consisting of an aerosol, a dry powder spray inhaler, a metered dose spray inhaler, and a nebulizer.
In another embodiment, the invention discloses a method of reducing inflammation in a subject comprising administering a therapeutically effective amount of a compound that inhibits MARCKS-related inflammatory mediator release, such that the release of inflammatory mediators is reduced in the subject compared to what would occur in the absence of the treatment. In one aspect of this embodiment, the compound is at least one active fragment of a MARCKS protein. In another aspect, the active fragment is at least 4 and preferably 6 amino acids in length. In another aspect, the compound is a MANS peptide or an active fragment thereof. In another aspect, the compound is an antisense oligonucleotide against the coding sequence of a MARCKS protein or an active fragment thereof. In another aspect, the active fragment is at least 4 and preferably 6 amino acids in length.
In another embodiment, the invention discloses a method of reducing inflammation in a subject comprising administering a therapeutically effective amount of a pharmaceutically active composition comprising a compound that inhibits MARCKS-related inflammatory mediator release, thereby causing inflammation in the subject to be reduced as compared to what would occur in the absence of the treatment. In one aspect of this embodiment, the compound is an active fragment of a MARCKS protein. In another aspect, the active fragment is at least 4 and preferably 6 amino acids in length. In another aspect, the compound is a MANS peptide or an active fragment thereof. In another aspect, the compound is an antisense oligonucleotide against the coding sequence of a MARCKS protein or an active fragment thereof. In another aspect, the active fragment is at least 4 and preferably 6 amino acids in length. The invention is intended to encompass compositions containing one or more of said MANS peptides or active fragments thereof and their use in therapy to inhibit the release of inflammatory mediators from particles or vesicles of inflammatory cells.
In another embodiment, the invention also discloses a method of reducing or inhibiting inflammation in a subject comprising administering a therapeutically effective amount of at least one peptide comprising a MANS peptide, or an active fragment thereof, effective to inhibit the release of inflammatory mediators at the site of inflammation. In one aspect of such embodiments, the active fragment is at least 4 and preferably at least 6 amino acids in length. In another aspect, the cells producing the inflammatory mediators are selected from the group consisting of neutrophils, basophils, eosinophils, monocytes and leukocytes. Preferably, the cell is a leukocyte, more preferably a granulocyte, and even more preferably a neutrophil, basophil, eosinophil, or a combination thereof. In another aspect, the medicament is administered orally, parenterally, intracavity, or via the airways. In another aspect, the composition further comprises a second molecule selected from the group consisting of an antibiotic, an antiviral compound, an antiparasitic compound, an anti-inflammatory compound, and an immunosuppressant.
An active fragment of MANS peptide is selected from the peptides in table 1. As described herein, these peptides contain optional chemical moieties at their N-terminal and/or C-terminal amino acids.
In another aspect of the invention, the methods of the invention may be practiced by using or administering a combination of the peptides of the invention disclosed in table 1, i.e., one or more of these peptides may be used or administered. Preferably, a single peptide is used or administered in the methods of the invention.
Degranulation of cells selected from the group consisting of neutrophils, eosinophils, monocytes/macrophages and lymphocytes can be reduced in response to protein kinase c (pkc) activation by inflammatory stimuli by preincubation or co-incubation with the same peptides as the N-terminal region of the MARCKS protein, wherein said peptides are selected from the group consisting of the MANS peptide fragments disclosed in table 1. In all cases, MANS peptide reduced PKC-induced degranulation, although time course and concentration may vary between cell types.
Having described the invention, the invention will be illustrated in conjunction with specific examples which are included herein for purposes of illustration only and are not intended to limit the scope of the invention.
Examples
Materials and methods
Radiolabeled immunoprecipitation assay Orthomson tag [ 2 ]32P]Phosphate when the cells were preincubated for 2h with phosphate-free DMEM medium (Dulbecco's modified Eagle's medium) containing 0.2% bovine serum albumin, followed by 0.1mCi/ml [ [ solution ] ]32P]Orthophosphate (9000Ci/mmol, Perkinelmer Life Sciences) was labeled for 2 h. To mark [ 2 ]3H]Myristic acid or3An H-amino acid, and incubating the cells with a medium containing 50. mu. Ci/ml [ alpha ], [ solution ] overnight3H]Myristic acid (49Ci/mmol, Perkinelmer Life Sciences) or 0.2mCi/ml [ 2 ], [ solution of3H]Leucine (159Ci/mmol, Perkinelmer Life Sciences) and 0.4mCi/ml [ 2 ]3H]Proline (100Ci/mmol, Perkinelmer Life sciences). After labeling, the cells were exposed to the stimulating agent for 5 minutes. If an inhibitor is used, the cells are preincubated with the inhibitor for 15 minutes prior to stimulation. After the treatment, the cells were lysed with a buffer containing 50mM Tris-HCl (pH7.5),150mM NaCl,1mM EDTA, 10% glycerol, 1% Nonidet P-40,1mM phenylmethylsulfonyl fluoride, 1mM benzamidine, 10. mu.g/ml pepstatin A and 10. mu.g/ml leupeptin. Trichloroacetic acid precipitation and scintillation counting can determine the radiolabelling efficiency within each culture. Immunoprecipitation of MARCKS proteins was performed using cell lysates (with same counts/min) following the method of Spizz and Blackshear. Spizz et al, J.biol.chem.271,553-562 (1996). The precipitated proteins were separated by electrophoresis on an 8% SDS-polyacrylamide gel and visualized by autoradiography. Anti-human MARCKS antibody (2F12) and non-immune control antibody (6F6) were used in the assay.
To analyze MARCKS or MARCKS-related protein complexes in different subcellular fractions, the radiolabeled and treated cells were scraped into homogenization buffer (50mM Tris-HCl (pH7.5),10mM NaCl,1mM EDTA,1mM phenylmethylsulfonyl fluoride, 1mM benzamidine, 10. mu.g/ml pepstatin A, 10. mu.g/ml leupeptin) and subsequently disrupted by the nitrogen cavitation (800 psi, 20 min, 4 ℃). The cell lysate was centrifuged at 600x g for 10 minutes at 4 ℃ to remove nuclei and unbroken cells. The enucleated supernatant was separated into membrane and cytoplasmic fractions by ultracentrifugation at 400,000x g for 30 minutes at 4 ℃. The membrane pellet was dissolved in lysis buffer by sonication. Immunoprecipitation was then performed as described above.
MARCKS-related peptides-myristoylated N-terminal sequence (MANS) and random N-terminal sequence (RNS) peptides were synthesized in Genemed Synthesis, inc. (San Francisco, Calif.) and subsequently purified by high pressure liquid chromatography (> 95% pure), which showed one single peak with appropriate molecular mass, respectively, as confirmed by mass spectrometry. The sequence constituting the MANS peptide is identical to the first 24 amino acids of MARCKS, the N-terminal region mediating myristoylation of MARCKS inserted into the membrane, MA-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1, where MA is the N-terminal myristoyl chain.) the amino acid composition of the corresponding control peptide (RNS) is identical to MANS, but arranged randomly, MA-GTAPAAEGAGAEVKRASAEAKQAF (SEQ ID NO: 232.) the hydrophobic myristate moiety contained in these synthetic peptides enhances their plasma membrane permeability, allowing the peptides to be readily taken up by cells.
Antisense oligonucleotides-MARCKS antisense oligonucleotides and their corresponding control oligonucleotides were synthesized at Biognostik GmbH (Gottingen, Germany). NHBE cells were treated with 5. mu.M antisense or control oligonucleotides on top for 3 days (2. mu.g/ml cationic liposomes (lipofectin) were present during the first 24 h). Cells were then incubated with secretagogues and assayed for mucin secretion by ELISA. Total RNA and protein were isolated from the treated cells. MARCKS mRNA was determined by Northern hybridization according to a conventional method using human MARCKS cDNA as a probe. MARCKS protein levels were determined by immunoblotting using purified anti-MARCKS IgG1 (clone 2F12) as the primary detection antibody.
