WO2008010218A1 - Peptides dérivés de l'histone h2a humaine et leurs procédés d'utilisation - Google Patents

Peptides dérivés de l'histone h2a humaine et leurs procédés d'utilisation Download PDF

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Publication number
WO2008010218A1
WO2008010218A1 PCT/IL2007/000897 IL2007000897W WO2008010218A1 WO 2008010218 A1 WO2008010218 A1 WO 2008010218A1 IL 2007000897 W IL2007000897 W IL 2007000897W WO 2008010218 A1 WO2008010218 A1 WO 2008010218A1
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peptide
amino acid
diseases
disease
seq
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PCT/IL2007/000897
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English (en)
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Uri Wormser
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Yissum Research Development Company Of The Hebrew University Of Jerusalem
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic

Definitions

  • the present invention relates to a peptide comprising the amino acid sequence of residues 36-44 of human H2A modified by a cysteine residue at the carboxy terminus of the peptide and to pharmaceutical compositions comprising same useful for treating burns, inflammatory, autoimmune and degenerative diseases.
  • the presence of superoxide radicals, hydrogen peroxide, and hydroxyl radicals has been demonstrated in phagocytic cells including neutrophils and monocytes. It has been shown that the oxygen free radicals not only cause direct damage to membranes and DNA, but also exert indirect effects such as de-regulation of cell proliferation and apoptosis, stimulation of angiogenesis, and modification of gene/protein expression.
  • the cellular enzymatic defense mechanism against superoxide and hydrogen peroxide includes superoxide dismutase (SOD), catalase, and glutathione peroxidase.
  • the pathologies and diseases which may be attributable to oxygen free radicals are numerous and include neuronal diseases such as brain infarction, brain edema, Parkinson's disease, and Alzheimer's disease; multiple sclerosis; lung diseases such as lung oxygen intoxication, chronic bronchitis, and adult respiratory distress syndrome; circulatory system diseases such as ischemic heart diseases (e.g., myocardial infarction and arrhythmia), and arteriosclerosis; and digestive organ diseases such as peptic ulcer, ulcerative colitis, and Crohn's disease.
  • neuronal diseases such as brain infarction, brain edema, Parkinson's disease, and Alzheimer's disease
  • multiple sclerosis lung diseases such as lung oxygen intoxication, chronic bronchitis, and adult respiratory distress syndrome
  • circulatory system diseases such as ischemic heart diseases (e.g., myocardial infarction and arrhythmia), and arteriosclerosis
  • digestive organ diseases such as peptic ulcer, ulcerative colitis, and Crohn's disease
  • MMPs matrix metalloproteinases
  • MMPs consist of five major groups of enzymes: gelatinases, collagenases, stromelysins, membrane-type MMPs, and matrilysins.
  • the activity of MMPs in normal tissue functions is strictly regulated by a series of complicated zymogen activation processes and inhibition by protein tissue inhibitors for matrix metalloproteinases ("TIMPs").
  • TIMPs matrix metalloproteinases
  • MMPs or an imbalance between MMPs and TIMPs have been suggested as factors in the pathogenesis of diseases characterized by the breakdown of extracellular matrix or connective tissues, including diseases such as rheumatoid arthritis, osteoarthritis, osteoporosis, periodontitis, multiple sclerosis, gingivitis, gastric ulceration, atherosclerosis, neointimal proliferation which leads to restenosis and ischemic heart failure, and tumor metastasis (see, for example, Massova, I. et al. FASEB J. 1998, 12, 1075-1095). It has been suggested that these and other diseases may be treated by inhibiting metalloproteinase enzymes, thereby curtailing and/or eliminating the breakdown of connective tissues that results in the disease states.
  • the catalytic zinc in matrix metalloproteinases has been the focal point for inhibitor design.
  • the modification of substrates by introducing zinc-chelating groups has generated potent inhibitors such as peptide and non-peptide hydroxamates and thiol-containing peptides.
  • Peptide hydroxamates and the natural endogenous inhibitors of MMPs have been used successfully to treat animal models of cancer and inflammation (see, for example, U.S. Pat. Nos. 5,300,501; 5,530,128; 5,455,258; and 5,552,419).
  • 2003/0021797 disclose peptides derived from nucleosomal histone proteins Hl, H2A, H2B, H3, and H4, which are useful for delaying the onset and progression of systemic lupus erythematosus (SLE).
  • U.S. Patent No. 6,468,537 claims methods of treating an animal having systemic lupus erythematosus (SLE) and SLE-associated manifestations comprising administering to the animal one of the disclosed peptides, wherein the peptide is capable of promoting immunological tolerance, thereby treating SLE and the SLE- associated manifestations. While U.S. patent No.
  • 6,468,537 discloses various peptides derived from different histones, among them a 15-mer peptide derived from H2A (amino acid residues 34 to 48), the peptides are disclosed solely as useful for promoting immunological tolerance in an animal having systemic lupus erythematosus.
  • WO 03/017920 assigned to the applicant of the present invention discloses peptides for protection against inflammatory processes.
  • the peptides were first isolated from the skin of guinea pigs exposed to a chemical or thermal burn and further exposed to iodine.
  • One of the peptides was found to have the amino acid sequence KGNYAERIA corresponding to residues 36-44 of guinea pig histone H2A designated peptide III.
  • WO 03/017920 further discloses human homologues of peptide III including peptide 3ml having the amino acid sequence KGHYAERVG, analogs and derivatives thereof, mainly methylated analogs.
  • the pharmaceutical compositions disclosed in WO 03/017920 are shown to protect against chemical and thermal burns.
  • the present invention provides peptides capable of reducing or ameliorating damage due to inflammatory processes including those due to thermal burns, chemical burns or other noxious stimuli.
  • the present invention provides peptides capable of treating diseases attributable to inflammatory processes including autoimmune diseases such as arthritis and multiple sclerosis.
  • the present invention further provides peptides that scavenge free radicals and chelate metals.
  • the peptides are effective for treating diseases attributable to the release of free radicals.
  • the peptides are also effective for treating diseases or disorders attributable to the breakdown of extracellular matrix.
  • HIM 1C of the amino acid sequence Lys-Gly-His-Tyr-Ala-Glu-Arg-Val-Gly-Cys (SEQ ID NO: 1) which is a specific modification derived from the amino acid sequence Lys-Gly-His-Tyr- Ala-Glu-Arg-Val-Gly (SEQ ID NO: 2; designated 3ml or IIIM1) of residues 36-44 of human histone H2A is highly effective in eliminating the neurological damages in animal models of multiple sclerosis.
  • IIIMIC peptide in eradicating the symptoms of multiple sclerosis is achieved whether the peptide is administered orally or intraperitoneally.
