Pharmaceutical compositions for inhibiting inflammatory cytokines

Abstract

To provide new safe and effective treatments that ameliorate inflammatory conditions by intervening in the inflammatory cascade.SOLUTION: The present invention provides pharmaceutical compositions comprising specific tetrapeptides for use in reducing the release or inhibiting the activity of inflammatory cytokines and mediators, and for treating diseases and disorders associated with these cytokines and mediators.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention provides a pharmaceutical composition and method for improving symptoms associated with the release of inflammatory cytokines in inflammatory conditions including, but not limited to, inflammatory eye disorders.

Background Art

[0002] Inflammation is part of a complex biological response of body tissues to harmful stimuli such as pathogens, damaged cells, or irritants, and is a defensive response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, sweep away necrotic cells and tissues damaged from the original injury and the inflammatory process, and initiate tissue repair.

[0003] The classical signs of inflammation are heat, pain, redness, swelling, and loss of function. Inflammation is a common reaction and is thus considered a mechanism of innate immunity.

[0004] Inflammatory diseases include various disorders and conditions characterized by inflammation. For example, allergies, asthma, transplant rejection, autoimmune diseases, and eye inflammation.

[0005] Inflammatory eye disorder Uveitis is a general term representing a group of inflammatory eye diseases that can slightly reduce vision or lead to severe blindness. The disease often affects a part of the eye called the uvea, but is not limited to this part of the eye. These diseases also affect the lens, retina, optic nerve, and vitreous body, causing vision loss or blindness. Uveitis may be caused by problems or diseases occurring in the eye, or may be part of an inflammatory disease affecting other parts of the body. Uveitis can be acute or chronic and is usually classified as infectious or non-infectious.

[0006] Dry eye disease (DED), also known as dry eye syndrome (DES), is a common eye disorder that millions of people seek ophthalmic treatment for. Regardless of the underlying cause, dry eye has been shown to be associated with abnormalities in the precorneal tear film and subsequent inflammatory changes throughout the ocular surface, including the adnexa, conjunctiva, and cornea. Activation of inflammatory cytokines, such as IL-6, plays an important role in the pathophysiology of DED.

[0007] Since the role of inflammation in dry eye has been recognized, many treatment methods designed to inhibit various inflammatory pathways have been studied, such as cyclosporine A, corticosteroids, tacrolimus, tetracycline derivatives, and autologous serum (Michelle He ssen M., and Karamursel Akpek E J Ophthalmic Vis Res. 2014, 9(2), 240 - 250).

[0008] U.S. Patent No. 4,619,916 teaches 13 tripeptides of the formula pGlu-X-Trp, where pGlu is cyclized glutamic acid (pyroglutamic acid) and X may be Gly, Val, Glu, Asp, Ser, Ala, Asn, Gln, Ile, Leu, Pro, Lys, and Arg. This reference does not describe or suggest the use of peptides for treating inflammation.

[0009] U.S. Patent No. 7,220,725 and International Patent Publication WO200212269 teach novel peptides including pGlu-Asn-Trp-Lys(octanoyl)-OH (ZEP3) and pGlu-Asn-Trp-Thr-OH (ZEP4), and their use for treating pain. This reference does not describe or suggest the use of peptides for treating inflammation.

[0010] U.S. Patent No. 9,012,397 and WO2012 / 131676 teach a topical pharmaceutical composition comprising the peptide ZEP3 or ZEP4 and its use for improving the symptoms of skin diseases. This reference does not describe or suggest the use of the peptide for treating inflammation.

[0011] Gaynes et al. (Invest. Ophthalmol. Vis. Sci. 54, E-Abstract 5416, 2013) describe the analgesic effect of the peptide ZEP4 in reducing ocular pain and altering the nociceptive pathway in a rat model of experimentally induced chemical corneal injury. This reference does not describe or suggest the anti-inflammatory activity of the peptide.

[0012] The present invention addresses the continuing need to improve and develop new safe and effective treatments for ameliorating inflammatory conditions by intervening in the inflammatory cascade. SUMMARY OF THE INVENTION

[0013] The present invention provides compositions and methods for improving symptoms associated with the release of inflammatory cytokines. These cytokines are known to be part of the etiology and pathology of many inflammatory diseases. Diseases and disorders suitable for treatment with the compositions of the present invention include, but are not limited to, inflammatory diseases of the eye, ear, lung, and intestine. Diseases and disorders suitable for treatment also include autoimmune diseases where the administration of medicine may be chronic in order to prevent exacerbation.

[0014] The present invention is based in part on the discovery that, in in vitro and in vivo models, the peptides represented by ZEP3 and ZEP4 exhibit unexpectedly strong activity in attenuating the expression and / or release of inflammatory and inflammation-inducing mediators, including the specific cytokines interferon gamma (IFN-gamma, IFNγ), interleukin 1 beta (IL-1 beta, IL-1β), interleukin 10 (IL-10), tumor necrosis factor alpha (TNF alpha, TNFα), and interleukin 6 (IL-6), as well as the mediator reactive oxygen species (ROS) and RANTES (regulated upon activation, normal T cell expressed and presumably secreted).

[0015] Surprisingly, it has been found that the activity of the peptides in inhibiting the inflammatory cascade is exerted even when the peptides are administered before the inflammatory reaction is initiated. Thus, the peptides can ameliorate or suppress the symptoms induced in various inflammatory diseases and disorders and can be useful for completely eliminating the inflammatory reaction. The compositions of the present invention are contemplated to be useful for preventing the exacerbation of chronic inflammatory diseases.

[0016] It should be clearly understood that the use of the compositions of the present invention for treating inflammatory diseases does not include the treatment of skin disorders or pain per se.

[0017] According to one aspect of the present invention, there is provided a pharmaceutical composition comprising, as an active ingredient, a peptide according to formula I: pGlu-X1-X2-X3-OH, wherein X1 is a polar amino acid residue, X2 is an aromatic or hydrophobic amino acid residue, and X3 is a positively charged amino acid residue and a polar amino acid residue, or a pharmaceutically acceptable salt or derivative thereof; and a pharmaceutically acceptable carrier, for use in reducing the release of inflammatory cytokines or inhibiting their activity.

[0018] According to some embodiments, X1 is selected from the group consisting of Asn, Gln, His, Ser, Thr, Tyr, and Cys; X2 is selected from the group consisting of Trp, Phe, Tyr, Ala, Ile, Leu, Met, Val, and Gly; and X3 is selected from the group consisting of Lys, Lys derivatives, Arg, His, Asn, Gln, His, Ser, Thr, and Tyr.

[0019] According to yet other embodiments, X1 is selected from Asn and Thr; X2 is selected from Trp, Phe, and Tyr; and X3 is selected from Lys, Lys derivatives, and Thr.

[0020] According to some embodiments, the peptide derivative comprises an alkyl group attached to a free functional group of the peptide sequence.

[0021] According to yet other embodiments, the alkyl group is attached to a side chain of the peptide or the free amino group at the N-terminus by an amide bond or a linkage.

[0022] According to some embodiments, the alkyl is C4-C30 alkyl.

[0023] According to some specific embodiments, an octanoyl group (octanoyl herein), a C8 alkyl group, is attached to the side chain of a Lys residue of the peptide sequence or the terminal amino group of the peptide by an amide linkage. According to some embodiments, the carboxy terminus of the peptide is modified to form, for example, an amide, alcohol, or ester terminus.

[0024] According to some specific embodiments, the peptide is selected from the group consisting of pGlu-Asn-Trp-Lys(octanoyl)-OH (SEQ ID NO: 1), pGlu-Asn-Trp-Thr-OH (SEQ ID NO: 2), and pharmaceutically acceptable derivatives and salts thereof.

[0025] According to some embodiments, the inflammatory cytokine is selected from the group consisting of TNF alpha, IL-1 beta, IL-6, IL-10 and IFN gamma.

[0026] According to yet another aspect, the present invention provides a pharmaceutical composition for use in reducing the release of inflammatory mediators or chemokines (mediators or chemokines), as an active ingredient, a peptide according to formula I: pGlu-X1-X2-X3-OH, wherein X1 is a polar amino acid residue; X2 is an aromatic or hydrophobic amino acid residue; X3 is selected from positively charged amino acid residues and polar amino acid residues, or a pharmaceutically acceptable salt or derivative thereof; and a pharmaceutically acceptable carrier.

[0027] According to some specific embodiments, the peptide is selected from the group consisting of pGlu-Asn-Trp-Lys(octanoyl)-OH (SEQ ID NO: 1), pGlu-Asn-Trp-Thr-OH (SEQ ID NO: 2), and pharmaceutically acceptable derivatives and salts thereof.

[0028] According to some embodiments, the inflammatory mediator or cytokine is selected from the group consisting of reactive oxygen species (ROS) and RANTES (regulated upon activation, normal T cell expressed and perhaps secreted).