Transient transfection-the Phosphorylation Site Domain (PSD) of MARCKS contains a PKC-dependent phosphorylation site and an actin filament binding site. To construct a PSD-deleted MARCKS cDNA, two fragments (encoding 25 amino acids) flanking the PSD sequence were generated by polymerase chain reaction and subsequently ligated by an XhoI site that was ligated to the 5' -end of an oligonucleotide primer designed for polymerase chain reaction. The resulting mutant cDNA and wild-type MARCKS cDNA were inserted into the mammalian expression vector pcDNA4/TO (Invitrogen, Carlsbad, Calif.), respectively. The isolated recombinant constructs were confirmed by restriction enzyme digestion and DNA sequencing.
HBE1 is in the form of a nippleA viral transformed human bronchial epithelial cell line, capable of secreting mucins when cultured in an air/liquid interface. HBE1 cells were transfected using the Effectene transfection reagent (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. Briefly, differentiated HBE1 cells grown in the air/liquid interface were dissociated by trypsin/EDTA and plated at 1x105Individual cell/cm2Cells were transfected with wild-type MARCKS cDNA, PSD-truncated MARCKS cDNA, or vector DNA after overnight incubation the cells were cultured for 48h for gene expression followed by exposure TO secretagogues and mucin secretion determined by ELISA all transfections were performed in the presence of pcDNA4/TO/lacZ plasmid (Invitrogen) (DNA ratio 6:1, total 1 μ g DNA, ratio of DNA TO efectene reagent 1:25) TO monitor changes in transfection efficiency.
Protein phosphatase Activity assay-PP 1and PP2A activity was determined using a protein phosphatase assay system known in the art (Life technologies, Inc.), with minor modifications in the method Huang et al, adv. exp. MedBiol.396,209-215 (1996). briefly, NHBE cells were treated with 8-Br-cGMP or medium only for 5 minutes, then cells were scraped into lysis buffer (50mM Tris-HCl (pH7.4), 0.1%. β. -mercaptoethanol, 0.1mM EDTA,1mM beneamidine, 10. mu.g/ml pepstatin A, 10. mu.g/ml leupeptin) and sonicated for 20 seconds to disrupt, 4. centrifugation of the cell lysate and collection of supernatant for phosphatase activity assay32The assay was performed with P-labeled phosphorylase A as a substrate. Released by scintillation counting32Pi. The protein concentration of each sample was determined by Bradford assay. PP2A activity was expressed as the total phosphatase activity of the sample minus the activity remaining in the presence of 1nM of roughcast acid. PP1 activity was expressed as the difference between the residual activity in the presence of 1nM and 1. mu.M respectively of roughhaired acid. P released by Per mg Total protein per minutei(nmol) to report protein phosphatase activity.
Cytotoxicity assay-cytotoxicity of various reagents used to treat NHBE cells was detected by measuring total lactate dehydrogenase released by the cells. The assay was performed using the Promega Cytotox96Kit according to the manufacturer's instructions. All experiments were performed with non-toxic concentrations of the reagents.
Statistical analysis-the significance of the data was analyzed using one-way anova with post-Bonferroni-test corrections (Bonferroni post-testcorrections). Differences between different treatments were considered significant when p < 0.05.
Isolation of PMNs from dog blood-the step involved in PMN isolation involved collection of 10ml of ACD anticoagulation. Then 5ml of the layer was spread over 3.5ml of PMN separation medium while ensuring that the PMN separation medium (IM) was at room temperature (RI). The blood was then centrifuged at room temperature for 30 minutes, 550X g, 1700 RPM. The lower white band layer was transferred to a 15ml conical centrifuge tube (CCFT). Subsequently, 2V HESS containing 10% fetal bovine serum (PBS) was added and centrifuged at room temperature for 10 minutes at 400X g, 1400 RPM. The pellet was resuspended in 5ml HESS containing PBS. The cell suspension was added to 50ml of ice-cold 0.88% NH containing 20ml4In CCFT of Cl, invert 2 to 3 times. The resulting product was centrifuged for 10 minutes at 800X g at 2000RPM, then aspirated and resuspended in 5ml HBSS containing FBS. Preparations are tested by counting and cytospin, and preferably, for whole blood, the cell number should be 109-1011Between cells, and for PMNs, the cell number should be between 2-4x107Between individual cells. See Wang et al, J.Immunol., "neutral-induced changes in the biological properties of endellial cells: rolls of ICAM-1and reactive oxygen species,"6487-94 (2000).
MPO enzyme chromogenic quantitative assay-samples were assayed for MPO activity in 96-well round-bottomed microtiter plates using an ELISA kit (R & D Systems, Minneapolis, Minn.). Briefly, 20. mu.l of sample was mixed with 180. mu.l of substrate mixture (containing 33mM potassium phosphate, pH6.0, 0.56% Triton X-100,0.11mM hydrogen peroxide and 0.36mM O-Diannidine dihydrate) in each microwell. The final concentration in the assay mixture was: 30mM potassium phosphate, pH6.0, 0.05% Triton X-100,0.1mM hydrogen peroxide and 0.32mM O-dianisidine dihydrate. Upon mixing, the assay mixture was incubated at room temperature for 5 minutes, and then the MPO enzyme activity was determined spectrophotometrically at 550 nm. Samples were assayed in duplicate.
Example 1: inflammatory mediator secretion study
Four different leukocyte types or models were used that secreted specific particulate components in response to activation of PKC induced by phorbol esters. Neutrophils were isolated from human blood and assayed for the release of MPO by these cells in vitro. Commercial human leukocyte cell lines were also evaluated for the release of membrane-bound inflammatory mediators. EPO release was analyzed using the early granulocyte cell line HL-60 clone 15 (Fischkoff SA. Graded increase in promoter region 1988; 12: 679. 686; Rosenberg HF, Ackerman S J, Tenen DG. Humanoenosinic promoter region 1989; molecular cloning of a cytotoxic and cytotoxic peptide with promoter region Exp. Med. 170: 163. 176; tifenberg HL, Li F, Rodgersin HF. Biocoding of promoter region 58. Biocoding AP 49. Biocoding J. 9. Biocoding of EPO, 2. 9. Biocoding protein, 2. 9. Biocoding of promoter region J. Biocoding protein, 2. 12. EPO, 2. 7. EPO. 7. origin, 2. Biocoding protein, 8. Biocoding protein, 2. 12. with the present. Lysozyme release was analyzed using the monocytic leukemia cell line U937 (Hoff T, Spenker T, EmmenoterferA., Goppelt-Struebe M.Effect of glucoortoids on the TPA-induced monoclonal differentiation. J Leukoc Biol 1992; 52: 173. sub.182; Balboa M A, Saez Y, Balsinde J.Calif. -indeno phospholyase A2is obtained for lysozyme precipitation in U.J. Immunol 2003; 170: 5276. sub.5280; Sundstrom C, Nilson K.Establysis and characteri of human husbandolitic cytolyies. J.Immunol 2003; 170: 5276. sub.79; Sundstrom C, Nilson K.Estaphylostimul and charactein J.577; 197577). Lymphocyte natural killer Cell line NK-92 was used to analyze granzyme release (Gong JH., Maki G, Klingemann HG. characteristics of a human Cell (NK-92) with photonic and functional characteristics of activated and natural killer cells Leukemia1994, 8: 652. invertebra 658; Maki G, Klingeman HG, Martinson JA, Tam YK. factor regulating the cytotoxic activity of the human naturai Cell line, NK-92.J promoter Stem Res 2001; 10: 369-383; Takakayama H, Trenng, Sitkovkoy. A novel cytotoxic T Cell 183. 198190. method 104. J. M. K. cells et al., K.K. K. K. In all cases, cells were preincubated with a range of concentrations of synthetic peptide identical to the N-terminus of MARCKS of 24 amino acids (MANS-myristoylated N-terminal sequence peptide; MA-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), where MA is myristoyl linked to the N-terminal amine of the peptide by an amide bond, or a missense control peptide (RNS: random N-terminal sequence peptide; MA-GTAPAAEGAGAEVKRASAEAKQAF, SEQ IDNO:232) consisting of 24 amino acids identical to the sequence of MANS peptide but arranged in a random order of these amino acids with sequence identity of less than 13% to the sequence of MANS peptide. Alternatively, the cells were treated with synthetic truncated peptides as listed in table 3 below.