  • IIIMIC peptide of SEQ ID NO:1 is highly effective as a hydroxyl radical scavenger.
  • the present invention discloses analogs, derivatives, and fragments of the IIIMIC peptide. It should be understood that the analogs, derivatives, and fragments of the IIIMIC peptide do not include any known peptide or any fragment of human H2A protein.
  • the present invention provides a peptide comprising the amino acid sequence KGH YAERVGC as set forth in SEQ ID NO:1, or an analog, derivative, or fragment thereof having anti-inflammatory activity, wherein the analog, derivative or fragment comprises at least four contiguous amino acid residues including the cysteine residue.
  • the fragment comprises at least five amino acid residues, alternatively at least six, seven, eight, or nine amino acid residues, including the cysteine residue.
  • the peptide has the amino acid sequence as set forth in SEQ ID NO:1.
  • the peptide comprises the amino acid sequence RVGC as set forth in SEQ ID NO:3.
  • the peptide comprises 11 amino acid residues, alternatively 12, 13, 14, 15 amino acid residues. According to further embodiments, the peptide comprises about 20 amino acid residues, 30, 40, or about 50 amino acid residues.
  • the present invention provides an isolated polynucleotide sequence encoding a peptide comprising the amino acid sequence
  • KGHYAERVGC as set forth in SEQ ID NO: 1, or an analog, derivative, or fragment thereof having anti-inflammatory activity, wherein the analog, derivative or fragment comprises at least four contiguous amino acid residues including the cysteine residue.
  • the isolated polynucleotide sequence encodes a peptide as set forth in SEQ ID NO:1. According to an additional embodiment, the isolated polynucleotide sequence encodes a peptide comprising the amino acid sequence as set forth in SEQ ID NO :3.
  • the present invention provides an expression vector comprising an isolated polynucleotide sequence encoding a peptide comprising the amino acid sequence as set forth in SEQ ID NO:1, or an analog, derivative, or fragment thereof having anti-inflammatory activity, wherein the analog, derivative or fragment comprises at least four contiguous amino acid residues including the cysteine residue.
  • the expression vector comprises an isolated polynucleotide sequence encoding a peptide of SEQ ID NOl. According to an additional embodiment, the expression vector comprises an isolated polynucleotide sequence encoding a peptide comprising the amino acid sequence as set forth in SEQ ID NO:3.
  • the present invention provides a host cell comprising an expression vector comprising an isolated polynucleotide sequence encoding a peptide comprising the amino acid sequence as set forth in SEQ ID NO:1, or an analog, derivative, or fragment thereof having anti-inflammatory activity, wherein the analog, derivative or fragment comprises at least four contiguous amino acid residues including the cysteine residue.
  • the host cell comprises an expression vector comprising an isolated polynucleotide sequence encoding a peptide as set forth in SEQ ID NO:1, or an analog, derivative, or fragment thereof having anti-inflammatory activity, wherein the analog, derivative or fragment comprises at least four contiguous amino acid residues including the cysteine residue.
  • the host cell comprises an expression vector comprising an isolated polynucleotide sequence encoding a peptide as set forth in SEQ ID NO:1, or an analog, derivative, or fragment thereof having anti-inflammatory activity, wherein the analog, derivative or fragment comprises at least four contiguous amino acid residues including the cysteine residue.
  • the host cell comprises an expression vector comprising an isolated polynucleotide sequence encoding a peptide comprising the amino acid sequence as set forth in SEQ ID NO:3.
  • the host cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, and a plant cell.
  • the present invention provides a pharmaceutical composition comprising as an active ingredient a peptide comprising the amino acid sequence KGHY AERVGC as set forth in SEQ ID NO:1, or an analog, derivative, or fragment thereof having anti-inflammatory activity, wherein the analog, derivative or fragment comprises at least four contiguous amino acid residues including the cysteine residue; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises as an active ingredient the peptide as set forth in SEQ ID NO:1 and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises as an active ingredient a peptide comprising the amino acid sequence as set forth in SEQ ID NO:3.
  • the pharmaceutical composition comprising the peptide of the invention is formulated for parenteral administration.
  • the pharmaceutical composition comprising the peptide of the invention is formulated for oral administration.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient an isolated polynucleotide sequence encoding a peptide comprising the amino acid sequence as set forth in SEQ ID NO:1, or an analog, fragment or derivative thereof having anti-inflammatory activity, wherein the analog, derivative or fragment comprises at least four contiguous amino acid residues including the cysteine residue; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises an isolated polynucleotide sequence encoding a peptide as set forth SEQ ID NO:1 and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient an expression vector comprising an isolated polynucleotide sequence encoding a peptide comprising the amino acid sequence as set forth in SEQ ID NO:1, or an analog, fragment or derivative thereof according to the principles of the present invention; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises as an active ingredient an expression vector comprising an isolated polynucleotide sequence encoding a peptide of SEQ ID NO:1 and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient a host cell comprising an expression vector comprising an isolated polynucleotide sequence encoding a peptide comprising the amino acid sequence as set forth in SEQ ID NO:1, or an analog, fragment or derivative thereof according to the principles of the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a host cell comprising an expression vector comprising an isolated polynucleotide sequence encoding a peptide of SEQ ID NO:1 and a pharmaceutically acceptable carrier.
  • the present invention provides a method for treating a disease or condition attributable to inflammation comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the principles of the present invention.
  • the subject is a human.
  • the pharmaceutical composition comprises as an active ingredient the peptide of SEQ ID NO:1 and a pharmaceutically acceptable carrier.
  • the route of administering the pharmaceutical composition is by parenteral administration.
  • the route of administering the pharmaceutical composition is by oral administration.
  • the pharmaceutical compositions of the present invention are useful for treating a diverse group of indications having an inflammatory or autoimmune mechanism involved in their etiology or pathogenesis.
  • the disease or condition is selected from the group consisting of inflammatory diseases, autoimmune diseases, degenerative neurological diseases, degenerative muscle diseases, wounds, hypersensitivity, infectious diseases, diseases associated with graft transplantation, allergic diseases, musculo-skeletal inflammations, and sepsis.
  • the inflammatory disease is selected from the group consisting of multiple sclerosis, arthritis including rheumatoid arthritis, inflammatory bowel disease (Crohn's disease), asthma, chronic bronchitis, sepsis, psoriasis, and systemic lupus erythematosus (SLE).
  • the degenerative neurological disease is selected from the group consisting of amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease.
  • the disease to be treated is multiple sclerosis.
  • the disease to be treated is Parkinson's disease.
  • the present invention provides a method for protecting against or treating tissue damage consequent to a noxious insult in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the principles of the present invention.
  • the subject is a human.