[0029] According to another aspect of the present invention, there is provided a method of reducing the release or inhibiting the activity of at least one inflammatory or inflammation-inducing cytokine, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide according to formula I: pGlu-X1-X2-X3-OH, wherein X1 is a polar amino acid residue; X2 is an aromatic or hydrophobic amino acid residue; X3 is selected from positively charged amino acid residues and polar amino acid residues, or a pharmaceutically acceptable salt or derivative thereof.

[0030] According to another aspect, there is provided a method of reducing the release of inflammatory mediators or chemokines, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide of formula I: pGlu-X1-X2-X3-OH, wherein X1 is a polar amino acid residue; X2 is an aromatic or hydrophobic amino acid residue; and X3 is selected from positively charged amino acid residues and polar amino acid residues, or a pharmaceutically acceptable salt or derivative thereof.

[0031] According to some embodiments, the inflammatory mediator or chemokine is selected from the group consisting of ROS and RANTES.

[0032] According to yet another aspect, the present invention provides a method of treating an inflammatory disease or disorder. According to certain embodiments, the inflammatory disease or disorder is selected from the group consisting of an inflammatory disease or disorder of the eye, an inflammatory disease or disorder of the ear, an inflammatory disease or disorder of the lung, an inflammatory disease or disorder of the intestine, or an inflammatory autoimmune disease or disorder, and the method comprises administering to a subject in need thereof a therapeutically effective amount of a peptide according to formula I: pGlu-X1-X2-X3-OH, or a pharmaceutically acceptable salt or derivative thereof, wherein X1 is a polar amino acid residue; X2 is selected from aromatic amino acid residues and hydrophobic amino acid residues; and X3 is selected from positively charged amino acid residues and polar amino acid residues.

[0033] According to some embodiments, the present invention provides a method of treating a chronic inflammatory disease. According to certain embodiments, the step of administering to a subject in need thereof a therapeutically effective amount of a peptide according to formula I is also carried out before symptoms appear or develop. These methods of treating chronic inflammatory diseases are effective in preventing exacerbation.

[0034] According to yet another aspect, the present invention provides a method for treating an inflammatory disease or disorder of the eye, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide according to Formula I: pGlu-X1-X2-X3-OH, or a pharmaceutically acceptable salt or derivative thereof, wherein X1 is a polar amino acid residue; X2 is an aromatic or hydrophobic amino acid residue; and X3 is selected from positively charged amino acid residues and polar amino acid residues.

[0035] In some embodiments, X1 is selected from the group consisting of Asn, Gln, His, Ser, Thr, Tyr, and Cys; X2 is selected from Trp, Phe, Tyr, Ala, Ile, Leu, Met, Val, and Gly; and X3 is selected from Lys, Lys derivatives, Arg, His, Asn, Gln, His, Ser, Thr, and Tyr.

[0036] In yet other embodiments, X1 is selected from Asn and Thr; X2 is selected from Trp, Phe, and Tyr; and X3 is selected from Lys, Lys derivatives, and Thr.

[0037] In some specific embodiments, the Lys derivative is Lys(octanoyl).

[0038] In some embodiments, the peptide derivative comprises an alkyl group attached to a functional group of the peptide sequence.

[0039] In yet other embodiments, the alkyl group is attached to the side chain or terminal amino group of the peptide by an amide bond.

[0040] In some embodiments, the alkyl is C4-C30 alkyl.

[0041] In some specific embodiments, an C8 alkyl group (octanoyl herein) is attached to the side chain of the Lys residue or the terminal amino group of the peptide sequence by an amide bond.

[0042] According to some specific embodiments, the peptide is selected from the group consisting of pGlu-Asn-Trp-Lys(octanoyl)-OH (SEQ ID NO: 1), pGlu-Asn-Trp-Thr-OH (SEQ ID NO: 2), and pharmaceutically acceptable salts or derivatives thereof.

[0043] According to some embodiments, the peptide has the amino acid sequence shown in SEQ ID NO: 1.

[0044] According to some embodiments, the peptide has the amino acid sequence shown in SEQ ID NO: 2.

[0045] According to some embodiments, the pharmaceutical composition comprises a derivative or salt of a peptide having an amino acid sequence represented by a sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.

[0046] According to some embodiments, the method comprises administering a derivative or salt of a peptide having an amino acid sequence represented by a sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.

[0047] According to some embodiments, the pharmaceutical composition comprises a sodium salt of the peptide shown in either SEQ ID NO: 1 or SEQ ID NO: 2.

[0048] According to some embodiments, the pharmaceutical composition is formulated for administration via a route selected from the group consisting of topical administration, ophthalmic administration, oral administration, nasal administration, and parenteral administration.

[0049] According to various embodiments, the formulation for topical administration can be selected from the form of cream, ointment, paste, lotion, gel, or eye drops.

[0050] According to some specific embodiments, a cream formulation comprising a peptide selected from SEQ ID NOs: 1 and 2 or a pharmaceutically acceptable salt thereof for use in topical administration is provided.

[0051] According to some embodiments, the cream composition comprises 0.1 to 5% w / w of a peptide. According to other embodiments, the composition comprises 0.5 to 2% of a peptide. According to still other embodiments, the composition comprises about 1% of a peptide.

[0052] According to some embodiments, the composition according to the present invention has a pH of about 4 to about 8. According to some embodiments, the composition has a pH of about 4.5 to about 6.5. According to still other embodiments, the composition has a pH of about 7 to about 9.

[0053] According to some embodiments, the pharmaceutical composition comprises at least one excipient selected from the group consisting of polysorbate 80, disodium edetate (EDTA), silicon dioxide, methylparaben, white petrolatum, isopropyl myristate, cetyl alcohol, and glyceryl monostearate.

[0054] According to other embodiments, a composition for administration to a subject having an eye disease or injury is formulated in a form selected from the group consisting of eye drops, emulsions, creams, ointments, sprays, and liquid solutions or suspensions for use as gels.

[0055] According to some embodiments, the eye disease or injury is an inflammatory disease or disorder selected from the group consisting of uveitis, dry eye syndrome, inflammatory symptoms associated with infectious eye diseases, allergic eye diseases, keratitis, conjunctivitis, meibomian gland dysfunction, and Sjogren's syndrome.

[0056] According to further embodiments, the pharmaceutical composition is formulated as eye drops, eye ointments, eye sprays, ophthalmic suspensions, ophthalmic emulsions, ophthalmic solutions, ophthalmic gels, or intravitreal injections.

[0057] According to further embodiments, the inflammatory disease or disorder is an autoimmune disease or disorder.

[0058] According to a further embodiment, the autoimmune disease or disorder is selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, lupus, and Sjögren's syndrome.

[0059] According to a further embodiment, the inflammatory disease or disorder is inflammatory bowel disease.

[0060] According to a further embodiment, the inflammatory bowel disease is selected from the group consisting of Crohn's disease, ulcerative colitis, and celiac disease.

[0061] According to a further embodiment, the inflammatory disease or disorder is an inflammatory disease or disorder of the lung.

[0062] According to a further embodiment, the inflammatory disease or disorder of the lung is selected from the group consisting of asthma, bronchitis, pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis, and cystic fibrosis.

[0063] According to a further embodiment, the inflammatory disease or disorder is an inflammatory disease or disorder of the ear.

[0064] According to a further embodiment, the inflammatory disease or disorder of the ear is selected from the group consisting of ear infections, otitis media, otitis externa, mastoiditis, and inflammatory symptoms associated with otomastoiditis.

[0065] According to an embodiment of a further feature, the administration is ophthalmic administration.

[0066] According to a further embodiment, the administration is oral administration.

[0067] According to a further embodiment, the administration is nasal administration.

[0068] According to a further embodiment, the administration is parenteral administration.

[0069] The present invention successfully addresses the drawbacks of the currently known configurations by providing a pharmaceutical composition for use in reducing the release or inhibiting the activity of inflammatory cytokines and mediators, and in treating diseases or disorders associated with inflammatory cytokines and mediators.

[0070] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting.

Brief Description of the Drawings

[0071] Some embodiments of the present invention are described herein by way of example only with reference to the accompanying drawings. Referring now particularly to the drawings in detail, it is emphasized that the details shown are for purposes of illustration and for the purpose of discussing embodiments of the present invention. In this regard, the description taken in conjunction with the drawings will make apparent to those skilled in the art how embodiments of the present invention may be practiced.

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DETAILED DESCRIPTION OF THE INVENTION

[0073] The present invention relates to a pharmaceutical composition comprising a specific peptide, its salts and derivatives for use in inhibiting the inflammatory cascade. Inhibition of inflammatory mediators is beneficial in the prevention and treatment of several inflammatory diseases and disorders, such as inflammatory diseases and disorders of the eye.

[0074] As exemplified herein, the inhibitory effect on inflammatory cytokine release has been demonstrated in vitro in several models including mouse macrophage cell lines, human monocyte cell lines, human keratinocyte cell lines, human peripheral blood monocytes (human PBMCs), and human corneal epithelial cells (HCECs), and in vivo in an inflammatory atopic dermatitis (AD) mouse model.

[0075] The beneficial effect of the peptide on experimental corneal inflammation has been demonstrated in a human corneal epithelial cell culture model.