MANS reduces inflammatory mediator release from various cell types in a concentration-dependent manner, whereas RNS does not. Useful observation periods are 0.5-3.0 hours. These results are consistent with the belief that the N-terminal region of MARCKS protein is involved in intracellular pathways leading to degranulation of leukocytes.
Human neutrophil isolation-these studies were approved by the human research Institute Review Board (IRB). Human neutrophils were isolated as previously described (see Takashi S, OkuboY, horie S. Contription of CD54to human eosinophil and neutrophile superoxideproduction. J Appl Physiol 2001; 91: 613-. Briefly, heparin-anticoagulated venous blood was obtained from normal healthy volunteers, diluted 1:1 with RPMI-1640 (Cellgro; Mediatech, Inc., Herndon, Va.), layered over Histopaque (density 1.077 g/ml; Sigma-Aldrich Co., St. Louis, Mo.) and centrifuged at 400g for 20 minutes, 4 ℃. The supernatant and mononuclear cells at the interface were carefully removed and the red blood cells in the pellet were lysed in ice cold distilled water. The isolated granulocytes were washed twice with Hanks Balanced Salt Solution (HBSS) and then resuspended in HBSS and placed on ice. The purity of neutrophils used in the experiment was > 98%, eosinophil contamination < 2%, and cell viability > 99% as determined by trypan blue dye exclusion.
Measurement of released neutrophil MPO Activity-to measure the release of MPO, purified human neutrophils suspended in HBSS were aliquoted as 4 × 106Cells/ml were placed in 15ml tubes and preincubated with 50 or 100. mu.M MANS, RNS, or a peptide of the invention for 10 minutes at 37 ℃ and then stimulated with 100nM (12-) phorbol myristate (-13-) acetate (PMA) for 3 hours A control reference (PMA control reference) was created by aliquoting purified human neutrophils 4 × 10 suspended in HBSS6Cells/ml were placed in 15ml tubes and stimulated with 100nM (12-) phorbol myristate (-13-) acetate (PMA) for the same time in the absence of test peptide. The reaction was terminated by placing the tube on ice and centrifuging at 400g for 5 minutes at 4 ℃.
The MPO activity in cell supernatants was analyzed using Tetramethylbenzidine (TMB) based on previously established techniques (Abdel-Latif D, Steward M, Macdonald DL, Francis GA., Dinauer MC, Lacy P. Rac2is diagnostic for neutrophilic coating exocytosis. BLOOD 2004; 104: 832-839). Briefly, 100 μ L of TMB substrate solution was added to 50 μ L of cell supernatant or human MPO standard (EMDBiosciences, inc., San Diego, CA) in a 96-well microplate, followed by incubation for 15 minutes at room temperature. 50 μ L of 1M H was added2SO4The reaction was terminated and absorbance at 450nm was measured using a spectrophotometric microplate reader (VERSA max, Molecular Devices, Sunnyvale, Calif.).
Study of leukocyte culture
Three human leukocyte cell lines, specifically the promyelocytic cell line HL-60 clone 15, the monocyte cell line U937, and the lymphocyte natural killer cell line NK-92, were purchased from American type culture Collection (ATCC; R)ockville, MD). HL-60 clone 15 cells (ATCC CRL-1964) were cultured in a medium consisting of RPMI1640 containing L-glutamine supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Invitrogen Co., Carlsbad, Calif.), 50IU/mL penicillin, 50. mu.g/mL streptomycin and 25mM HEPES buffer, pH7.8 at 37 ℃ with 5% CO2In the environment of (2). Cells were cultured by washing at 5X10 in the above medium containing 0.5mM butyric acid (Sigma-Aldrich Co.)5Each cell/ml was cultured for 5 days to finally differentiate into an eosinophil-like phenotype as described previously (Tiffany HL, Li F, Rosenberg HF. Hyperglycosylation of eosinophiribosomal nuclei in a myelocytic leukemia cell line and in a differential viral population promoter cell J Leucoc Biol 1995; 58: 49-54; Tiffany HL, Alkhatib G, Combadiere C, Berger EA, Murphy PM. CC chemokinesin receptors1and3 imaging regulation by IL-5 discrete mutation information of eosinophii HL-60cells J1998; 160: 1385. 1392). U937 cells (ATCC CRL-1593.2) contained 5% CO at 37 ℃2NK-92 cells (ATCC CRL-2407) were cultured in α -MEM medium (Sigma-Aldrich Co.) supplemented with 20% FBS, 100U/mL interleukin-2 (IL-2) (Chemicon International, Inc., Temecula, Calif.), 5x10-5M2-mercaptoethanol, 50IU/mL penicillin, and 50. mu.g/mL streptomycin, incubated at 37 ℃ with 5% CO2In the environment of (2). Cells were analyzed by Wright-Giemsa staining to assess cell morphology. Viability of the cells collected for the experiment was determined by trypan blue dye exclusion and used>95% of the cell population.
Incubating cells for degranulation assays
HL-60 clone 15, U937 and NK-92 cells were washed with 2.5 × 106The density of individual cells/ml was resuspended in phenol red free RPMI-1640 (Cellgro; Mediatech, Inc.) and used in all degranulation assays. Aliquots of cells were placed in 15ml tubes and preincubated with the indicated concentrations of MANS, RNS, or test peptide for 10 minutes at 37 ℃. Subsequently usePMA stimulation of cells for 2 hours control (PMA control) was established for each cell type using HL-60 clone 15, U937, and NK-92 cells, respectively, which were washed and washed with 2.5 × 106The density of individual cells/ml was resuspended in phenol red-free RPMI-1640 and stimulated with PMA for the same time in the absence of MANS, RNS, or test peptide. The reaction was terminated by placing the tube on ice and centrifuging at 400g for 5 minutes at 4 ℃.
For the determination of MPO released from neutrophils and lysozyme released from U937 cells, we can quantify the secretion by using human MPO and ovalbumin, respectively, as standards. There were no standards for quantification of EPO released from HL-60 clone 15 cells and granzyme released from NK-92 cells. Thus, both the EPO and granzyme levels released and intracellular (from lysed cells) were determined and expressed as a percentage of the total amount of each (intracellular plus released). To determine intracellular EPO of HL-60 clone 15 cells and intracellular granzymes of NK-92 cells, an appropriate amount of 0.1% triton X-100-lysed cells were taken for quantification of intracellular granule proteins, as described below. All treatments were expressed as a percentage of the control in order to minimize culture-to-culture variability.
Determination of HL-60EPO Release
The EPO activity released by HL-60 clone 15 cells was determined by TMB according to a previously established technique (Lacy P, Mahmudi-Azer S, Bablitz B, Hagen SC, Velazquez JR, Man SF, Moqbel R. Rapid diagnosis of intracellular stored RANTES in response to interference-gamma human eosinophiles. Blood 1999; 94: 23-32). Thus, 100 μ L of TMB substrate solution was added to 50 μ L (μ L ═ μ L) samples in 96-well microplates and incubated for 15 minutes at room temperature (min ═ min). 50 μ L of 1.0M H was added2SO4The reaction was terminated and absorbance at 450nm (nm ═ nm) was measured using a spectrophotometric microplate reader. The amount of EPO secreted is expressed as a percentage of the total content (the amount obtained using the same number of triton X-100-lysed cells).