  • the pharmaceutical composition comprises as an active ingredient the peptide of SEQ ID NO:1 and a pharmaceutically acceptable carrier.
  • the route of administering the pharmaceutical composition comprising a peptide of the invention is by parenteral administration.
  • the route of administering the pharmaceutical composition comprising a peptide of the invention is by oral administration.
  • the noxious insults include, but are not limited to, heat stimuli, cold stimuli, chemical stimuli, electric stimuli, ultraviolet irradiation, ionizing radiation, non-ionizing irradiation, and ultrasound.
  • the present invention provides a method for scavenging free radicals in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the principles of the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition to be administered for scavenging free radicals in a subject comprises as an active ingredient the peptide of SEQ ID NO:1 and a pharmaceutically acceptable carrier.
  • the subject is a human.
  • the free radicals are reactive oxygen species
  • ROS selected from the group consisting of superoxide radicals, hydrogen peroxide, and hydroxyl radicals.
  • the free radicals are carbon tetra chloride radicals. It will be appreciated that the peptides of the present invention are capable of scavenging other toxic radicals as are known in the art.
  • the present invention provides a method for protecting against or treating a disease or condition attributable to free radicals in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the principles of the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition to be administered for protecting against or treating a disease or condition attributable to free radicals in a subject comprises as an active ingredient the peptide of SEQ ID NO:1 and a pharmaceutically acceptable carrier.
  • the subject is a human.
  • the free radicals are reactive oxygen species selected from the group consisting of superoxide radicals, hydrogen peroxide, and hydroxyl radicals.
  • the subject to be treated is a human.
  • the disease or condition attributable to reactive oxygen species to be treated by the pharmaceutical compositions of the invention are selected from the group consisting of neuronal diseases, lung diseases, cardiovascular diseases, and digestive organ diseases.
  • the disease or condition attributable to free radicals is selected from the group consisting of brain infarction, brain edema, Parkinson's disease, Alzheimer's disease, multiple sclerosis, lung oxygen intoxication, chronic bronchitis, adult respiratory distress syndrome, ischemic diseases (e.g., myocardial infarction and arrhythmia), arteriosclerosis, peptic ulcer, ulcerative colitis, and Crohn's disease. It will be appreciated that biological damages caused by infections can also be treated with the pharmaceutical compositions of the invention.
  • the present invention provides a method for protecting against or treating a disease or condition attributable to altered metalloproteinase (MMP) activity in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the principles of the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition to be administered for protecting against or treating a disease or condition attributable to altered MMP activity in a subject comprises a peptide of SEQ ID NO: 1.
  • the subject is human.
  • the disease or condition which are attributable to MMP activity are selected from the group consisting of multiple sclerosis, atherosclerosis, restenosis, aortic aneurysm, cardiovascular disease, periodontal disease, corneal ulceration, burns, decubital ulcers, chronic ulcers or wounds including gastric ulcers, osteoporosis, rheumatoid arthritis, osteoarthritis, renal disease, neurological disorders, or any other connective tissue disease.
  • the present invention provides a method for protecting against or treating a condition attributable to metal poisoning in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the principles of the present invention.
  • the pharmaceutical composition to be administered for protecting against or treating a condition attributable to metal poisoning in a subject comprises a peptide of SEQ ID NO: 1.
  • the subject is human.
  • the present invention provides a method for protecting against or treating a T cell mediated disease comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the principles of the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition to be administered for protecting against or treating a T cell mediated disease in a subject comprises a peptide of SEQ ID NO:1.
  • the subject is human.
  • T cell mediated disease that can be treated by a pharmaceutical composition of the invention is selected from the group consisting of psoriasis; allergy; T cell lymphomas and other malignancies; graft versus host disease; prevention of transplant rejection; bronchitis; asthma; autoimmunity; sarcoidosis; bone marrow depression; bone marrow stimulation; depression or other mood or psychotic disorders; sepsis; Parkinson's disease; skin disorders and irritation; arthritis; multiple sclerosis; neurodegenerative disorders (amyotrophic lateral sclerosis, chorea, Alzheimer disease); atherosclerosis; fibrosis; pain; chronic or acute inflammation.
  • the peptides and pharmaceutical compositions of the present invention can be used in combination therapy with standard medicaments for the diseases listed herein above.
  • the present invention provides use of the peptides of the invention in the preparation of a medicament for treating a disease or condition attributable to inflammatory processes according to the principles of the present invention.
  • the present invention provides use of the peptides of the invention in the preparation of a medicament for protecting against tissue damage consequent to noxious stimuli according to the principles of the present invention.
  • the present invention provides use of the peptides of the invention in the preparation of a medicament for treating a disease attributable to free radicals according to the principles of the present invention. According to yet further aspect, the present invention provides use of the peptides of the invention in the preparation of a medicament for treating a disease attributable to altered metalloproteinase activity according to the principles of the present invention.
  • FIG. 1 shows the effect of oral administration of HIM 1, HIM 1C, or a cyclic analog of IIIMl peptide on experimental autoimmune encephalitis in mice.
  • FIG. 2 shows the effect of intraperitoneal (i.p.) administration of HIM 1C peptide on experimental autoimmune encephalitis in mice.
  • FIG. 3 shows the effect of IIIMl C peptide on hydroxy radical scavenging.
  • the present invention relates to a peptide designated HIM 1C having the sequence KGHYAERVGC as set forth in SEQ ID NO:1 derived from amino acid residues 36 to 44 of human histone H2A.
  • the present invention further relates to analogs, derivatives and fragments of peptide IIIMIC having anti-inflammatory activity and/or free radical scavenging activity and/or metal chelating activity and/or metalloprotease inhibitory activity.
  • the peptides of the invention are useful for treating or protecting a subject against noxious stimuli, inflammatory conditions or diseases, diseases associated with free radicals, and diseases associated with altered metalloproteinase activity.
  • the present invention demonstrates that administration of peptide IIIMIC to animals reduces the degree of inflammatory processes.
  • the peptides of the present invention are also capable of scavenging oxygen free radicals and hence can prevent or reduce biological damage caused by free oxygen radicals, particularly hydroxyl radicals.
  • the peptides of the present invention are, therefore, useful for treating diseases such as inflammatory, autoimmune, neurological, cardiovascular, and connective tissue diseases.
  • WO 03/017920 WO 2005/090387
  • U.S. Patent Application Publication No. 2007/0093426 a series of peptides derived from human and guinea pig histone H2A corresponding to amino acid residues 36-44 of histone H2A (Table 1).
  • Administration of these peptides to animal models ameliorated diseases or conditions attributable to inflammatory processes such as multiple sclerosis, arthritis, asthma, sepsis, inflammatory bowel disease as well as Parkinosn's diseases in the animals treated (see WO 03/017920; WO 2005/090387; and U.S. Patent Application Publication No. 2007/0093426, the content of which is incorporated by reference as if fully set forth herein).