[0076] The principles and operations of the present invention can be better understood with reference to the drawings and the accompanying description.

[0077] Before explaining at least one embodiment of the present invention in detail, it is to be understood that the present invention is not necessarily limited to the details described in the following explanation or illustrated by the examples in its application. The present invention can have other embodiments or can be practiced or carried out in various ways. Also, it is to be understood that the expressions or terms used herein are for the purpose of description and should not be construed as limiting.

[0078] During the practice of the present invention, the inventors surprisingly found that the peptides ZEP3 and ZEP4, and their sodium salt forms, reduced the expression of the inflammatory cytokines TNF alpha, IL-1 beta, and IL-6 by macrophages and corneal epithelial cells induced by inflammation (Examples 1-7 below), and decreased the expression of IFNγ, IL-10, IL-1β, and RANTES in inflamed human PBMCs (Example 12 below). Furthermore, in an inflammatory model of atopic dermatitis in mice, ZEP4, ZEP3, and its sodium salt formed ZEP3Na and significantly reduced clinical lesions in vivo (Example 13 below). The results indicate that the peptides of the present invention can inhibit or prevent inflammation independently.

[0079] Accordingly, according to one aspect of the present invention, there is provided a pharmaceutical composition for reducing the release of or inhibiting the activity of at least one cytokine or mediator involved in the etiology or condition of an inflammatory disease or disorder. The pharmaceutical composition comprises a peptide as an active ingredient and a pharmaceutically acceptable carrier. The peptide has an amino acid sequence shown in any of SEQ ID NO: 1 and 2, or a pharmaceutically acceptable salt thereof.

[0080] Accordingly, the present invention provides a method for improving and treating inflammatory conditions associated with the excessive release or activity of specific cytokines and mediators.

[0081] According to some embodiments, the inflammatory condition is a disease or disorder associated with the excessive release or activity of at least one cytokine or mediator, and the at least one cytokine or mediator is selected from the group consisting of IFN-gamma, IL-1 beta, IL-10, TNF alpha, IL-6, ROS, and RANTES.

[0082] As used herein, the phrase "inflammatory disease or disorder" refers to a disease, condition, or disorder associated with inflammation. As used herein, the term "inflammation" refers to the process by which the immune system of a subject regulates its response to tissue damage, infection, antigen load, etc. Inflammation can be associated with an increase in blood supply to the tissue, an increase in capillary permeability of the tissue, and / or an increase in the migration of white blood cells to the tissue.

[0083] Inflammatory cytokines are mainly produced by activated macrophages and are involved in the upregulation of the inflammatory response.

[0084] Inflammation can be diagnosed by an increase in the levels of inflammatory cytokines such as, but not limited to, TNFα, IL-1-alpha, IL-1-beta, IFN-gamma, and IL-6 in a biological sample obtained from a subject. The biological sample can be, for example, tears, blood, serum, plasma, urine, sputum, saliva, feces, semen, cerebrospinal fluid, bone marrow, lymph, or CSF, external secretions of the skin, respiratory, intestinal, and urogenital systems, milk, human organs or tissues, samples obtained by washing, multiple exudates, samples of in vitro or ex vivo cell cultures, and cell culture components.

[0085] Reactive oxygen species (ROS) are important signaling molecules that play an important role in the progression of inflammatory diseases. Increased ROS production by polymorphonuclear neutrophils (PMNs) at the site of inflammation causes endothelial dysfunction and tissue damage. In inflammatory conditions, oxidative stress generated by PMNs leads to the opening of endothelial junctions and promotes the migration of inflammatory cells through the endothelial barrier.

[0086] According to some embodiments of the present invention, the inflammatory disease or disorder is an inflammatory disease or disorder of the eye. The inflammatory eye disease or disorder can be, but is not limited to, uveitis, dry eye syndrome, inflammatory symptoms associated with viral, bacterial, or fungal eye infections, allergic eye diseases, keratitis, conjunctivitis, meibomian gland dysfunction, and Sjogren's syndrome.

[0087] In one embodiment, the inflammatory disease or disorder of the eye is uveitis. As used herein, the term "uveitis" refers to inflammation of the uvea, which is the layer between the sclera and the retina, including the iris, ciliary body, and choroid. Uveitis is generally also called iritis, pars planitis, choroiditis, chorioretinitis, anterior uveitis, and posterior uveitis. The most common form of uveitis is anterior uveitis, which usually involves inflammation of the anterior part of the eye that is isolated to the iris. This condition is often called iritis.

[0088] In one embodiment, the inflammatory disease or condition of the eye is dry eye syndrome. As used herein, the phrase "dry eye syndrome (DES)" refers to a condition of altered tear composition resulting from an affected or dysfunctional lacrimal gland functional unit. Evidence suggests that inflammation causes structural changes in the tear-secreting glands. Changes in tear composition resulting from lacrimal gland insufficiency, increased evaporation, and / or poor clearance exert an inflammation-inducing effect on the surface of the eye. This inflammation is, in part, responsible for irritation symptoms, ocular surface epithelial diseases, and changes in the corneal epithelial barrier function of dry eye. Anti-inflammatory therapy for DES targets one or more of the inflammatory mediators / pathways identified in dry eye. DES is also known to those skilled in the art as dry eye disease (DED), keratoconjunctivitis sicca, and keratitis sicca.

[0089] In one embodiment, the inflammatory disease or condition of the eye is associated with Sjögren's syndrome. As used herein, the phrase "Sjögren's syndrome" refers to a systemic autoimmune inflammatory disorder characterized by decreased tear production, dry mouth, and other mucosal dryness, and is often associated with autoimmune rheumatic disorders such as rheumatoid arthritis. Dryness of the eyes and mouth are the most common symptoms of this syndrome. The symptoms may occur alone or may be accompanied by symptoms associated with rheumatoid arthritis or other connective tissue diseases. Enlargement of the associated salivary glands may also occur. Other organs may be affected. This syndrome may also be associated with rheumatoid arthritis, systemic lupus erythematosus (SLE), scleroderma, polymyositis, and other diseases.

[0090] In some embodiments, the inflammatory disease or condition of the eye is due to a procedure involving the eye, such as corneal transplantation / keratoplasty, corneal prosthesis surgery, lamellar grafting, or selective endothelial transplantation.

[0091] In some embodiments, the inflammatory disease or condition of the eye affects the surface of the eye, including, but not limited to, keratitis such as kerato-ocular surface inflammatory states, corneal neovascularization, peripheral ulcerative keratitis, and microbial keratitis, conjunctivitis, or pemphigoid syndrome.

[0092] In some embodiments, the ocular inflammatory disease or condition is, but not limited to, an autoimmune disorder affecting the eye such as sympathetic ophthalmoplegia, Vogt-Koyanagi-Harada (VKH) syndrome, birdshot retinochoroidopathy, ocular cicatricial pemphigoid (OCP), Sjogren's syndrome or Fuchs' heterochromic iridocyclitis.

[0093] According to some embodiments of the present invention, the inflammatory disease or disorder is an autoimmune disease or disorder. The autoimmune disease or disorder may be, but not limited to, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, pemphigus vulgaris, ankylosing spondylitis, juvenile idiopathic arthritis, lupus and Sjogren's syndrome.

[0094] In one embodiment, the autoimmune disease or disorder is rheumatoid arthritis. As used herein, the term "rheumatoid arthritis" refers to a chronic autoimmune disorder in which multiple joints become inflamed. Inflammation of the synovial joint lining is accompanied by joint pain and stiffness and usually results in bone and joint destruction, deformity, dysfunction, and even death.

[0095] In one embodiment, the autoimmune disease or disorder is lupus. As used herein, the term "lupus" refers to a chronic inflammatory autoimmune disorder called erythematosus that can affect many organ systems including the skin, joints, and internal organs. Lupus is a general term that includes several specific types of lupus, including systemic lupus, lupus nephritis, and lupus encephalitis. In systemic lupus erythematosus (SLE), the body's natural defenses turn against the body and rogue immune cells attack the body's tissues. Antibodies may be produced that can react against the body's blood cells, organs, and tissues. This reaction results in immune cells that attack the affected system and cause a chronic disease. Lupus nephritis, also known as lupus glomerulonephritis, is a kidney disorder that is usually a complication of SLE and is characterized by damage to the glomeruli and progressive loss of kidney function. Lupus encephalitis refers to another complication of SLE that is inflammation of the brain and / or central nervous system.

[0096] In one embodiment of the present invention, the inflammatory disease or disorder is osteoarthritis (OA). OA is also referred to as hypertrophic osteoarthritis, degenerative arthritis, and degenerative joint disease. OA is a chronic degenerative disease of the skeletal joints, affecting specific joints in adults of all ages, usually the knees, lower back, hand joints, and spine. OA is characterized by several symptoms including degeneration and thinning of the articular cartilage with associated development of "ulcers" or craters, osteophyte formation, bone hypertrophy at the margins, and synovial changes and swelling of the affected joints. Furthermore, osteoarthritis is accompanied by pain and stiffness, especially after prolonged activity. The peptides, salts, and derivatives of the present invention, as well as pharmaceutical compositions containing them, can be used for treating osteoarthritis. Characteristic radiographic features of osteoarthritis include joint space narrowing, subchondral sclerosis, osteophytosis, subchondral cyst formation, and loose bodies (or "joint mice").