Measurement of monocyte lysozyme secretion
The lysozyme secreted by U937 cells was determined spectrophotometrically, as described above, with minor modifications (Balboa M A, Saez Y, Balsamide J.calcium-independent phosphoipase A2is requiredfor lysozyme section in U937promonocytes. J Immunol 2003; 170: 5276-. Thus, 100. mu.L of the sample was mixed with 100. mu.L of a suspension of Micrococcus lysodeikticus (Sigma-Aldrich Co.) dissolved at 0.3mg/ml in 0.1M sodium phosphate buffer, pH7.0 in a 96-well microplate. The decrease in absorbance at 450nm was measured at room temperature. A calibration curve was established using egg white lysozyme (EMD Biosciences, Inc.) as a standard.
Measurement of NK cell granzyme secretion
The granzymes secreted by NK-92 cells were determined by determining the hydrolysis of N α -benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) essentially as described previously (Takayama H, Trenn G, Sitkovsky MV. A novel cytoxic T lysine catalysis assay. J Immunol Methods 1987; 104: 183-. Briefly, 50. mu.L of the supernatant was transferred to a 96-well plate, and 150. mu.L of a BLT solution (0.2mM BLT; EMDBiosciences, Inc., and 0.22mM DTNB; Sigma-Aldrich Co.) (mM. millimolar) dissolved in phosphate buffered saline (PBS, pH7.2) was added to the supernatant. After incubation at room temperature for 30 minutes, the absorbance at 410nm was measured. Results are expressed as a percentage of total intracellular enzyme content (using the amount obtained in the same number of triton X-100-lysed cells).
Statistical analysis
One-way anova was used to evaluate the statistical significance of the differences between the different treatment groups. P values <0.05 were considered significant.
Inhibition of human neutrophil release of MPO
It was found that 100nM PMA (as a stimulator of inflammatory mediator release) increased MPO release from human neutrophils in the PMA control reference by approximately 3-fold at 30 minutes relative to control levels, with a 5-6 fold increase in MPO release after 3 hours. At 30 minutes, the MPO activity of the PMA control reference was approximately 275%, the PMA plus 50 μ M MANS approximately 275%, and the 100 μ M MANS approximately 305%, relative to the control MPO activity (100%) in the absence of PMA and in the absence of PMA plus MANS, RNS, or test peptide. Therefore, the MANS peptide had no detectable effect at 30 minutes. However, by 1 hour, higher concentrations of MANS (100. mu.M) produced significant inhibition (260% of the control) or a reduction of approximately 25% in MPO release relative to the PMA control reference level (approximately 340% of the control). The 50 μ M MANS sample was about 290% of the control or about 15% reduction relative to the PMA control reference. At 2 hours and up to 3 hours, MANS peptide significantly attenuated MPO activity in a concentration-dependent manner. At 2 hours, the PMA control MPO activity was approximately 540% of the control, and 50 μ M MANS (approximately 375% of the control) resulted in an approximately 30% reduction in MPO release relative to the PMA control; whereas 100 μ M MANS (approximately 295% of the control) resulted in an approximately 45% reduction in MPO release relative to the PMA control reference. At 3 hours, the PMA control MPO activity was about 560% of the control, and 50 μ M MANS (about 375% of the control) resulted in about a 33% reduction in MPO release relative to the PMA control; 100 μ M MANS (approximately 320% of the control) resulted in an approximately 40% reduction in MPO release relative to the PMA control reference. The RNS peptide did not affect PMA-induced MPO release at any time point or concentration tested. The data presented in the table below represent the test peptide at a concentration of 100. mu.M and incubated with 100nM PMA for 2 hours.
Inhibiting HL-60cell release of EPO
EPO activity in the supernatants of HL-60 clone 15 cells was significantly enhanced at 1and 2 hours post PMA stimulation. At 1 hour, the PMA control reference was approximately 110% relative to control EPO activity (100%); approximately 95% of samples containing 10 μ M MANS resulted in approximately 15% reduction in EPO activity relative to the PMA control reference; approximately 78% for the 50 μ M MANS containing sample, resulting in an approximately 30% reduction in EPO activity relative to the PMA control reference; whereas the 100 μ M MANS containing sample was approximately 65%, resulting in an approximately 40% reduction in EPO activity relative to the PMA control reference. At 2 hours, the PMA control reference was approximately 145% relative to control EPO activity (100%); approximately 130% for the 10 μ M MANS-containing sample, resulting in an approximately 10% reduction in EPO activity relative to the PMA control reference; approximately 70% for the 50 μ M MANS containing sample, resulting in an approximately 50% reduction in EPO activity relative to the PMA control reference; whereas the 100 μ M MANS containing sample was about 72%, resulting in an approximate 50% reduction in EPO activity relative to the PMA control reference. Thus, at both 1and 2 hours, 50 or 100 μ M MANS significantly attenuated EPO release. The RNS peptide did not affect PMA-enhanced EPO release at any time point or concentration tested. The data presented in the table below represent the test peptide at a concentration of 50. mu.M and incubated with 100nM PMA for 2 hours.
Inhibition of lysozyme release from U937 cells
PMA stimulation increased lysozyme secretion from U937 cells 1 hour after incubation, and increased more at 2 hours. At 1 hour, the PMA control reference was approximately 210% relative to lysozyme secretion (100%) of the control U937 cells; the sample containing 10 μ M MANS was about 170%, resulting in a reduction of lysozyme secretion by U937 cells of about 20% relative to the PMA control reference; the sample containing 50 μ M MANS was about 170%, resulting in a reduction of lysozyme secretion by U937 cells of about 20% relative to the PMA control reference; whereas the sample containing 100. mu.M MANS was about 115%, resulting in a reduction of lysozyme secretion by U937 cells of about 45% relative to the PMA control reference. At 2 hours, the PMA control reference was approximately 240% relative to lysozyme secretion (100%) of control U937 cells; the sample containing 10 μ M MANS was about 195%, resulting in a reduction in lysozyme secretion by U937 cells of about 20% relative to the PMA control reference; approximately 185% of the sample containing 50 μ M MANS resulted in approximately 25% reduction in lysozyme secretion from U937 cells relative to the PMA control reference; whereas the sample containing 100. mu.M MANS was about 140%, resulting in a reduction of lysozyme secretion by U937 cells of about 40% relative to the PMA control reference. Thus, 100 μ M MANS significantly attenuated lysozyme secretion 1and 2 hours after stimulation, while 50 or 10 μ M MANS did not. The RNS peptide did not affect PMA-enhanced lysozyme secretion at any time point or at the concentrations tested. The data presented in the table below represent the test peptide at a concentration of 50. mu.M and incubated with 100nM PMA for 2 hours.
Inhibition of NK-92 cell release granzyme
The lymphocyte natural killer Cell line NK-92 was used to evaluate granzyme release (Gong JH, Maki G, Klingemann HG. characterization of a human Cell line (NK-92) with photonic and functional characteristics of activated natural killer cells. Leukemia8: 652. though.1994; Maki G, Klingeman HG, Martinson JA, Tam YK. factors regulating the cytotoxic activity of the human mammalian Cell line, NK-92.J. promoter. Stem Res. 10: 369. 383, 2001; Takayama H, Trenn G, Sitkovyyyya. A novel cytotoxic T Cell J. 183. medium J. 198190. No. M. medium J. No. 9. 198190. No. 9. M. medium J. Cell. No. 9. No. 5. medium J. No. 5. medium J. No. 8. No. 5. medium J. No. 9. No. 5. medium J. 1987. No. 8, 1987. No. 8, No. 5. No. 5, 2, No..