  • the present invention discloses a new peptide designated IIIMIC of the amino acid sequence Lys-Gly-His-Tyr-Ala-Glu-Arg-Val-Gly-Cys as set forth in SEQ ID NO: 1, and fragments, derivatives and analogs thereof derived from amino acid residues 36-44 of human histone H2A.
  • peptide refers to a linear series of natural, non-natural and/or chemically modified amino acid residues connected one to the other by peptide bonds.
  • amino acid residues are represented throughout the specification and claims by either one or three-letter codes, as is commonly known in the art.
  • analogs and derivatives refer to a peptide comprising at least one altered amino acid residue by an amino acid substitution, addition, deletion, or chemical modification, as compared with the native peptide.
  • Peptide derivatives particularly include amino acid substitutions and/or additions with naturally occurring amino acid residues, and chemical modifications such as, for example, enzymatic modifications, typically present in nature.
  • Peptide analogs particularly include amino acid substitutions and/or additions with non-natural amino acid residues, and chemical modifications which do not occur in nature.
  • the present invention encompasses both peptide derivatives and analogs of the IIIMIC peptide as set forth in SEQ ID NO: 1. According to the principles of the present invention the peptide derivatives or analogs of IIIMIC peptide do not include the intact H2A protein or any known fragments thereto.
  • amino acid substitutions By using “amino acid substitutions”, it is meant that functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • the term “functionally equivalent” means, for example, a group of amino acids having similar polarity, similar charge, or similar hydrophobicity.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs.
  • the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such substitutions are known as conservative substitutions. Additionally, a non-conservative substitution can be made in an amino acid that does not contribute to the biological activity of the peptide.
  • amino acid substitution Such non-conservative substitutions are also encompassed within the term "amino acid substitution", as used herein. It will be appreciated that the present invention further encompasses peptide IIIMIC derivatives or analogs, wherein at least one amino acid is substituted by another amino acid to produce a peptide derivative or analog having increased stability or higher half life as compared to IIIMIC peptide. The present invention encompasses peptides of which at least one amino acid has been chemically modified.
  • Chemical modifications of amino acid residues include, but are not limited to, amidation, methylation, acetylation, glycosylation, oxidation, reduction, myristylation, sulfation, acylation, ADP-ribosylation, cyclization, hydroxylation, iodination, derivatization by protecting/blocking groups, or any other derivatization method known in the art.
  • Such alterations, which do not destroy, but may improve the biological activity of IIIMIC peptide can occur anywhere along the sequence of the peptide, including at the peptide backbone, the amino acid side-chains, and at the amino or carboxyl termini.
  • fragment refers to a portion of a peptide, peptide derivative or peptide analog having an anti-inflammatory activity and may have an additional biological activity selected from the group consisting of free radical scavenging activity, metal chelating activity, and metalloproteinase inhibitory activity.
  • the present invention encompasses peptide hydrates.
  • hydrate includes, but is not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, and the like.
  • Peptide IIIMIC and analogs, derivatives and fragments thereof can be produced by various methods known in the art, including recombinant production or synthetic production. Recombinant production can be achieved by the use of an isolated polynucleotide encoding peptide IIIMIC, or a fragment, derivative or analog thereof, the isolated polynucleotide operably linked to a promoter for the expression of the polynucleotide. Optionally, a signal peptide and a regulator of the promoter are added.
  • the construct comprising the polynucleotide encoding the peptide IIIMIC, or a fragment, derivative or analog thereof, the promoter, and optionally the regulator can be placed in a vector, such as a plasmid, virus or phage vector.
  • the vector can be used to transfect or transform a host cell, e.g., a bacterial, yeast, insect, or mammalian cell.
  • the vector can also be introduced into a transgenic animal such as, for example, a transgenic mouse.
  • the peptide can by produced synthetically. Synthetic production of peptides is well known in the art.
  • the IIIMIC peptide, derivatives, analogs and/or fragments thereof can be synthesized using standard direct peptide synthesis (see, for example, Bodanszky, 1984, Principles of Peptide Synthesis, Springer-Verlag, Heidelberg), such as via solid-phase synthesis (see, for example, Merrifield, 1963, J. Am. Chem. Soc.
  • solid phase peptide synthesis methods include, but are not limited to, the BOC method, which utilizes tert-butyloxcarbonyl as the ⁇ -amino protecting group, and the FMOC method, which utilizes 9-fluorenylmethyloxcarbonyl to protect the ⁇ -amino of the amino acid residues, both methods are well-known by those of skill in the art.
  • peptide derivatives, analogs, and fragments of the present invention can be synthesized using standard solution methods (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer-Verlag, 1984, the content of which is hereby incorporated by reference in its entirety).
  • the present invention also encompasses peptide derivatives and analogs in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonylamino groups, carbobenzoxyamino groups, t-butyloxycarbonylamino groups, chloroacetylamino groups or formylamino groups.
  • Free carboxyl groups may be derivatized to form, for example, salts, methyl and ethyl esters or other types of esters or hydrazides.
  • the imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine.
  • peptide derivatives which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues.
  • 4-hydroxyproline can be substituted for proline
  • 5-hydroxylysine can be substituted for lysine
  • 3-methylhistidine can be substituted for histidine
  • homoserine can be substituted or serine
  • ornithine can be substituted for lysine.
  • the peptide analogs can also contain non-natural amino acids.
  • non-natural amino acids include, but are not limited to, sarcosine (Sar), norleucine, ornithine, citrulline, diaminobutyric acid, homoserine, isopropyl Ly s, 3-(2'-naphtyl)-Ala, nicotinyl Ly s, amino isobutyric acid, and 3-(3'-pyridyl- AIa).
  • the peptide analogs can contain other derivatized amino acid residues including, but not limited to, methylated amino acids, N-benzylated amino acids, O- benzylated amino acids, N-acetylated amino acids, O-acetylated amino acids, carbobenzoxy-substituted amino acids and the like.
  • Specific examples include, but are not limited to, methyl-Ala (MeAIa), MeTyr, MeArg, MeGIu, MeVaI, MeHis, N-acetyl-Lys, O- acetyl-Lys, carbobenzoxy-Lys, Tyr-O-Benzyl, Glu-O-Benzyl, Ben2yl-His, Arg-Tosyl, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, and the like.
  • MeAIa methyl-Ala
  • MeTyr MeArg
  • MeGIu MeVaI
  • MeHis N-acetyl-Lys
  • O- acetyl-Lys carbobenzoxy-Lys
  • Tyr-O-Benzyl Glu-O-Benzyl
  • Ben2yl-His Arg-Tosyl
  • the invention further includes peptide HIM 1C analogs, which can contain one or more D-isomer forms of the amino acids.