[0097] According to some embodiments of the present invention, the inflammatory disease or disorder is an inflammatory bowel disease. The inflammatory bowel disease may be, but is not limited to, Crohn's disease, ulcerative colitis, and celiac disease.

[0098] According to some embodiments of the present invention, the inflammatory disease or disorder is an inflammatory disease or disorder of the lung. The inflammatory disease or disorder of the lung may be, but is not limited to, asthma, bronchitis, pleurisy, pneumonia, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonia, idiopathic pulmonary fibrosis, and cystic fibrosis.

[0099] According to some embodiments of the present invention, the inflammatory disease or disorder is an inflammatory disease or disorder of the ear. The inflammatory disease or disorder of the ear may be, but is not limited to, inflammatory symptoms associated with ear infections, otitis media, otitis externa, mastoiditis, and otomastoiditis.

[0100] The present invention also provides a pharmaceutical composition for use in treating diseases or disorders associated with the over-release of inflammatory mediators and chemokines including, but not limited to, ROS, RANTES, and monocyte chemoattractant protein-1 (MCP-1). Inflammatory diseases associated with the release of ROS treatable with the compositions of the present invention include, but are not limited to, lung diseases, cardiovascular diseases, kidney disease-related vascular calcification, and inflammatory metabolic diseases.

[0101] The chemokines RANTES and MCP-1 are involved in the recruitment of eosinophils in the development of lung allergic inflammation, lung leukocyte infiltration, bronchial hypersensitivity, and asthma.

[0102] The terms "treating" or "treatment" may be used interchangeably herein and refer to inhibiting, preventing or arresting the onset of a disease or disorder, and / or causing the reduction, remission or regression of a disease or disorder.

[0103] The peptides, derivatives and salts used in the compositions and methods of the present invention can be synthesized using any method known in the art including, but not limited to, solid phase and liquid phase peptide synthesis. Some of the peptides used in the compositions of the present invention can be generated using recombinant methods or a combination of recombinant and synthetic methods.

[0104] In one embodiment of the present invention, the peptide is the peptide of pGlu-Asn-Trp-Lys(octanoyl)-OH (SEQ ID NO: 1; hereinafter referred to as "ZEP3") and pGlu is pyroglutamic acid. The peptide ZEP3 can be generated, for example, by the procedure described in U.S. Patent No. 7,220,725.

[0105] In one embodiment of the present invention, the peptide is pGlu-Asn-Trp-Thr-OH (SEQ ID NO: 2; hereinafter referred to as "ZEP4"). The peptide ZEP4 can be generated, for example, by the following procedure.

[0106] The synthesis of peptide ZEP4 is carried out by the sequential synthesis of 9-fluorenylmethoxycarbonyl (Fmoc) amino acids on a solid support of chlorotrityl chloride resin (CTC). Fmoc-threonine (t-butyl; 79 gr) is loaded onto the CTC resin (125 gr), and diisopropylamine (DIPEA; 160 gr) is used as a coupling agent for the amino acid to the solid support. The Fmoc protecting group is removed with a mixture of 25% piperidine, and the resin-peptide is filtered and washed with dimethylformamide (DMF). The second amino acid, Fmoc-Trp (85 gr), is activated with a mixture of 3-[bis(dimethylamino)methyliminyl]-3H-benzotriazol-1-oxide, hexafluorophosphate (HBTU) / hydroxybenzothiazole (OHBT), and coupled to the first amino acid by the addition of DIEA. The Fmoc group is removed as described above, the resin-peptide is filtered, and washed with dimethylformamide (DMF). The third amino acid, Fmoc-Asn(trt) (119 gr), is activated with HBTU / HOBT and coupled by the addition of DIEA. The Fmoc group is removed as described above, the resin-peptide is filtered, and washed with dimethylformamide (DMF). The fourth amino acid, pGlu (26 gr), is activated with HBTU / HOBT and coupled with DIEA.

[0107] After the peptide-resin is thoroughly washed with DMF, it is washed with IPA and dried under reduced pressure. The peptide is cleaved from the resin and the protecting groups of Thr and Asn with trifluoroacetic acid (TFA) (95%) and triisopropylsilane (TIS) (5%) at room temperature for 2 hours. The peptide is precipitated by adding methyl tert-butyl ether (MTBE), filtered, and dried (yield 46 gr).

[0108] The crude product (46 gr) is dissolved in a mixture of acetonitrile (ACN) / water and loaded onto a preparative HPLC system (4", RP C-18 100 - 120A pore size), and purified using a gradient system consisting of phase A - 0.1% TFA in water and phase B - ACN. Elution is carried out by gradually increasing phase B over 45 minutes (from 3% to 33%). Fractions with a purity exceeding 97% are collected. The combined fractions are eluted on the same HPLC system using a gradient consisting of phase A: 0.2% acetic acid and phase B: ACN. Elution is carried out by gradually increasing phase B over 30 minutes (from 10% to 40%). Fractions with a purity exceeding 97% are collected, combined, and lyophilized (yield 29 gr). The final product has an M.W (MS) of 530.5 and a purity (HPLC) of 97.3%.

[0109] Also included within the scope of the present invention are salts and derivatives of the peptides used in the disclosed compositions and methods.

[0110] As used herein, the term "salt" refers to both salts of the carboxyl groups of the peptide molecule and acid addition salts of the amino or guanidino groups. Salts of carboxyl groups can be formed by means known in the art, including, for example, inorganic salts such as sodium, calcium, ammonium, ferric or zinc salts, and salts with organic bases such as salts formed with amines, for example, triethanolamine, piperidine, procaine, etc. Acid addition salts include, for example, salts with mineral acids such as acetic or oxalic acid. Here, the salt also describes the ionic component added to the peptide solution to enhance hydrogel formation and / or mineralization of calcium minerals.

[0111] As used herein, "derivatives" of the peptides of the present invention include derivatives that can be prepared by means known in the art from residues or functional groups arising as side chains of the N - terminal or C - terminal groups, and are included in the present invention as long as they remain pharmaceutically acceptable, i.e., as long as they do not destroy the activity of the peptide, do not impart toxic properties to the composition containing it, and do not adversely affect its immunogenic properties.

[0112] These derivatives may include, for example, aliphatic esters of carboxyl groups, amides of carboxyl groups formed by reaction with ammonia or primary or secondary amines, N-acyl derivatives of the free amino groups of amino acid residues formed by reaction with an acyl moiety (e.g., an alkanoyl or aroyl group), or O-acyl derivatives of free hydroxyl groups (e.g., those of seryl or threonyl residues) formed by reaction with an acyl moiety.

[0113] In one embodiment of the present invention, the peptide is the sodium salt of the peptide shown in SEQ ID NO: 1 (pGlu-Asn-Trp-Lys(octanoyl)-OH nNa, where n is 1 or 2, hereinafter referred to as "ZEP3 sodium salt" or ZEP3Na).

[0114] The ZEP3 sodium salt can be produced, for example, by the following procedure. ZEP3 (3.1 g) is dissolved in NaHCO3 (100 mM) in water (50 g / l). The solution is injected into an HPLC ion exchange column (2.5×22 cm Luna C18, 100A, 15 micron) and eluted with a gradient consisting of mobile phase A: 2 mM NaHCO3 in H2O; mobile phase B: 2 mM NaHCO3 in CH3CN / H2O (8 / 2); and mobile phase C: 100 mM NaHCO3 in water. Load per run: maximum 5% (W / W% peptide / stationary phase). Flow rate: 4.8 cm / min (24 ml / min). The gradient procedure is as follows: phase C for 20 minutes; phase A for 5 minutes; phase B for 18 minutes; and phase C for 7 minutes. The fractions containing the product are collected, concentrated under reduced pressure to remove acetonitrile (110 g / l), and then freeze-dried [yield 2.2 g (71%)]. The final product has a purity of 99.7% (HPLC), a sodium content of 3.1%, and a solubility in water of 50 mg / ml.

[0115] In one embodiment of the present invention, the peptide is the sodium salt of the peptide set forth in SEQ ID NO: 2 (pGlu-Asn-Trp-Thr-OH·nNa, where n is 1 or 2; hereinafter referred to as "ZEP4 sodium salt" or ZEP4Na). The ZEP4 sodium salt can be produced, for example, by the following procedure. ZEP4 (5 g) is dissolved in NaHCO3 (100 mM) in water (50 g / l). The solution is injected into an HPLC ion exchange column (2.5×22 cm Luna C18, 100A, 15 micron) and eluted with a gradient consisting of mobile phase A: 2 mM NaHCO3 in H2O; mobile phase B: 2 mM NaHCO3 in CH3CN / H2O (8 / 2); and mobile phase C: 100 mM NaHCO3 in water. Load per run: maximum 5% (W / W% peptide / stationary phase). Flow rate: 4.8 cm / min (24 ml / min). The gradient procedure is as follows: phase C for 20 minutes, then phase A for 5 minutes, then phase B for 20 minutes, and phase C for 10 minutes. The fractions containing the product are collected, concentrated under reduced pressure to remove acetonitrile (110 g / l), and then freeze-dried [yield 4 g (80%)]. The final product has a purity of 97.5% (HPLC), a sodium content of 2.5%, and a solubility in water of 50 mg / ml.