Measurement of granzyme secretion by NK cells: the granzymes secreted by NK-92 cells were assayed by measuring the hydrolysis of N α -benzyloxycarbonyl-L-lysine thiobenzyl alcohol ester (BLT, EMBBIOSCENCE, Inc.) essentially as described previously (TakayamaH, Trenn G, Sitkovsky MV.A novel cytoxic T lymphocyte activity say.J.Immunol.Methods104:183-190, 1987). An equal amount of 50. mu.L of the supernatant was transferred to a 96-well plate, and 150. mu.L of a 0.2mM BLT solution in phosphate buffered saline (PBS, pH7.2) and 0.22mM DTNB (Sigma-Aldrich Co.) were added to the supernatant. After incubation at room temperature for 30 minutes, the absorbance at 410nm was measured. Results are expressed as a percentage of total intracellular enzyme content (using the amount obtained in the same number of triton X-100-lysed cells).
Since there is no NK-92 cellular granzyme standard available for quantification, we measured both the level of released and intracellular (from lysed cells) granzymes and expressed the released granzymes as a percentage of the respective total amount (intracellular and released). To determine the intracellular granzyme of NK-92 cells, an appropriate amount of 0.1% triton X-100-lysed cells was taken for quantification of the enzyme, as described above. All treatments were expressed as a percentage of the control in order to minimize culture-to-culture variability. The data presented in the table below represent the test peptide at a concentration of 50. mu.M and incubated with 100nM PMA for 2 hours.
Cytotoxicity
None of the above treatments were found to produce toxic reactions in cells by measuring retention/release of LDH (results not shown) (see also Park J-A, He F, Martin LD, Li Y, Adler KB. human neutral electrophoresis semiconductors hyper section of music from human brouchi epithelial cells in vitro via PKC-mediated mechanism. am J Pathol 2005; 167: 651-661).
In preliminary experiments, the peptides given in the table below showed a certain percentage of inhibitory effect on the release of MPO by human neutrophils, EPO by HL-60 clone 15 cells, lysozyme by U937 cells and granzyme by NK-92 cells, where MA-indicates the presence of a myristoyl substituent at the alpha-N-terminal position of the peptide; ac-indicates the presence of an acetyl substituent at the alpha-N-terminal position of the peptide; h represents no group attached to the peptide; NH2 indicates the presence of an amide group at the C-terminal position. Inhibition data are the average of multiple experiments. The qualitative solubility of the peptide at 0.5N saline pH6.5 is shown in mg/mL in Table 3 below. Altering the myristoyl group of the N-terminal chemical moiety can alter the solubility of the peptides disclosed herein in aqueous media. For example, as shown in table 3, changing myristoyl to acetyl resulted in increased water solubility.
Table 3: enzyme inhibition assay results and solubilities of representative and substituted peptides
-------------------------
1N-as N-terminal group
2C ═ C-terminal group
30.5N saline, pH6.5
Example 2: MANS and related peptides inhibit Lipopolysaccharide (LPS) -induced pulmonary inflammation in vivo
This example was carried out essentially as described in the following documents: cox, G, Crossley, j., and Xing, z.; macro enzyme of amorphous neutral lipids to thermal solution of acid pulmony inventory in vivo; am.J.Respir.CellMol.biol.12:232-237, 1995; hirano S., Quantitative time-coarse profiles of branched organic laboratory cells of branched organic insulation of a polar polymeric carbohydrate in mice, Ind. Health35:353-358, 1997; and Ulich TR, Watson LR, YinSM, Guo KZ, Wang P, Thang H, and del Castillo, J.Am.J.Pathol.138: 1485-.
Thus, 6 to 7 week old CD1 female mice weighing 15-20g were obtained from Charles River Laboratories with 5 mice per cage being housed in groups. Animals received standard rodent diet and filtered water ad libitum. Animals were kept under standard temperature (64 ° to 79 ° F) and relative humidity 30 to 70% conditions following NIH guidelines.
5 groups of experimental mice, 5 mice per group, were given the following treatments: PBS followed by PBS; PBS followed by LPS; (myristoylated) MANS peptide followed by LPS; acetylated peptide SEQ ID NO 1 followed by LPS; or the acetylated peptide SEQ ID NO 106 followed by LPS.
Intranasal instillation peptide pretreatment: the peptides of the invention to be analyzed for their ability to inhibit or reduce LPS-induced lung inflammation in vivo are dissolved in PBS at a concentration of 1 mM. Animals were anesthetized by inhalation of 0.8% isoflurane and 2x10 μ L bolus of peptide solution was intranasally instilled into one nostril and LPS was instilled after 30 minutes.
Intranasal instillation of LPS: lipopolysaccharide (LPS) endotoxin (E.coli serotype 011: B4-derived endotoxin; Sigma, St Louis, Mo; see Sigma product information page L4130, titled: Lipopolysaccharides from Escherichia coli011: B4) was dissolved in Phosphate Buffered Saline (PBS) at a concentration of 2,500. mu.g/mL. To expose the animals to endotoxin, 10 μ L of an intranasal bolus of 2,500 μ g/ml endotoxin solution was administered to animals anesthetized by inhalation of 0.8% Isofluorane. A bolus of 10 μ L was applied to one nostril. Animals were monitored for vigorous respiration, lethargy, and decreased water/food intake following endotoxin instillation.
Bronchoalveolar lavage (BAL): 6 hours after the last instillation, the animals were anesthetized (90mg/kg sodium pentobarbital) and sacrificed by exsanguination. Lungs were irrigated 2 times in succession with 1.0mL aliquots of PBS. The collected BAL fluid was centrifuged to extract cells for subsequent counting and sorting analysis. The recovered lavage fluid was used for analysis of total protein, Myeloperoxidase (MPO), LDH and hemoglobin.
And (3) analysis: equal amounts of BAL solution were immediately used to determine LDH, total protein or hemoglobin levels using a COBASS FARA II automated analyzer (Roche Diagnostic Systems Inc., Montclair, NJ). An aliquot of the BAL solution was frozen at-80 ℃ for subsequent quantification of Myeloperoxidase (MPO) by a mouse specific ELISA assay (CellSciences, Inc., Canton, Mass). BAL data were analyzed by standard techniques to check for differences between control and treatment groups. The results presented in the tables below demonstrate that the test peptides can inhibit or reduce inflammation.
Table 4: average value of inflammatory markers in the Presence of MANS peptide MA-GAQFSKTAAKGEAAAERPGEAAVA, SEQ ID NO 1
Table 5: average value of inflammatory markers in the presence of the N-terminal acetylated analog of MANS peptide Ac-GAQFSKTAAKGEAAAERPGEAAVA, SEQ IDNO:1
Table 6: average value of inflammatory markers in the presence of acetylated peptide Ac-GAQFSKTAAK, SEQ ID NO 106
Table 7: MANS peptide (Myr-SEQ ID NO: 1); test peptide (Ac-GAQFSKTAAKGEAAAERPGEAAVA), SEQ ID NO: 1; and Ac-GAQFSKTAAK, SEQ ID NO 106 inhibition of inflammatory markers as compared to PBS/LPS treatment
Treatment protocol Inhibition of neutrophil migration Inhibition of MPO
MANS/LPS 41.4％ 67.2％
SEQ ID NO:1/LPS 35.9％ 18.75％
Ac-SEQ ID NO:106/LPS 88.5％ 79.1％
PBS/PBS represents PBS control alone administered, without LPS endotoxin addition to stimulate chemotactic neutrophil migration; PBS/LPS represents stimulation of chemotactic neutrophil migration by addition of LPS (endotoxin); MANS/LPS representative was pretreated with MANS peptide in PBS followed by LPS stimulation in order to induce neutrophil migration. After MANS peptide treatment, the percentage of neutrophils in the LPS-treated group in the total number of cells was reduced from 41.7% to 30.9%; peptide Ac-GAQFSKTAAKGEAAAERPGEAAVA, SEQID NO 1 treatment reduced it from 65.5% to 41.47%; 106 treatment of the peptide Ac-GAQFSKTAAK, SEQ ID NO reduced it from 24.4% to 3.0%. MPO levels in the LPS-treated group were determined to decrease from 28.98ng/mL to 9.49ng/mL after MANS peptide treatment; peptide treatment with acetylated SEQ ID NO 1 reduced it from 37.9ng/mL to 30.79 ng/mL; whereas treatment of the peptide with acetylated SEQ ID NO 106 reduced it from 7.19ng/mL to 1.50 ng/mL.