  • Production of retro-inverso D-amino acid peptides where at least one amino acid, and perhaps all amino acids, is D-amino acids is well known in the art.
  • the result is a molecule having the same structural groups being at the same positions as in the L-amino acid form of the molecule.
  • the molecule is more stable to proteolytic degradation and is therefore useful in many of the applications recited herein.
  • peptide conjugates comprising a IIIMIC peptide derivative, analog, or fragment thereof joined at its amino or carboxy- terminus or at one of the side chains via a peptide bond to an amino acid sequence of a different protein.
  • a IIIMIC peptide derivative, analog, or fragment thereof can be joined to another moiety such as, for example, a fatty acid, a sugar moiety, arginine residues, and any known moiety that facilitate membrane or cell penetration.
  • Conjugates comprising peptides of the invention and a protein can be made by protein synthesis, e.
  • the present invention provides a peptide designated HIM 1C having the amino acid sequence:
  • a peptide derivative, analog, or fragment can be prepared and tested in one of the assays disclosed herein below: assays for anti-inflammatory activity (see the assay of experimental autoimmune encephalitis in Examples 1 and 2), and assays for free radical scavenging (see Example 3).
  • a peptide derivative, analog, or fragment which is active in one of these assays or in any assay aimed at evaluating an anti-inflammatory activity, metal chelating activity, or metalloproteinase inhibiting activity as known in the art falls under the scope of the invention.
  • the present invention provides an isolated polynucleotide sequence encoding the IIIMIC peptide, or a fragment, derivative, analog, or a conjugate thereof, the IIIMIC peptide, fragment, derivative, analog, or conjugate thereof has anti-inflammatory activity and can have free radical scavenging activity, metal chelating activity, metalloproteinase inhibiting activity, and/or T cell inhibitory activity.
  • polynucleotide means a polymer of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a combination thereof, which can be derived from any source, can be single- or double-stranded, and can optionally contain synthetic, non-natural, or altered nucleotides, which are capable of being incorporated into DNA or RNA polymers.
  • isolated polynucleotide refers to a polynucleotide segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to polynucleotides, which have been substantially purified from other components, which naturally accompany the polynucleotide in the cell, e.g., RNA or DNA or proteins.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA, which is part of a hybrid gene encoding additional polypeptide sequence, and RNA such as mRNA.
  • encoding refers to the inherent property of specific sequences of nucleotides in an isolated polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a peptide or protein if transcription and translation of mRNA corresponding to that gene produces the peptide or protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the peptide or protein or other product of that gene or cDNA.
  • polynucleotide may encode any given peptide or protein in view of the degeneracy of the genetic code and the allowance of exceptions to classical base pairing in the third position of the codon, as given by the so-called "Wobble rules.” It is intended that the present invention encompass polynucleotides that encode peptide IIIMIC as set forth in SEQ ID NO: 1 as well as any derivative, analog, and fragment thereof.
  • a polynucleotide of the present invention can be expressed as a secreted peptide where the peptide IIIMIC, or a derivative, analog or fragment thereof is isolated from the medium in which the host cell containing the polynucleotide is grown, or the polynucleotide can be expressed as an intracellular peptide by deleting the leader or other peptides, in which case the peptide IIIMIC, derivative, analog or fragment thereof is isolated from the host cells. The peptide IIIMIC, derivative, analog or fragment thereof so isolated is then purified by standard protein purification methods known in the art.
  • the IIIMIC peptide, analogs, derivatives, or fragments thereof can also be provided to the tissue of interest by transferring an expression vector comprising an isolated polynucleotide encoding the IIIMIC peptide, an analog, derivative, or fragment thereof to cells associated with the tissue of interest.
  • the cells produce the peptide such that it is suitably provided to the cells within the tissue to exert a biological activity such as, for example, to reduce or inhibit inflammatory processes within the tissue of interest.
  • the expression vector according to the principles of the present invention further comprises a promoter.
  • the promoter must be able to drive the expression of the peptide within the cells.
  • Many viral promoters are appropriate for use in such an expression vector (e.g., retroviral ITRs, LTRs, immediate early viral promoters (IEp) (such as herpes virus IEp (e.g., ICP4-IEp and ICPO-IEp) and cytomegalovirus (CMV) IEp), and other viral promoters (e.g., late viral promoters, latency- active promoters (LAPs), Rous Sarcoma Virus (RSV) promoters, and Murine Leukemia Virus (MLV) promoters).
  • IEp immediate early viral promoters
  • CMV cytomegalovirus
  • promoters are eukaryotic promoters, which contain enhancer sequences (e.g., the rabbit ⁇ -globin regulatory elements), constitutively active promoters (e.g., the ⁇ -actin promoter, etc.), signal and/or tissue specific promoters (e.g., inducible and/or repressible promoters, such as a promoter responsive to TNF or RU486, the metallothionine promoter, etc.), and tumor-specific promoters.
  • enhancer sequences e.g., the rabbit ⁇ -globin regulatory elements
  • constitutively active promoters e.g., the ⁇ -actin promoter, etc.
  • signal and/or tissue specific promoters e.g., inducible and/or repressible promoters, such as a promoter responsive to TNF or RU486, the metallothionine promoter, etc.
  • tumor-specific promoters eukaryotic promoters, which contain enhancer sequences (
  • the expression vector can include more than one gene, such as multiple genes separated by internal ribosome entry sites (IRES).
  • IRS internal ribosome entry sites
  • the expression vector can optionally include other elements, such as splice sites, polyadenylation sequences, transcriptional regulatory elements (e.g., enhancers, silencers, etc.), or other sequences.
  • the expression vectors are introduced into the cells in a manner such that they are capable of expressing the isolated polynucleotide encoding the IIIMIC peptide, a fragment, derivative or analog thereof contained therein.
  • Any suitable vector can be so employed, many of which are known in the art.
  • examples of such vectors include naked DNA vectors (such as oligonucleotides or plasmids), viral vectors such as adeno-associated viral vectors (Berns et al, 1995, Ann. N. Y. Acad. Sci. 772:95-104, the contents of which are hereby incorporated by reference in their entirety), adenoviral vectors, herpes virus vectors (Fink et al., 1996, Ann. Rev. Neurosci.
  • the vector can also include other genetic elements, such as, for example, genes encoding a selectable marker (e.g., ⁇ -gal or a marker conferring resistance to a toxin), a pharmacologically active protein, a transcription factor, or other biologically active substance.
  • a selectable marker e.g., ⁇ -gal or a marker conferring resistance to a toxin
  • a pharmacologically active protein e.g., a transcription factor, or other biologically active substance.