[0116] The peptide of the present invention can be used for treatment by itself or as part of a pharmaceutical composition (active ingredient).

[0117] As used herein, "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein, including other chemical components such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to a living being.

[0118] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" may be used interchangeably and refer to a carrier or diluent that does not cause significant irritation to a living being and does not inhibit the biological activity and properties of the administered compound. Adjuvants are included in these terms.

[0119] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to facilitate the administration of the active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starches, polysaccharides, cyclodextrins, suspending agents such as polyvinylpyrrolidone, polyvinyl alcohol, ionic or nonionic surfactants, solubilizing agents, emulsifying agents such as lecithin, penetration enhancers, polycarboxylic acids, cellulose derivatives, gelatin, vegetable oils, waxes, mineral oils, propylene glycol, and polyethylene glycol.

[0120] Techniques for formulation and administration of drugs can be found in the latest edition of "Remington’s Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

[0121] Suitable routes of administration include, for example, intravitreal, topical, oral, rectal, transmucosal, nasal, enteral, or parenteral delivery (including intramuscular, subcutaneous and intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intravitreal injections).

[0122] The pharmaceutical compositions of the present invention can be manufactured by processes well known in the art, for example, by conventional mixing, dissolving, suspending, solubilizing, granulating, tablet coating, levigating, emulsifying, encapsulating, entrapping, spray drying, or freeze drying processes.

[0123] Accordingly, the pharmaceutical compositions for use according to the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries that facilitate the processing of the active ingredient into a preparation that can be used pharmaceutically. Appropriate formulations depend on the chosen route of administration.

[0124] The pharmaceutical composition described in this specification can be formulated for ophthalmic administration. As used herein, the phrase "ophthalmic administration" refers to the administration of the peptide or pharmaceutical composition of the present invention into the external eye (e.g., the conjunctival sac) or into the vitreous body. Dosage forms suitable for ophthalmic administration may be, but are not limited to, eye drops, eye ointments, eye sprays, ophthalmic suspensions, ophthalmic emulsions, ophthalmic solutions, ophthalmic gels, or intravitreal injections.

[0125] The pharmaceutical composition described in this specification can be formulated for topical administration. Dosage forms suitable for topical administration may be, but are not limited to, droplets, liquid detergents, gels, ointments, emulsions, suspensions, lotions, sprays, spray liquids, suppositories, creams, powders, foams, crystals, and liposomes.

[0126] In the case of injection, the active ingredient of the pharmaceutical composition can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. One route of administration suitable for the pharmaceutical composition of the present invention is subperiosteal injection as described in U.S. Patent No. 6,525,030 to Erikkson. In the case of transmucosal administration, a penetration enhancer suitable for the permeation barrier is used in the formulation. Such penetration enhancers are generally known in the art.

[0127] In the case of oral administration, the pharmaceutical composition can be easily formulated by combining the active compound with pharmaceutically acceptable carriers well-known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, emulsions, etc. for oral ingestion by the patient. Pharmacological preparations for oral use can be made using solid excipients, the resulting mixture can be optionally comminuted, and after adding suitable auxiliaries if desired, the mixture of granules can be processed to obtain tablet or dragee cores. Suitable excipients are, in particular, sugars including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.; and / or fillers such as physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). Optionally, disintegrants such as crosslinked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, may be added. As used herein, the term "oral administration" includes the administration of a pharmaceutical compound to any oral surface, including the tongue, gums, palate, sublingual, or other buccal surfaces.

[0128] The cores of dragees are provided with suitable coatings. For this purpose, concentrated sugar solutions which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures can be used. Dyes or pigments may be added to the tablets or dragee coatings for identification purposes or to characterize different combinations of the active compound dosage.

[0129] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft-sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. Push-fit capsules may contain an active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Additionally, a stabilizer may be added. All formulations for oral administration need to be in a dosage suitable for the selected route of administration.

[0130] For buccal administration, the composition can take the form of tablets or lozenges formulated by conventional methods.

[0131] For administration by nasal inhalation, the active ingredient for use according to the present invention is conveniently delivered in the form of an aerosol spray from an overpressure pack or nebulizer by the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, a dosage unit can be determined by providing a valve for delivering a metered amount. Capsules and cartridges, for example made of gelatin, for use in a dispenser can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0132] The pharmaceutical compositions described herein may be formulated for parenteral administration, for example by bolus injection or continuous infusion. Injectable formulations may be in unit dosage form, for example provided in ampoules or multi-dose containers, optionally with the addition of a preservative. The composition may be a suspension, solution or emulsion in an oily or aqueous vehicle, and may also contain formulating agents such as solubilizers, suspending agents, stabilizers and / or dispersing agents.

[0133] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form. Additionally, suspensions of the active ingredient can be prepared as suitable oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglyceride emulsions or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredient to enable the preparation of highly concentrated solutions.

[0134] Alternatively, the active ingredient may be in powder form for constituting a suitable vehicle, such as a sterile water-based solution free of pyrogens, before use.

[0135] The pharmaceutical compositions of the present invention may be formulated into rectal compositions such as suppositories or retention enemas using conventional suppository bases such as cocoa butter or other glycerides.

[0136] Pharmaceutical compositions suitable for use in connection with the present invention include compositions in which the active ingredient is included in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of the active ingredient effective to prevent, alleviate, or ameliorate the symptoms of the disease or disorder of the subject being treated.

[0137] Determination of a therapeutically effective amount is well within the ability of one of ordinary skill in the art, especially in light of the detailed disclosure provided herein.

[0138] For any preparation used in the methods of the present invention, a therapeutically effective amount or dose can initially be estimated from in vitro and cell culture assays. For example, the dose can be formulated in an animal model to achieve the desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0139] The toxicity and therapeutic efficacy of the active ingredients described in this specification can be determined by standard pharmaceutical procedures in vitro in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal tests can be used in formulating various dosages for use in humans. The dosage may vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician taking into account the patient's condition. (See, e.g., Fingl, E. et al. (1975), "The Pharmacological Basis of Therapeutics", Ch. 1, p. 1.)

[0140] Dosage and interval can be adjusted individually to a level of the active ingredient sufficient to achieve the minimum effective concentration (MEC). The MEC varies for each preparation but can be estimated from in vitro data. The dosage required to achieve the MEC depends on the individual characteristics and the route of administration. Detection assays can be used to determine plasma or tissue concentrations.

[0141] Depending on the severity and responsiveness of the condition being treated, the dosing may be by a single or multiple administrations, the course of treatment may last from several days to several weeks, or a diminution of the disease state may be achieved.

[0142] The amount of the composition 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, etc.

[0143] Accordingly, the compositions and / or articles of some embodiments of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may include, for example, a metal or plastic foil such as a blister pack. Instructions for administration may be attached to the pack or dispenser device. The pack or dispenser may also be adapted to a notice associated with a container in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, the notice reflecting the agency's approval of the composition or the form of administration in human or veterinary medicine. Such notice may be, for example, a label approved by the U.S. Food and Drug Administration for prescription pharmaceuticals, or the package insert of an approved product. Compositions containing preparations of the present invention formulated in a pharmaceutically acceptable carrier may also be prepared for the treatment of an indication, placed in a suitable container (e.g., a lyophilized vial), and labeled as further detailed above.

[0144] The peptides or pharmaceutical compositions of the present invention can be used to inhibit the release or activity of inflammatory cytokines and mediators and to treat inflammatory diseases or disorders by providing a therapeutically effective amount of the peptide or pharmaceutical composition to a subject in need thereof.

[0145] As used herein, the phrase "a subject in need thereof" refers to a mammalian male or female subject (e.g., human) diagnosed with an inflammatory disease or disorder. In certain embodiments, the term includes individuals at risk of developing an inflammatory disease or disorder. The subject can be of any gender or age and includes neonates, infants, juveniles, young adults, adults, and the elderly.

[0146] As used herein, the term "about" refers to ±10%.

[0147] The terms "comprises", "comprising", "includes", "including", "has" and their variations mean "including but not limited to".

[0148] The term "consisting of" means "including and limited to".

[0149] The term "consisting essentially of" means that a composition, method or structure may include additional components, steps, and / or parts, provided that the additional components, steps, and / or parts do not substantially change the basic and novel characteristics of the claimed composition, method or structure.

[0150] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "compound" or "at least one compound" may include a plurality of compounds including mixtures thereof.