Example 3: ozone-induced COPD mouse model
Oxidative stress caused by chemical stimuli such as ozone is a widely recognized feature of chronic obstructive respiratory disease (COPD). See: reine JE, Bast A, Lankhorst I, and the Oxidative Stress student group, am.J.Respir.Crit.Care Med.156:341-357, 1997; and also Harkema JR and Hotchkiss JA, Toxicology Letters,68:251-263, 1993.
10 week old Balb/C female mice were obtained from Charles River Laboratories and 5 mice per cage were housed in groups following the NIH guidelines. Animals received standard rodent diet and filtered water ad libitum. Mice from 3 treatment groups, 5 per group, were anesthetized by intraperitoneal injection of ketamine (100mg/kg) and xylazine (20mg/kg), respectively, followed by pretreatment by intratracheal administration of 25 μ L of the following treatments: PBS only; or 1.0mM MANS peptide in PBS; or a 1.0mM solution of acetylated MANS fragment peptide Ac-GAQFSKTAAK (designated acetylated SEQ ID NO:106) in PBS. After 30 minutes, the animals were placed in a suitable custom room and exposed to ozone or forced air. Animals were exposed to ozone for 2 hours (ozone concentrations 1-10ppm) with minor modifications using the method of Haddad et al,1995 (Haddad E-B, Salmonon M, Sun J, Liu S, DasA, Adcock I, Barnes PJ, and Chung KF, FEBS Letters,363: 285-. Ozone was generated using ozone generating apparatus OL80F/B (Ozonelab, Burton, British Columbia, Canada). Ozone concentration was continuously monitored by a Teledyne Photometric O3Analyzer (model400E, Teledyne Instruments, City of industry, Calif.). The additional two groups of mice were not pretreated and were exposed to ozone under the same conditions or to forced air under conditions similar to the ozone-treated group but lacking ozone, respectively. After exposure the animals were sacrificed by exsanguination and the lungs were irrigated 2 times with 1.0mL aliquots of PBS. The collected bronchoalveolar lavage (BAL) fluid was centrifuged to extract cells for subsequent enumeration and classification analysis. The recovered lavage fluid was used for protein analysis and additional analysis of IL-6, IFN γ, and KC (murine IL-8 analogs) by ELISA assays (kit from R & D Systems, Minneapolis, MN).
The table below gives the percent inhibition of migration of neutrophils to BAL fluid as a function of each treatment group compared to the control group treated with PBS alone.
Table 8: MANS peptide and acetylated SEQ ID NO 106 peptide Ac-GAQFSKTAAK inhibit ozone-induced neutrophil migration
Treatment group Inhibition of neutrophil migration into BAL fluid (%)
MANS + ozone 93.0
Ac-SEQ ID NO 106+ ozone 81.2
PBS + ozone Not applicable to
Forced ventilation only Not applicable to
IL-6 concentration (pg/mL) in BAL fluid as a function of intratracheal injection pretreatment followed by ozone treatment was obtained as follows. IL-6 levels were: approximately 364.5pg/mL in the group of mice pretreated with MANS peptide followed by exposure to ozone; approximately 130.4pg/mL in the group of mice pretreated with the acetylated MANS fragment peptide Ac-GAQFSKTAAK (SEQ ID NO:106) followed by exposure to ozone; approximately 1041.3pg/mL in the group of mice pretreated with PBS followed by exposure to ozone; approximately 43.2pg/mL in the group of mice exposed directly to forced air without any pretreatment.
The KC concentration in BAL solution (pg/mL) as a function of intratracheal injection pretreatment followed by ozone treatment was obtained as follows. The KC level is: approximately 183.6pg/mL in the group of mice pretreated with MANS peptide followed by exposure to ozone; approximately 159.7pg/mL in the group of mice pretreated with the acetylated MANS fragment peptide Ac-GAQFSKTAAK (SEQ ID NO:106) followed by exposure to ozone; approximately 466.6pg/mL in the group of mice pretreated with PBS followed by exposure to ozone; approximately 36.3pg/ml in the group of mice exposed to forced air without pretreatment.
The IFN γ concentration (pg/mL) in BAL fluid as a function of intratracheal injection pretreatment followed by ozone treatment was obtained as follows. IFN γ levels were: approximately 7.4pg/mL in the group of mice pretreated with MANS peptide followed by exposure to ozone; approximately 3.6pg/ml in the group of mice pre-treated with the acetylated MANS fragment peptide Ac-GAQFSKTAAK (SEQ ID NO:106) followed by exposure to ozone; approximately 8.6pg/mL in the group of PBS pretreated mice subsequently exposed to ozone; and approximately 5.0pg/mL in the group of mice exposed to forced air.
Ozone administration to mice significantly increased numbers of infiltrating neutrophils in BAL as well as IL-6 and KC levels. The MANS peptide pre-treated group and the acetylated peptide Ac-GAQFSKTAAK, i.e., acetylated SEQ ID NO:106 pre-treated group, respectively, showed reduced neutrophil infiltration in BAL fluid after exposure to ozone as compared to the control group of PBS pre-treated mice (e.g., 93% + -10% and 81% + -10%, respectively, as compared to the PBS control). Consistent with this, acetylation of MANS peptide and acetylated peptide of seq id NO:106 also significantly reduced KC concentration after exposure to ozone (e.g., 65.8% ± 10% and 71.3% ± 10%, respectively, compared to PBS control) and IL-6 levels (e.g., 67.8% ± 15% (MANS) and 91.3% ± 15% (acetylated seq id NO:106), compared to PBS control), but had little effect on interferon- γ levels. Taken together, these data indicate that MANS peptide and acetylated SEQ ID NO:106 peptide significantly reduce or inhibit ozone-induced neutrophil migration into the airways and selectively reduce chemokines and cytokines. IL-6 levels in BAL fluid of animals pretreated with MANS peptide or acetylated peptide SEQ ID NO 106 showed approximately 68% and 91% inhibition, respectively, relative to animals pretreated with PBS. In addition, KC levels in BAL fluid of animals pretreated with MANS peptide or acetylated peptide SEQ ID NO 106 also showed approximately 65% and 71% inhibition relative to PBS-pretreated animals.
Example 4: model of chronic bronchitis
The mouse model of chronic bronchitis was established by the method described in Voynow JA, Fischer BM, Malarkey DE, Burch LH, Wong T, Longphre M, HoSB, Foster WM, Neutrophil Elastase indices cells meta plastics in mouse lung, am.J.Physiol.Lung Cell mol.Physiol.287: L1293-L1302,2004. Specifically, goblet cell proliferation, which is a hallmark pathological feature of chronic bronchitis, is induced by instillation of human Neutrophil Elastase (NE) into the airways of mice for a long period of time.
Human NE was inhaled into the bronchi by male Balb/c mice. A total of 30 mice (approximately 25-30g in body weight) were purchased from commercial suppliers such as Jackson Laboratories, Bar Harbor, ME. Mice were housed on a 12-hour day cycle with food and water ad libitum. Animals received NE by oropharyngeal inhalation on days 1, 4, and 7. Immediately after the animal is anesthetized by inhalation of IsoFlo from Abbott Laboratories and Open-Circuit Gas anesia System from storage, a liquid volume of human NE [50ug (43.75 units)/40 μ LPBS ] (Elastin Products, Owensville, Mo.) is delivered to the distal portion of the oropharynx along the animal's pulled tongue, with its upper incisors suspended above the 60 ° inclined plate. This volume of liquid is sucked into the respiratory tract as the animal cannot swallow it because the tongue is pulled.