  • Methods for manipulating a vector comprising an isolated polynucleotide are well known in the art (e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, the contents of which are hereby incorporated by reference in their entirety) and include direct cloning, site specific recombination using recombinases, homologous recombination, and other suitable methods of constructing a recombinant vector.
  • an expression vector can be constructed such that it can be replicated in any desired cell, expressed in any desired cell, and can even become integrated into the genome of any desired cell.
  • the expression vector comprising the polynucleotide of interest is introduced into the cells by any means appropriate for the transfer of DNA into cells. Many such methods are well known in the art (e.g., Sambrook et al., supra; see also Watson et al., 1992, Recombinant DNA, Chapter 12, 2d edition, Scientific American Books, the contents of which are hereby incorporated by reference in their entirety). Thus, in the case of prokaryotic cells, vector introduction can be accomplished, for example, by electroporation, transformation, transduction, conjugation, or mobilization. For eukaryotic cells, vectors can be introduced through the use of, for example, electroporation, transfection, infection, DNA coated microprojectiles, or protoplast fusion.
  • Examples of eukaryotic cells into which the expression vector can be introduced include, but are not limited to, ovum, stem cells, blactocytes, and the like.
  • Cells into which the polynucleotide has been transferred under the control of an inducible promoter if necessary, can be used as transient transformants. Such cells themselves may then be transferred into a subject for therapeutic benefit therein.
  • the cells can be transferred to a site in the subject such that the peptide of the invention is expressed therein and secreted therefrom and thus reduces or inhibits, for example, inflammatory processes so that the clinical condition of the subject is improved.
  • the cells can first be subjected to several rounds of clonal selection (facilitated usually by the use of a selectable marker sequence in the vector) to select for stable transformants.
  • Such stable transformants are then transferred to a subject, preferably a human, for therapeutic benefit therein.
  • the polynucleotide encoding the IIIMIC peptide, an analog, derivative or fragment thereof is expressed, and optionally is secreted.
  • Successful expression of the polynucleotide can be assessed using standard molecular biology techniques (e.g., Northern hybridization, Western blotting, immunoprecipitation, enzyme immunoassay, etc.).
  • IIIMIC peptide, an analog, derivative or fragment thereof produced by recombinant techniques can be purified so that the peptides will be substantially pure when administered to a subject.
  • substantially pure refers to a compound, e.g., a peptide, which has been separated from components, which naturally accompany it.
  • a peptide is substantially pure when at least 50%, preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the peptide of interest. Purity can be measured by any appropriate method, e.g., in the case of peptides by
  • compositions and administration routes The present invention provides pharmaceutical compositions comprising as an active ingredient a therapeutically effective amount of a source of IIIMIC, and a pharmaceutically acceptable carrier.
  • IIIMIC refers herein to IIIMIC peptide, derivative, analog or fragment thereof, to an isolated polynucleotide encoding the IIIMIC peptide, a derivative, analog or fragment thereof, to an expression vector comprising an isolated polynucleotide encoding the IIIMIC peptide, a derivative, analog or fragment thereof, or to cells transfected with the expression vector as described herein above.
  • compositions of the invention can be formulated in the form of a pharmaceutically acceptable salt of the peptides of the present invention or their analogs, derivatives or fragments thereof.
  • Pharmaceutically acceptable salts include those salts formed with free amino groups such as salts derived from non-toxic inorganic or organic acids such as hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those salts formed with free carboxyl groups such as salts derived from non-toxic inorganic or organic bases such as sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylarnine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • pharmaceutically acceptable means suitable for administration to a subject, e.g., a human.
  • pharmaceutically acceptable can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.
  • compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, gels, creams, ointments, foams, pastes, sustained-release formulations and the like.
  • the compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in: Remington's Pharmaceutical Sciences" by E.W. Martin, the contents of which are hereby incorporated by reference herein.
  • Such compositions will contain a therapeutically effective amount of a source of IIIM1, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • the amount of a source of HIM 1C, which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition and on the particular HIM 1C source, and can be determined by standard clinical techniques known to a person skilled in the art.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the nature of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in-vitro or in- vivo animal model test bioassays or systems.
  • a source of IIIMIC can be supplied in any manner suitable for the provision of the peptide to cells within the tissue of interest.
  • a composition containing a source of IIIMIC i.e., a IIIMIC peptide derivative, analog or fragment thereof, or an isolated polynucleotide encoding the IIIMIC peptide, a derivative, analog or fragment thereof, or an expression vector comprising an isolated polynucleotide encoding the IIIMIC peptide, a derivative, analog or fragment thereof, or cells transfected with the expression vector as described herein above
  • a source of IIIMIC i.e., a IIIMIC peptide derivative, analog or fragment thereof, or an isolated polynucleotide encoding the IIIMIC peptide, a derivative, analog or fragment thereof, or cells transfected with the expression vector as described herein above
  • a source of IIIMIC i.e., a IIIMIC peptide derivative, analog or fragment thereof, or an isolated polynucleotide encoding the IIIMIC
  • composition containing a source of IIIMIC can be applied topically to the tissue of interest (e.g., injected, or pumped as a continuous infusion, or as a bolus within a tissue, applied to all or a portion of the surface of the skin, etc.).
  • the route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art. Although the bioavailability of peptides administered by other routes can be lower than when administered via parenteral injection, by using appropriate formulations it is envisaged that it will be possible to administer the compositions of the invention via oral, transdermal, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment.
  • intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer. It may be desirable to administer the pharmaceutical composition of the invention locally to the area in need of treatment; this can be achieved by, for example, and not by way of limitation, local infusion, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non- porous, or gelatinous material. According to some preferred embodiments, administration can be by direct injection e.g., via a syringe, at the site of a damaged tissue.
  • IIIMIC peptide, or a derivative, analog or fragment thereof can be combined with a pharmaceutically acceptable carrier so that an effective dosage is delivered, based on the desired activity.
  • the peptide of the invention is applied to the skin for treatment of diseases such as psoriasis.
  • the carrier can be in the form of, for example, and not by way of limitation, an ointment, cream, gel, paste, foam, aerosol, suppository, pad or gelled stick.
  • the pharmaceutical composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; or a glidant such as colloidal silicon dioxide.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose
  • a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate
  • a glidant such as colloidal silicon dioxide.
  • dosage unit form can contain, in addition to the ingredients of the above type, a liquid carrier such as fatty oil.
  • dosage unit forms can contain various other materials which modify the physical form of the dosage unit,
  • the IIIMIC peptide derivative, analog or a fragment thereof can be delivered in a controlled release system.
  • an infusion pump can be used to administer the peptide such as the one that is used, for example, for delivering insulin or chemotherapy to specific organs or tumors.