[0151] Throughout this application, various embodiments of the invention may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all the possible sub-ranges and the individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within that range such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0152] When a numerical range is indicated in this specification, it always means including any recited number (fraction or integer) within the indicated range. The expressions "ranging / ranges" between a first recited number and a second recited number and "ranging / ranges" from the first recited number "to" the second recited number are used interchangeably herein and mean including the first and second recited numbers, and all fractional and integral numbers therebetween.

[0153] As used herein, the term "method" refers to a manner, means, technique and procedure for accomplishing a given task, and includes, but is not limited to, manners, means, techniques and procedures known to, or readily developed from those known to, practitioners in the fields of chemistry, pharmacy, biology, biochemistry and medicine.

[0154] It is understood that certain features of the invention that are described in connection with separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in connection with a single embodiment may be provided separately, or in any suitable partial combination, or in any other described embodiment of the invention. Certain features described in connection with various embodiments should not be considered essential features of those embodiments, except where an embodiment would not function without those elements.

[0155] The various embodiments and aspects of the invention as described above and claimed in the following claims are experimentally verified in the following examples.

Examples

[0156] Reference is now made to the following examples, which illustrate the invention in a non-limiting manner in conjunction with the above description.

[0157] Example 1 - Effect of ZEP4 on the Expression of TNF Alpha by Inflammatory Macrophage Cells Materials and Methods The peptide pGlu-Asn-Trp-Thr-OH (hereinafter referred to as "ZEP4", SEQ ID NO: 2) was synthesized as described above.

[0158] Expression of TNF Alpha by B6 Macrophage Cells Macrophages derived from C57BL / 6 mice were cultured in 24-well plates (500 μl / well) at a density of 200,000 cells / mL and incubated overnight at 37 °C and 5% CO2. After incubation, the culture medium of a specific experimental group was replaced with fresh medium containing 100 ng / mL of LPS and then incubated for 3 hours. Subsequently, the medium of a specific experimental group (with or without LPS) was replaced with fresh medium containing ZEP4 at concentrations ranging from 3.125 to 100 μg / ml and then incubated for 1 hour. Then, palmitic acid (PA, 200 μM) was added to the appropriate wells, and the plates were incubated for an additional 24 hours. Next, the amount of TNF alpha in each well was measured. Table 1 shows various treatment groups and control groups. The reduction in TNF alpha production in the treatment groups was calculated compared to the cells treated with LPS and PA (CLP200).

[0159] Results Figures 1 and Table 1 below show that ZEP4 significantly reduced the amount of TNF alpha produced by inflammatory macrophage cells treated with LPS and palmitic acid for 24 hours. A ZEP4 concentration of 3.125 μg / ml resulted in a 70.6% reduction in TNF alpha expression. The most effective concentration of ZEP4 was 50 μg / ml, which resulted in a 77.0% reduction in TNF alpha expression (p < 0.0001).

Table 1

[0160] Example 2 - Effect of ZEP4 on TNF-alpha expression by inflammation-induced macrophage cells Materials and Methods The peptide ZEP4 (SEQ ID NO: 2) was synthesized as described above. Expression of TNF-alpha by B6 macrophage cells Macrophages derived from C57BL / 6 mice were cultured in 24-well plates (500 μl / well) at a density of 200,000 cells / mL and incubated overnight at 37 °C and 5% CO2. After incubation, the medium was replaced with fresh medium containing 100 ng / mL of LPS and incubated for 3 hours. Then, the LPS-containing medium in the treatment group was replaced with fresh medium containing 50 μg / ml of ZEP4 and subsequently incubated for 1 hour. Next, an aliquot of palmitic acid (100, 200, or 400 μM) was added to the appropriate wells. The plates were incubated for an additional 24 hours, and the amount of TNF-alpha in each well was measured. Table 2 shows the various treatment groups and control groups. The reduction of TNF-alpha production in the treatment group was calculated compared to the cells treated with LPS and PA according to the PA concentration.

[0161] Results Figures 2 and Table 2 below show that palmitic acid concentrations of 100, 200, and 400 μM induced macrophage TNF-alpha expression of 416, 462, and 593 pg / mL, respectively. ZEP4 at a concentration of 50 μg / ml substantially reduced TNF-alpha expression by macrophages at all palmitic acid concentrations (****p < 0.0001).

Table 2

[0162] Example 3 - Effect of ZEP3 on TNF-alpha expression by inflammation-induced macrophage cells Materials and Methods The peptide pGlu-Asn-Trp-Lys(octanoyl)-OH (hereinafter referred to as "ZEP3", SEQ ID NO: 1) was synthesized as described in U.S. Patent No. 7,220,725.

[0163] Expression of TNF-alpha by B6 macrophage cells Macrophage cells derived from C57BL / 6 mice were cultured in 24-well plates (500 μl per well) at a density of 200,000 cells / mL and incubated overnight at 37 °C and 5% CO2. After incubation, the medium of a specific experimental group was replaced with fresh medium containing 100 ng / mL of LPS and then incubated for 3 hours. Subsequently, the medium of a specific experimental group (with or without LPS) was replaced with fresh medium containing ZEP3 at concentrations ranging from 3.125 to 100 μg / ml and incubated for 1 hour. Next, an aliquot of palmitic acid (200 μM) was added to the appropriate wells, and the plates were incubated for an additional 24 hours. Then, the amount of TNF alpha in each well was measured. Table 3 shows various treatment groups and control groups. The reduction of TNF alpha production in the treatment groups was calculated compared to the cells treated with LPS and PA.

[0164] Results Figures 3 and 3 below and Table 3 show that ZEP3 significantly reduced TNF alpha expression by macrophage cells treated with LPS and palmitic acid. The most effective concentration of ZEP3 under the experimental conditions was 3.125 μg / ml (82.9% reduction in TNF alpha expression; p < 0.0001). The ZEP3 ED50 was estimated to be 51.94 μg / mL.

Table 3

[0165] Example 4 - Effect of ZEP4 on the expression of IL-1 beta by nigericin-activated macrophage cells Materials and methods Peptide ZEP4 (SEQ ID NO: 2) was synthesized as described above. Expression of IL-1 beta by nigericin-activated macrophages. Macrophage cells derived from C57BL / 6 mice were cultured in 24-well plates (500 μl / well) at a density of 200,000 cells / mL and incubated overnight at 37 °C and 5% CO2. After overnight incubation, the medium was replaced with fresh medium containing 100 ng / mL of LPS. After 3 hours, the LPS-containing medium was replaced with fresh medium containing 50 μg / ml of ZEP4 and transferred to the treatment wells. After 1 hour of incubation, an aliquot of nigericin (20 mM) was added to the ZEP4-treated and LPS control wells. The plates were incubated for an additional 24 hours, and the amount of IL-1 beta in each well was measured. Table 4 shows various control and treatment groups. Reduction of TNF alpha production in the treatment group was calculated compared to cells treated with LPS and nigericin (LPS+N).

[0166] Results Figures 4 and Table 4 below show that ZEP4 at a concentration of 50 μg / ml reduced the release of IL-1 beta by nigericin-activated macrophages by 53.8% (****p<0.0001).

Table 4

[0167] Example 5 - Effect of ZEP3 on the viability of inflammation-induced macrophage cells Materials and methods The peptide ZEP3 (SEQ ID NO: 1) was synthesized as described in U.S. Patent No. 7,220,725. Effect of ZEP3 on the viability of macrophages activated by LPS and palmitic acid Macrophages derived from C57BL / 6 mice were cultured in 24-well plates (500 μl / well) at a density of 200,000 cells / mL and incubated overnight at 37 °C and 5% CO2. After overnight incubation, the medium was replaced with fresh medium containing 100 ng / mL of LPS and then incubated for 3 hours. Subsequently, the LPS-containing medium in the treatment group was replaced with fresh medium containing ZEP3 at a concentration of 50 μg / ml and then incubated for 1 hour. Then, palmitic acid (100, 200, or 400 μM) was added to the appropriate wells. The plates were incubated for an additional 24 hours, and the viability of the cells was estimated using an LDH cytotoxicity assay kit. Table 5 shows the various treatment groups and control groups.

[0168] Results Figures 5 and 5 below and Table 5 show that ZEP3 had no significant effect on the viability of inflammation-induced macrophage cells under the experimental conditions.

Table 5

[0169] Example 6 - Effect of ZEP3 pretreatment on TNF-alpha expression by inflammatory-induced macrophage cells Materials and Methods Peptide ZEP3 (SEQ ID NO: 1) was synthesized as described in U.S. Patent No. 7,220,725. Expression of TNF-alpha by B6 macrophage cells Macrophages derived from C57BL / 6 mice were cultured in 24-well plates (500 μl wells) at a density of 200,000 cells / mL and placed in an incubator at 37 °C and 5% CO2 overnight. After overnight incubation, the medium was replaced with fresh medium containing 50 μg / ml of ZEP3. After 1 hour of incubation, 100 ng / mL of LPS was added to the appropriate wells, and the plates were incubated for an additional 24 hours, and then the amount of TNF-alpha in each well was measured.

[0170] Results Table 6 below shows that pretreatment of macrophage cells with ZEP3 prior to LPS induction reduced TNF-alpha produced by the cells by 46.7% (**p < 0.05).