On day 7 after the last exposure to NE, when goblet cell proliferation maximally mimics the airways of chronic bronchitis (see Voynow et al,2004), mice (5 animals per group) were instilled intratracheally with 50 μ L of PBS (as control) or 100 μ M MANS peptide solution, RNS peptide solution, or peptide solution such as acetylated peptide SEQ ID NO:106 dissolved in PBS. After 15 minutes, mucus secretion was induced by methacholine administration, using a Buxco system nebulizer to provide a fine aerosol, delivering approximately 60mM of methacholine for 3 minutes. 15 minutes after methacholine administration, by inhalation of 100% CO2The mice were sacrificed by gas.
Histology-after the above exposure, the lungs of the animals are washed to remove blood, then filled with OCT (optical Cutting Temperature medium (Sakura Finetck, Torrance, CA) diluted in half with saline-immersion in 10% formaldehyde in PBS overnight at 4 ℃ and then paraffin-embedded the 5 μ M sections are treated with periodic acid-Schiff/hematoxylin to stain mucin in the airways, see, for example, Singer M, Vargaft BB, Martin LD, Park JJ, Gruber AD, LiY, AdKB, A MARCKS-related peptide blocks tissue hybridization in a muramidation of 193 asthe, Nature Medicine10: 196, 2004.
Histological mucus indemutex-histological mucus indices were performed on AB/PAS stained sections including central and peripheral airways (Whittaker L, Niu N, Temann U-A, Stoddard A, Flavelra RA, Ray A, Homer RJ, and cohn L, Interleukin-13media a functional pathway for air epithelial emutexpressed by CD4T cells and Interleukin-9, am. J. Respir. cell mol. biol.27: 593-. The slide was examined with a 10X objective lens and an image was taken with a digital camera. At least 4 representative transverse or radial airway cross-sectional images were acquired for each animal. Only those airways where a complete airway circumference can be observed and can be incorporated into the image are analyzed. Those airways that open directly into the alveolar space are not analyzed. The extent of PAS-positive staining in each airway in the image can be semi-quantified by an examiner blinded to the treatment conditions of each section, using the following grade 5 system: grade 0, no PAS staining; grade 1, 25% or less of airway epithelium has PAS staining; grade 2, 26-50% of airway epithelium has PAS staining; grade 3, 51-75% of airway epithelium has PAS staining; and grade 4, > 75% of airway epithelium has PAS staining. This grading system was used to calculate the mucus index score for each group, and the results were expressed as mean ± SE.
All results are expressed as mean ± sem (n ═ 5 animals, 10-20 sections per animal). Significance levels (═ between data significance with a threshold of p <0.05) will be calculated using SPSS6.1 software using one-way anova followed by Scheffe's test.
Example 5: in vivo analysis
The purpose of the following experiments was to clarify the effect on inflammation of the peptides of the present invention administered in vivo by local instillation into the site of inflammation or intravenous injection compared to control peptides such as RNS. Two models can be used for this purpose: (i) a murine model of balloon (airmouth) inflammation and (ii) a murine model of thioglycollate-induced peritonitis. Both are well characterized models of inflammation in which neutrophils have a critical role. The balloon model enables the determination of the effect of peptides on short-term inflammation (about 4 hours), whereas the peritonitis model can be used for longer-term inflammation (about 24 hours).
Overall experimental design:
4 studies, two per model, one to test intravenous delivery of the peptide and the other to test local delivery of the peptide, can be used to study the effect of the peptides of the invention. Each study included 2 experimental groups, one non-inflammatory control (treated with vehicle) and the other inflammatory group (i.e., treated with inflammatory stimuli). Each component is 5 and optionally 6 processing subgroups, each subgroup n ═ 6. The treatment subgroups are for example: a vector, MANS, RNS, a test peptide, optionally a peptide having a scrambled sequence of the test peptide (the scrambled sequence is referred to as "peptide-SCR"), and dexamethasone. Dexamethasone served as the reference anti-inflammatory agent. The selection of an appropriate dose for intravenous injection or topical instillation is determined from preliminary dose-response experiments. The experimental doses based on the inhibitory activity of MANS on human neutrophils were: 1mg/kg for intravenous delivery for single administration or a final concentration of 50. mu.M for local delivery (to the balloon or peritoneal cavity). The intravenous dose selected assumed a volume of distribution of 2L/kg.
Balloon inflammation model:
neutrophil infiltration and inflammation analysis within the mouse air sac was performed as described in the literature: clish CB, O' BrienJA, Gronert K, Stahl GL, Petasis NA, Serhan CN.local and system delivery of soluble aspin-branched lipoxin preservation in vivo.ProcNatl Acad Sci U S.1999Jul 6; 96(14):8247-52. Therefore, white male BALB/c mice (6-8 wk) were anesthetized with Isofluran and dorsal air sacs were created by subcutaneous injection of 3ml of sterile air on day 0 and day 3. On day 6, mice were anesthetized with Isoflurane while vehicle, MANS, RNS, test peptide, or optionally peptide-SCR was delivered intravenously as a bolus into the tail vein in 100 μ Ι _ sterile 0.9% Saline or locally into the balloon in 900 μ Ι _ PBS-/(dubco's Phosphate Buffered Saline without magnesium or calcium ions, BioWhittaker). Dexamethasone (Sigma) was delivered either intravenously at 0.1mg/kg in 100. mu.l sterile 0.9% saline or locally at 10. mu.g in 900. mu.L PBS-/-to serve as a reference anti-inflammatory agent. Inflammation was induced in the air sac by local injection of recombinant murine tumor necrosis factor alpha (TNF-. alpha., 20ng) (Boehringer Mannheim) dissolved in 100. mu.L sterile PBS. Mice were anesthetized with Isoflurane while the balloon was irrigated with 3mL sterile PBS 4 hours after the first TNF- α injection. The extract was centrifuged at 2,000rpm for 15 minutes at 23 ℃. The supernatant was removed and the cells were suspended in 500. mu.L of PBS. Equal amounts of supernatant were taken for determination of inflammatory mediator concentration (optionally excluding TNF α), MPO activity and lipid peroxidation.
Total leukocytes in the cell suspension were counted using a hemocytometer and light microscopy. Resuspended aspirate cells (50. mu.L) were added to 150. mu.L of 30% BSA and centrifuged at 2,200rpm for 4 minutes in a centrifuge onto microscope slides. Differential white blood cell counts were determined on Wright Giemsa dye stained cytospin smears and used to calculate the absolute number of each type of white blood cell in each air sac exudate. For microscopic analysis, tissues were harvested with a 6-mm tissue biopsy needle (Acu-Punch, Acuderm) and fixed in 10% buffered formaldehyde. Samples were embedded in paraffin, sectioned and stained with hematoxylin-eosin. Neutrophils in the tissue sections were counted by counting cells per high power field. The distant dermis was used as a control for the inflammation balloon dermis.
Data are expressed as the total number of neutrophils, monocytes, eosinophils, basophils and lymphocytes per exudate or as the number of neutrophils per tissue high field of view. The values given are in the form of mean ± SEM (n ═ 6). The significance of the effect of either treatment on migration was determined by ANOVA. P <0.05 was considered significant.