  • the peptide of the invention is administered in combination with a biodegradable, biocompatible polymeric implant, which releases the peptide over a controlled period of time at a selected site.
  • polymeric materials include, but are not limited to, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (See, Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, FIa., the contents of which are hereby incorporated by reference in their entirety).
  • a controlled release system can be placed in proximity to a therapeutic target, thus requiring only a fraction of the systemic dose.
  • compositions of the present invention can be placed on a stent.
  • the present invention provides a method for protecting a subject against noxious insults.
  • the present invention further provides a method for preventing or treating diseases or disorders attributable to inflammatory processes in a subject.
  • the present invention also provides a method for treating a disease or condition attributable to free radicals in a subject.
  • the present invention further provides a method for treating T cell mediated disease in a subject.
  • the methods comprise the step of administering to a subject in need thereof a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of a HIM 1C source and a pharmaceutically acceptable carrier.
  • the IIIMIC source according to the present invention includes the IIIMIC peptide, or a derivative, analog or fragment thereof according to principles of the present invention; an isolated polynucleotide sequence encoding the IIIMIC peptide, or a derivative, analog or fragment thereof; an expression vector comprising the isolated polynucleotide sequence encoding the IIIMIC peptide, or a derivative, analog or fragment thereof; and a host cell transfected with the expression vector comprising the isolated polynucleotide sequence of the invention.
  • the IIIMIC source is the IIIMIC peptide or an analog, derivative or fragment thereof.
  • the peptide is IIIMIC peptide of SEQ ID NO: 1.
  • a "therapeutically effective amount" of the IIIMIC source is that amount of the
  • IIIMl source which is sufficient to provide a beneficial effect to the subject to which the source is administered. More specifically, a therapeutically effective amount means an amount of the source effective to prevent, alleviate or ameliorate tissue damage or symptoms of a disease of the subject being treated.
  • Noxious stimuli include, but are not limited to, heat stimuli, cold stimuli, chemical stimuli, electric stimuli, ultraviolet irradiation, ionizing or non-ionizing irradiation, irradiation of all kinds including electromagnetic and ultrasound, and oxidation.
  • the noxious stimulus is sulphur mustard (SM).
  • SM sulphur mustard
  • SM toxicity results from inflammatory responses and involvement of inflammatory mediators, namely the synthesis and secretion of inflammatory mediators are involved in the evolution and creation of tissue damage caused by SM.
  • the peptides of the invention have proven to be particularly efficacious against inflammatory processes associated with experimental autoimmune encephalitis, the peptides of the invention are therefore very useful as bona fide anti-inflammatory agents.
  • the peptides of the invention are expected to be efficacious in all diseases, disorders, or conditions that involve inflammation or inflammatory activity. Therefore, this invention relates to the protective effect of the IIIMIC source against all disorders or diseases that are related to or involve inflammation.
  • Inflammatory diseases that can be treated by the IIIM1 source include autoimmune diseases including, but not limited to, arthritis including rheumatoid arthritis, asthma, chronic bronchitis, inflammatory bowel disease (Crohn's disease), psoriasis, sepsis, and systemic lupus erythematosus (SLE).
  • arthritis including rheumatoid arthritis, asthma, chronic bronchitis, inflammatory bowel disease (Crohn's disease), psoriasis, sepsis, and systemic lupus erythematosus (SLE).
  • Inflammation is also associated with chronic neurological degenerative diseases and muscle degenerative diseases.
  • the degenerative diseases include, but are not limited to, multiple sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, muscle dystrophy, and amyotrophic lateral sclerosis.
  • Hypersensitivity includes, but is not limited to, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and delayed type hypersensitivity.
  • Inflammation is also associated with an infectious disease.
  • Infectious diseases that can be treated with the pharmaceutical compositions of the invention include, but are not limited to, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, and mycoplasma diseases.
  • the compositions comprising the IIIMIC source of the invention can be used as cosmetics to eliminate skin infections.
  • Inflammation can also be associated with transplantation of a graft, such as, for example, in conditions of graft rejection. Inflammation can also be associated with an allergic disease and with musculoskeletal inflammation.
  • the musculo-skeletal inflammation is selected from the group consisting of arthritis, muscle inflammation, myositis, a tendon inflammation, tendinitis, a ligament inflammation, a cartilage inflammation, a joint inflammation, a synovial inflammation, carpal tunnel syndrome and a bone inflammation.
  • the peptides of the invention are useful for protecting against or treating T cell mediated disease.
  • T cell mediated diseases include, but are not limited to, psoriasis; allergy; T cell lymphomas and other malignancies; graft versus host disease; prevention of transplant rejection; bronchitis; asthma; autoimmunity; sarcoidosis; bone marrow depression; bone marrow stimulation; sepsis; Parkinson's disease; skin disorders and irritation; arthritis; multiple sclerosis; neurodegenerative disorders (amyotrophic lateral sclerosis, chorea, Alzheimer disease); atherosclerosis; fibrosis; pain; chronic or acute inflammation.
  • the protective effect of the peptides of the invention can be achieved by prophylactic treatment.
  • the protective effect can also be achieved by post-exposure treatment with the peptide.
  • the protective effect of the peptides is achieved against inflammatory processes as exemplified herein below.
  • the term "protecting" relate to reduction of degree of lesion or biological damage as measured by gross pathology or histopathological evaluation, subjective burning sensation or other accepted parameters for tissue damage, lesion, discomfort and pain.
  • the pharmaceutical compositions of the invention can be used for accelerated healing of or prevention of development of wounds including decubitus, ulcers (also induced by drugs), internal and external wounds, abscesses and various bleedings.
  • the pharmaceutical compositions of the invention are useful for treatment and protection of the central and peripheral nervous systems against noxious stimuli caused by, but not limited to, chemicals, drugs, all kinds of irradiation and mechanical stress.
  • the peptides may also serve in treatment of a variety of mental diseases and mental-related syndromes.
  • the pharmaceutical compositions of the invention are useful for treatment or protection against tissue damage including, but not limited to, neuronal, neurological, skin, hepatic, nephrologic, urologic, cardiac, pulmonary, gastrointestinal, lower and upper airways, visual, audiologic, spleen, bone, and muscle damage.
  • Treatment or protection against tissue damage can be accomplished in the fetus, newborn, child, adolescent as well as in adults and old persons, whether the condition or disorder to be treated is spontaneous, of traumatic etiology or as a congenital defect.
  • compositions of the invention can be used for treatment or protection against metal poisoning, particularly heavy metal poisoning.
  • the peptides can be used as antidote against toxic metals.