Table 6

[0171] Example 7 - Anti-inflammatory effect of ZEP3 in a corneal epithelial cell culture dry eye disease model The anti-inflammatory effect of the peptide in experimental corneal inflammation was tested in a cell culture model that models dry eye disease (DED) using various inflammasome inducers such as LPS, 4-HNE, and nigericin. A dose-response model was used to pretreat human primary corneal epithelial cells with the peptide and then expose them to DED inducers. The expression of IL-6, and cell viability and ROS production were measured.

[0172] Materials and Methods Human primary corneal epithelial cells (PCS-700-010; HCEC) were treated with ZEP3 at concentrations ranging from 3 to 12 μg / ml for 2 hours and then exposed to LPS, 4-HNE, or nigericin for 24 hours. After incubation, the levels of the pro-inflammatory cytokine IL-6, cell viability, and reactive oxygen species (ROS) production in cell culture were measured.

[0173] Result As shown in Figure 7, the level of IL-6 in cell culture decreased from 52.45 ± 0.9121 pg / mL in the untreated cell culture to 38.08 ± 1.368 pg / mL in the cells treated with ZEP3 (a 27.4% reduction; P < 0.05).

[0174] As shown in Figure 8, the level of ROS in cell culture (reported as relative fluorescence units; RFU) decreased from 27043 ± 952 RFU in the untreated cell culture to 13000 ± 800 RFU in the cell culture treated with ZEP3 (a 51.9% reduction; P < 0.0001).

[0175] As shown in Figure 6, the cell death density decreased from 21.125 ±.942% in the untreated cell culture to 3.625 ± 1.092% in the cultured cells treated with ZEP3 (an 82.8% reduction; P < 0.0001). The ZEP3 peptide is effective in reducing corneal inflammation, restoring the viability of HCECs, and decreasing ROS production, and is concluded to be an important factor in DEA severity. The results provide a basis for the use of this peptide and similar peptides in the prevention and treatment of DED caused by various pathological conditions.

[0176] Examples 1 - 7 above show that peptides ZEP3 (SEQ ID NO: 1) and ZEP4 (SEQ ID NO: 2) reduced the expression of inflammatory cytokines TNF alpha, IL-1 beta, and IL-6 in inflammatory macrophages and corneal epithelial cells. Furthermore, the peptides were able to restore the viability of cells exposed to inflammatory stress. The results indicate that the peptides of the present invention can effectively inhibit or prevent inflammation in macrophages and corneal epithelial cells.

[0177] Example 8 - Effects of ZEP3, ZEP3 sodium salt, and ZEP4 on endotoxin-induced uveitis (EIU) in rats The model is run according to De Vos et al., Exp Eye Res 61:667-675, 1995. In male Lewis rats weighing 180 - 200 g, EIU is induced by injecting 200 μg of LPS diluted in 0.1 mL of sterile water into the plantar surface of the foot. The animals are then randomized into (4 peptides) × (3 concentrations) = 12 groups. An untreated induced group is used as the induction control.

[0178] Twenty-four hours after induction, corresponding to the peak severity of EIU, the animals are examined with a slit-lamp biomicroscope. Clinical eye inflammation is scored in each eye using a scale of 0 - 7 as follows: Iris congestion and cells in the anterior chamber are scored 0 - 2 (0 = no sign; 1 = mild; 2 = severe), flare, miosis, and hypopyon are scored 0 if no sign is present and 1 if present. The maximum possible score is 7 (the sum of the 5 parameter scores).

[0179] Example 9 - Effects of ZEP3, ZEP3 sodium salt, and ZEP4 in a controlled environmental chamber (CEC) mouse model of dry eye The model procedure is described by Barabino et al. (IOVS 46:2766-2771, 2005). Briefly, 8 - 12-week-old BALB / c mice are used in a controlled environmental chamber (CEC) where relative humidity (RH), temperature (T), and air flow (AF) are controlled and monitored. The mice are randomized and treated with ZEP3, ZEP sodium salt, and ZEP4 (test peptides). An untreated induced group is used as the control. The mice are then placed in the CEC and exposed to specific environmental control conditions (RH = 18.5% ± 5.1%, AF = 15 L / min, T = 21 - 23°C) for 3, 7, 14, and 28 days. Control mice are maintained for the same period in a normal environment (RH = 50% - 80%, no AF, T = 21 - 23°C). Cotton thread test, corneal fluorescein staining (score, 0 - 15), and aqueous tear production based on goblet cell density in the upper and lower conjunctiva are measured by masked observers.

[0180] Example 10 - Effects of ZEP3, ZEP3 Sodium Salt, and ZEP4 in a Scopolamine-Induced Rat Model of Dry Eye Male Sprague-Dawley rats weighing 300 g to 350 g are used. Scopolamine (Sigma-Aldrich, St. Louis, Mo.) is delivered continuously and systemically to the animals via an osmotic pump (2ML4 Alzet®; CedarLane, Burlington, Ontario) filled with scopolamine and is subcutaneously implanted in the mid-back between the shoulder blades to induce dry eye. The wound is closed with 2 to 3 wound clips. Postoperatively and again the next day, the animals are subcutaneously injected with carprofen (0.5 mg / 100 g), a non-steroidal anti-inflammatory drug and a potent long-acting analgesic for rodents. The animals are anesthetized before implantation of the surgical pump and before all endpoint tests in an isoflurane 99.9% USP (Abraxis Bioscience, Richmond Hill, Ontario) chamber. Scopolamine is delivered at 12.5 mg / day, and the data are evaluated on day 14.

[0181] A sterile solution of scopolamine hydrobromide (Sigma-Aldrich, St. Louis Mo.) at 0.175 g / mL is prepared in physiological saline (0.9%) and filtered through a 0.22 µm syringe end filter (Millex-GC, Millipore Corp., Bedford, Mass.). According to the manufacturer's instructions, 2 mL of the 0.175 g / mL scopolamine solution is filled into a 2ML4 Alzet® pump.

[0182] The groups of eyes of the rats tested were as follows: Group 1: Control rats (n = 12 eyes from 6 rats). Group 2: Dry eye was induced in rats (n = 12 eyes from 6 rats) by systemic administration of scopolamine, and measurement of fluorescein staining was performed on day 14. Group 3: Dry eye was induced in rats (n = 14 eyes from 7 rats) by systemic administration of scopolamine, and they were treated locally once with physiological saline on day 8. Group 4: Dry eye was induced in rats (n = 14 eyes from 7 rats) by systemic administration of scopolamine, and they were treated locally once with the test peptide on day 8.

[0183] The endpoints were as follows: (i) Reduction of corneal fluorescein staining measured on day 13 compared to the physiological saline-treated control; (ii) Aqueous tear production and aqueous tear turnover rate measured on day 13 compared to the untreated or scopolamine-treated control.

[0184] Example 11 - Effects of ZEP3, ZEP3 sodium salt, and ZEP4 in a rabbit model of dry keratoconjunctivitis Dry keratoconjunctivitis (KCS) was induced in the right eyes of 8 New Zealand white rabbits by surgically closing the lacrimal excretory ducts and removing the nictitating membrane, nictitating gland, and Harder's gland. All rabbits were left untreated for 8 weeks, and KCS was confirmed by collecting 0.1 - 0.4 μL of tear samples and measuring the increase in tear film osmotic pressure as described by J. Gilbard et al. (Ophthalmol. 96:677, 1978). A 3.0 mmol solution of UTP or analog was prepared with the preserved isotonic buffer. The rabbits were randomized and treated with ZEP3, ZEP sodium salt, and ZEP4 (test peptide). The untreated group was used as a control. After the start of treatment, 0.1 - 0.4 μL of tear samples were collected from all rabbits for osmotic pressure measurement before the first administration. The animals were sacrificed at week 20, and the goblet cell density was measured by staining with alcian blue and periodic acid Schiff reagent (D. Dartt, et al., Exp. Eye Res. 67:27, 1996).

[0185] Example 12 - Effects of ZEP3 and its sodium salt (ZEP3Na) on cytokine production in an in vitro model of inflammation in human PBMCs The effect of the peptide ZEP3 (SEQ ID NO: 1) on cytokine production was evaluated in an in vitro human model of inflammation. Human peripheral blood mononuclear cells (PBMCs) were stimulated with lipopolysaccharide (LPS) to produce cytokines.

[0186] Methods: Briefly, cryopreserved human PBMCs from four healthy non-smokers who had not been administered any corticosteroids or other anti-inflammatory drugs were grown in RPMI 1640 with 10% heat-inactivated fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / mL streptomycin (complete growth medium, CGM).

[0187] The human PBMCs were thawed, seeded into all groups (1 - 6, Table 7), and allowed to stand for 1 hour before treatment. The cell groups were treated with the test item ZEP3 (SEQ ID NO: 1) at concentrations of 40, 100, and 150 μg / ml, or dexamethasone for 1 hour, followed by stimulation with LPS for 24 hours. Almar Blue was added 4 hours before supernatant collection. Twenty-four hours after LPS addition, Almar Blue, which fluoresces at 585 nm (correlated with cell viability), was measured, and the supernatant was collected for cytokine measurement. Each group was repeated three times.