Example 6: inflammatory peritoneal model
Male BALB/c mice (6-8 wk) were used to establish a thioglycolate-induced peritonitis model as described in the following references: tedder TF, Steeber DA, Pizcueta P.L-selectin-specific mica have inpatirmylvanilloid sites.J Exp Med.1995Jun 1; 181(6):2259-64. The vehicle, MANS, RNS, test peptide, and optionally peptide-SCR were delivered as a bolus into the tail vein in 100 μ L sterile 0.9% saline or locally in 900 μ L PBS-/-into the peritoneal cavity immediately followed by intraperitoneal injection of thioglycolate. Dexamethasone (Sigma) was delivered either intravenously at 0.1mg/kg in 100. mu.l sterile 0.9% saline or locally at 10. mu.g in 900. mu.L PBS-/-to serve as a reference anti-inflammatory agent. Inflammation was induced by intraperitoneal injection of 1mL thioglycolate solution (3% wt/vol; Sigma Immunochemicals) into mice. Mice were euthanized 24 hours after induction of inflammation and 5mL of warm (37 ℃) medium (RPMI1640, 2% FCS, and 2mM EDTA) was injected intraperitoneally, followed by gentle massaging of the abdomen. The peritoneal lavage aspirate was centrifuged at 2,000rpm for 15 minutes at 23 ℃. The supernatant was removed and the cells were suspended in 500. mu.L of PBS. Equal amounts of supernatant were taken for determination of MPO activity, inflammatory mediator concentration and lipid peroxidation.
Total leukocytes in the cell suspension were counted using a hemocytometer and light microscopy. Resuspended aspirate cells (50. mu.L) were added to 150. mu.L of 30% BSA and centrifuged at 2,200rpm for 4 minutes in a centrifuge onto microscope slides. Differential white blood cell counts were determined on Wright Giemsa dye stained cytospin smears and used to calculate the absolute number of each type of white blood cell in each air sac aspirate.
Data are expressed as the total number of neutrophils, monocytes, eosinophils, basophils and lymphocytes per exudate. The values given are in the form of mean ± SEM (n ═ 6). The significance of the effect of either treatment on migration was determined by ANOVA. P <0.05 was considered significant.
Removing particles:
myeloperoxidase was used as a marker for degranulation. Myeloperoxidase activity in cell supernatants from air sacs or peritoneal lavage were determined and analyzed using the TMB method as described above.
Concentration of inflammatory mediators:
the concentrations of the key proinflammatory mediators TNF α, IL-1 β, IL-10, IL-6, KC and PGE2 in the air sac and peritoneal lavage fluid were determined using a commercial ELISA kit (R & D Systems) according to the manufacturer's instructions.
Lipid peroxidation:
f2-isoprostane (F2-isoprostane) concentration is a sensitive and specific measure of oxidative damage caused by reactive oxygen intermediates released by neutrophils and other cells (Milne GL, Musiek ES, Morrow JD. F2-isoprostanes as markers of oxidative stress in vivo: anoverview. Biomarkers.2005 Nov; 10Suppl1: S10-23). The concentration of F2-Isoprostane in the balloon and peritoneal exudate supernatant was determined using a commercial ELISA kit (8-Isoprostane EIA, Cayman Chemical) according to the manufacturer's instructions.
End point:
this experiment is considered successful if local delivery or systemic delivery of the test peptide reduces inflammation by one or more of the above-described means of inhibiting the release of inflammatory mediators.
The active fragment peptides of the present invention inhibit neutrophil entry into and degranulation of the air sac or abdominal cavity, thereby resulting in decreased MPO activity, decreased lipid peroxidation, and decreased production of inflammatory mediators.
The above-described embodiments are illustrative of the present invention and therefore should not be construed as limiting the invention. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims (18)

1. Use of a peptide for the manufacture of a medicament for inhibiting the release of at least one inflammatory mediator from a granule in at least one inflammatory cell in a tissue and/or a body fluid of a subject, wherein the at least one inflammatory mediator is Eosinophil Peroxidase (EPO), wherein the peptide is selected from the group consisting of: acetyl-GAQFSKTAAK (SEQ ID NO:106), acetyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), acetyl-GAQFSKTAAKGEAAAERPGE (SEQ ID NO:11), acetyl-GAQFSKTAAKGEAAAE (SEQ ID NO:37), acetyl-AKGEAAAERPGEAAVA (SEQ ID NO:45), acetyl-GAQFSKTAAKGE (SEQ ID NO:79), acetyl-AAAERPGEAAVA (SEQ ID NO:91), acetyl-GAQFSKTAA (SEQ ID NO:121), acetyl-TAAKGEAA (SEQ ID NO:143), acetyl-RPGEAAVA (SEQ ID NO:153), acetyl-AKGE (SEQ ID NO:219), acetyl-GAQFSKTAAAGE (SEQ ID NO:239), acetyl-GAQFSKTAAA (SEQ ID NO:248), acetyl-GAQFSKTAAKGA (SEQ ID NO:241), acetyl-GAQFSATAAA (SEQ ID NO:249), acetyl-AAGE (SEQ ID NO:251), acetyl-AAKGEA (SEQ ID NO:179), acetyl-GAQFSKTAAKGE-NH2(SEQ ID NO:79), acetyl-AQFSKTAAKGE-NH2(SEQ ID NO:93), acetyl-QFSKTAAKGE-NH 2(SEQ ID NO:108), acetyl-FSKTAAKGE-NH2(SEQ ID NO:124), acetyl-SKTAAKGE-NH2(SEQ ID NO:141) and acetyl-AKGE-NH2(SEQ ID NO:219)；
And wherein the subject has pulmonary inflammation.
2. The use of claim 1, wherein the pulmonary inflammation is associated with Adult Respiratory Distress Syndrome (ARDS), acute pulmonary inflammation, or acute thermal injury.
3. The use of claim 1 or 2, wherein said peptide in a therapeutically effective amount to reduce the release of inflammatory mediators reduces the amount of inflammatory mediators released from at least one inflammatory cell as compared to the release of said inflammatory mediators from at least one inflammatory cell of the same type that occurs in the absence of said peptide.
4. The use of claim 1 or 2, wherein the peptide consists of acetyl-peptide 106(SEQ ID NO: 106).
5. The use of claim 1 or 2, wherein the peptide consists of acetyl-peptide 121(SEQ ID NO: 121).
6. The use of claim 1 or 2, wherein the peptide is in combination with a pharmaceutically acceptable carrier.
7. The use of claim 1 or 2, wherein the inflammatory cell is a granulocyte, monocyte, macrophage or combination thereof.
8. The use of claim 7, wherein said granulocytes are selected from the group consisting of neutrophils, basophils, and eosinophils.
9. The use of claim 1 or 2, wherein the medicament further inhibits the release of an inflammatory mediator selected from the group consisting of: myeloperoxidase (MPO), lysozyme, granzyme and combinations thereof.
10. The use of claim 9, wherein the medicament further inhibits the release of Myeloperoxidase (MPO).
11. The use of claim 9, wherein the medicament further inhibits the release of lysozyme.
12. The use of claim 3, wherein said amount of said peptide effective to reduce the release of inflammatory mediators comprises a degranulation-inhibiting amount of peptide that reduces the amount of inflammatory mediators released from at least one inflammatory cell by 1% to 99% as compared to the amount released from at least one inflammatory cell in the absence of said peptide.
13. The use of claim 3, wherein said amount of said peptide effective to reduce the release of inflammatory mediators comprises a degranulation-inhibiting amount of peptide that reduces the amount of inflammatory mediators released from at least one inflammatory cell by 5% to 99% as compared to the amount released from at least one inflammatory cell in the absence of said peptide.
14. The use of claim 1 or 2, wherein the peptide is administered by parenteral, rectal or oral administration.
15. The use of claim 1 or 2, wherein the peptide is administered by nasal administration or pulmonary administration.
16. The use of claim 14 or 15, wherein the peptide is administered by aerosol.
17. The use of claim 16, wherein the aerosol is produced by a dry powder inhaler, a metered dose inhaler or a nebulizer.
18. The use of claim 1 or 2, wherein the medicament further comprises a second molecule selected from the group consisting of an antibiotic, an antiviral compound, an antiparasitic compound, an anti-inflammatory compound, and an immunomodulator.
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