  • compositions of the present invention can comprise the HIM 1C peptide, or a derivative, analog or fragment thereof or any other source of IIIM1C, or all possible combinations of two or more of these peptide derivatives, analogs, or fragments or other sources of IIIMIC.
  • pharmaceutical compositions can comprise one or more isolated polynucleotides, one or more expression vectors, or one or more host cells or any combination thereof, according to the principles of the present invention.
  • the IIIMIC source particularly the peptides of the invention can inhibit or significantly reduce metalloproteinase activity.
  • the peptides will reduce at least 10%, preferably at least 40%, and more preferably at least 80%, of the hydrolytic activity of at least one matrix metalloproteinase that is naturally occurring in a mammal.
  • MMP matrix metalloproteinases
  • MMP-1 and gelatinase A are the more abundant members of the family.
  • Other members include fibroblast collagenase (MMP-I), neutrophil collagenase (MMP-8), gelatinase B (92 kDa gelatinase) (MMP-9), stromelysin-2 (MMP-10), stromelysin-3 (MMP-Il), matrilysin (MMP-7), collagenase 3 (MMP-13), TNF-alpha converting enzyme (TACE).
  • the present invention encompasses IIIMl peptide derivatives, analogs or fragments thereof capable of inhibiting any known membrane-associated matrix metalloproteinases (see, for example, Sato H., Takino T., Okada Y., Cao J., Shinagawa A., Yamamoto E., and Seiki M., Nature, 1994, 370:61-65, the contents of which are hereby incorporated by reference in their entirety).
  • the peptides, derivatives and analogs of the present invention can also be used in combination with one or more known peptide-based or non-peptide- based MMP inhibitors, such as peptide or non-peptide hydroxamates or thiol-containing peptides.
  • altered MMP activity refers to MMP activity, which is higher than the normal activity of the enzyme at that cell or tissue.
  • Inflammatory diseases can affect the central nervous system (brain and spinal cord). Some of the best characterized disorders are multiple sclerosis (MS) and various forms of meningitis and encephalitis. A common feature of these diseases is a disruption of the blood-brain barrier (BBB) followed by inflammatory perivascular infiltration and eventual demyelination and astrogliosis. MMPs play a key role in allowing inflammatory cell access to the CNS. Both MMP-2 and MMP-9 are found in the CSF of patients with MS, and MMP-9 immunoreactivity can be detected in active MS lesions.
  • MS central nervous system
  • BBB blood-brain barrier
  • MMPs also play a role in other neurological disorders, which are not generally considered to be inflammatory in nature.
  • MMPs are probably responsible for the opening of the BBB in focal ischemia and hemorrhagic brain injury leading to secondary injury from vasogenic edema.
  • the peptides of the present invention are useful in the treatment of brain injury.
  • Toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC 50 (the concentration which provides 50% inhibition) and the LD 50 (lethal dose causing death in 50 % of the tested animals) for a subject compound.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, for example, Fingl et al., 1975, in The Pharmacological Basis of
  • dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • the amount of a composition to be administered will, of course, depend on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors. Determination of the exact dose to be administered is conducted by methods known to a person of skill in the art.
  • peptides of the invention can be formulated or administered together with additional active ingredients as required to treat the condition of the patient.
  • HIM 1 or HIM 1C peptides Effect of oral administration of HIM 1 or HIM 1C peptides on experimental autoimmune encephalitis
  • mice Female C57BL/6 mice (20-23 g) were injected subcutaneously into 4 sites on the back, adjacent each of the forelimbs and hindlimbs (total volume 200 ⁇ l), with 200 ⁇ g myelin oligodendrocyte glycoprotein (MOG) 35-55 emulsified with lOO ⁇ l complete Freund's adjuvant, 800 ⁇ g Mycobacterium tuberculosis H37RA (Difco, Detroit, MI) and 80 ⁇ l phosphate buffered saline. Thereafter, each animal was injected intraperitoneally (i.p.) with pertusis toxin (PTX; 200ng/mouse) and an additional PTX injection was repeated two days later.
  • PTX pertusis toxin
  • IIIMIC peptide having the amino acid sequence KGHYAERNGC (SEQ ID NO:1) or IIIM1 peptide having the amino acid sequence KGHYAERVG of SEQ ID NO:2 were orally administered on the 9 day (day of onset of neurological symptoms), 11, 14, 18, 21, 23, 25 and 28 days after MOG administration (marked by arrows) at a dose of 10 mg/kg for each administration.
  • FIG. 1 shows that IIIM1 or IIIMIC when administered orally to EAE mice reduced the neurological symptoms associated with the disease.
  • mice Female C57BL/6 mice (20-23 g) were injected subcutaneously into 4 sites on the back, adjacent each of the forelimbs and hindlimbs (total volume 200 ⁇ l), with 200 ⁇ g myelin oligodendrocyte glycoprotein (MOG) 35-55 emulsified with lOO ⁇ l complete
  • FIG. 2 shows that the neurological score was significantly improved after multiple i.p. administrations of IIIMIC.
  • FIG. 3 shows the activity of the IIIMIC peptide in scavenging free hydroxyl radicals as a function of the concentration of the peptide.

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Abstract

La présente invention concerne un peptide comprenant la séquence d'acides aminés des résidus 36-44 de la H2A humaine modifiée par un résidu cystéine à l'extrémité terminale carboxy du peptide. L'invention concerne également des analogues, des dérivés et des fragments actifs dudit peptide, ainsi que des compositions pharmaceutiques le comprenant. Les compositions de l'invention sont utiles pour le traitement des brûlures et des maladies inflammatoires, auto-immunes et dégénératives.
PCT/IL2007/000897 2006-07-17 2007-07-17 Peptides dérivés de l'histone h2a humaine et leurs procédés d'utilisation WO2008010218A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002092784A2 (fr) * 2001-05-15 2002-11-21 Emory University Polynucleotides et polypeptides lies a la modulation de sirp $g(a)-cd47
WO2005090387A2 (fr) * 2004-03-23 2005-09-29 Yissum Research Development Company Of The Hebrew University Of Jerusalem Derives de peptide d'histone h2a et analogues, et methodes d'utilisation
US20070093426A1 (en) * 2004-03-23 2007-04-26 Uri Wormser Histone H2A peptide derivatives and analogs and methods of use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002092784A2 (fr) * 2001-05-15 2002-11-21 Emory University Polynucleotides et polypeptides lies a la modulation de sirp $g(a)-cd47
WO2005090387A2 (fr) * 2004-03-23 2005-09-29 Yissum Research Development Company Of The Hebrew University Of Jerusalem Derives de peptide d'histone h2a et analogues, et methodes d'utilisation
US20070093426A1 (en) * 2004-03-23 2007-04-26 Uri Wormser Histone H2A peptide derivatives and analogs and methods of use thereof

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