Table 7

[0188] Detailed method: Cells were thawed according to the manufacturer's instructions, washed with CGM, and viability was assessed using trypan blue staining. A stock solution of 2×10^6 cells / mL was prepared in CGM. 100 μL of the stock solution was added to the appropriate wells of a 96-well black-wall plate (1 plate per donor), and 2×105 cells per well were seeded. The cells were incubated at 37 °C, 5% CO2 for 1 hour. 100 μL of 2X test item, dexamethasone, or 1X CGM was added to the appropriate wells to bring the final volume to 200 μL / well. The plate was incubated at 37 °C, 5% CO2 for an additional 1 hour. After 1 hour, 20 μL of LPS was added to groups 2 - 9 to bring the final volume to 220 μL. 20 μL of IM was added to group 1. The cells were incubated at 37 °C, 5% CO2 for 20 hours. At the 20-hour time point, 20 μL of Alamar Blue was added to all wells. The plate was incubated at 37 °C, 5% CO2 for an additional 4 hours. At 24 hours post-LPS stimulation, the plate was read for cell viability assessment, the cell culture supernatant was collected and stored at -80 °C for cytokine analysis. The remaining cells were washed once with sterile PBS. The cells were incubated in trypsin solution to detach them from the plate, centrifuged to pellet, and the trypsin solution was removed. The cells were then stored at -80 °C for optional further analysis. The concentrations of GM-CSF, IFN-g, IL-10, IL-12p40, IL-17a, IL-1β, IL-4, IL-6, RANTES, and TNF alpha present in the supernatant samples collected after the Alamar Blue assay were determined using the Luminex assay. In the use of the cytokine assay, the manufacturer's protocol was followed. The percentage of cytokine production and sample stimulation relative to the mean of the stimulation control was calculated.

[0189] Results: As shown in Figures 9 - 12, ZEP3 was shown to elicit a significant reduction in the major mediators of inflammation, including interferon gamma (IFNγ, Figure 9), IL-10 (Figure 10), IL-1β (Figure 11), and RANTES (Figure 12), thus supporting the concept of its anti-inflammatory effect. A dose-dependent response was observed for some of the cytokines measured. After treatment with the peptide at all concentrations tested, no significant changes in cell viability or any donor trends were observed, indicating that the peptide is not cytotoxic to PBMC. The levels of IL-4 and IL-17a were below the limit of detection in all test groups.

[0190] The reduction in cytokine production was directly related to ZEP3, confirming the anti-inflammatory effect of ZEP3 in human blood cells.

[0191] Example 13 - In Vivo Atopic Dermatitis Model The effects of topical administration of ZEP3 (SEQ ID NO: 1), and its sodium salt form (ZEP3Na), and ZEP4 (SEQ ID NO: 2) were evaluated in an oxazolone-induced atopic dermatitis (AD) mouse model. This model is widely used and is also well described in the literature as an accepted animal model for evaluating drugs for the treatment of human AD (Avci et al. 2013 Expert Opin Drug Discov. 8, 331 - 355; Petersen T.K. 2006, Basic Clin. Pharmacol. Toxicol. 99, 104 - 115).

[0192] Method: On the back of the animals on day 0 after shaving and hair removal, 2% oxazolone in ethanol was topically administered to sensitize the animals (sensitization period). Then, one week later, 1% oxazolone in ethanol was topically administered 10 times every 2 days to the back and right ear for a total of 3 weeks to challenge. The test compound was applied during the challenge phase every 2 days via the topical route as an immediately available cream formulation. The application area was the same as the challenge (back + ear). 100 μL was placed on the back and the back was smoothly massaged. Then, the formulation remaining in the finger gloves was adhered to the inner surface of the ear. Every other day, the skin thickness of the back and ear was measured with calipers, and the following clinical scores were recorded from the back skin: erythema and scaling. The body weight of the animals was also measured every other day (the approximate body weight of the mice at the start was about 20 g). Corrected digital photographs of the back skin area were taken on days 8, 18, and 28. The experiment was terminated on day 28, blood was collected from the animals, and plasma specimens were separated. For the euthanized animals, samples of the right ear and back skin were collected and frozen at -80 °C (samples from the back skin and plasma) or stored in formalin for further analysis (samples from the back skin and right ear). Table 8 describes the dosing groups.

Table 8

[0193] The formulations used for topical administration of the test compounds were as follows: ZEP3, a 1% cream at pH 7.1, containing propylene glycol, polysorbate 80, disodium edetate, silicon dioxide (syloid), methylparaben, white petrolatum, isopropyl myristate, cetyl alcohol, and glyceryl monostearate. ZEP3Na, a 1% cream at pH 8.0, containing propylene glycol, polysorbate 80, disodium edetate, silicon dioxide (syloid), methylparaben, white petrolatum, isopropyl myristate, cetyl alcohol, and glyceryl monostearate. ZEP4, a 1% cream at pH 4.5, containing propylene glycol, glycerin, polysorbate 80, disodium EDTA, xanthan gum, silicon dioxide (syloid), methylparaben, white petrolatum, isopropyl myristate, cetyl alcohol, stearyl alcohol, and glyceryl monostearate (kolliwax).

[0194] Results: The test compounds ZEP3, ZEP3Na, and ZEP4 administered at 50 mg / kg to atopic dermatitis-induced mice had good tolerability. All peptides significantly reduced the clinical lesions of atopic dermatitis, dorsal skin thickness (Figures 13A and 13B), ear thickness (Figures 14A and 14B), and erythema (Figures 15A and 15B). Scaling decreased from day 18 to day 24 in mice treated with ZEP3 (Figure 16A), and from day 14 to day 24 in mice treated with ZEP3Na (Figure 16A) or ZEP4 (Figure 16B), showing a delayed effect. The body weights of mice treated with ZEP3, ZEP3Na, or ZEP4 showed normal behavior compared to the vehicle (Figures 17A and 17B).

[0195] Specifically, the body weights of ZEP3, ZEP3Na, and ZEP4 were very stable during the first few days of the test and showed an increase of about 3 - 4% on day 28 (Figures 17A and 17B). In the group treated with betamethasone, as expected, a body weight loss of about 8 - 10% was observed.

[0196] Except for the betamethasone-treated mice, the dorsal skin thickness increased during the first few days of the test and then remained very stable until the end of the test (Figures 13A and 13B). On day 28, an increase of approximately 110% was observed in the negative control group administered the vehicle, 90% in group 3 administered 1% ZEP3, 105% in group 4 administered 1% ZEP3Na, and approximately 90% in group 5 administered 1% ZEP4, while a 40% decrease was observed in the positive control group 2 administered betamethasone. In the graph, all of the treatment groups (groups 3, 4, and 5) showed a tendency for the dorsal skin thickness to be lower than that of the negative control group 1. For ZEP3 on days 20 (-25.5%) and 24 (-16.6%), a significant difference in the dorsal thickness compared to control group 1 was roughly shown. For ZEP4, a significant difference in the dorsal thickness compared to control group 1 was roughly shown on day 14 (-13.5%).

[0197] Right ear thickness: As is normally observed in this model, all groups showed significant thickening from the first few days until the end of the test, except that the positive control group 2 showed only a slight increase of about 15 - 20% throughout the test (Figures 14A and 14B). Significant differences were roughly shown for ZEP3 on days 20, 22, and 28 (-10.1 to -18.0% compared to the control), for ZEP3 Na on days 18, 20, and 22 (-18.4 to -22.7% compared to the control), and for ZEP4 on days 20, 22, and 28 (-12.3 to -16.0% compared to the control).

[0198] The erythema scoring was maximal on day 16 in the negative control group (group 1) and decreased slightly to 0 on days 26 - 28. The erythema scoring recorded in test groups 3, 4, and 5 was the same as that of the negative control group 1 (Figures 15A and 15B). Significant differences were shown for ZEP3 on days 22 and 24, for ZEP3Na on day 24, and for ZEP4 on days 12, 16, 22, and 24.

[0199] Scaling scores increased gradually during the first few days, then decreased slightly, and remained very stable in the second half of the test (Figures 16A and 16B). All treatment groups 3, 4, and 5 showed lower scaling scores than the negative control group 1 from day 14 to day 28.

[0200] The total score (the sum of the erythema and scaling scores) was also lower in treatment groups 3, 4, and 5 than in the negative control group 1 from day 14 to day 28. The significant differences in the total scores were shown at days 20, 24, and 28 for ZEP3, at days 14, 18, 20, and 22 for ZEP3Na, and at days 12 - 20, 24, and 28 for ZEP4.

[0201] Conclusion: Compounds ZEP3 and ZEP4 and their sodium salts administered to atopic dermatitis - induced mice at 50 mg / kg had good tolerability and significantly reduced the clinical lesions of atopic dermatitis as shown by the reduction in the skin thickness of the back, ear thickness, erythema, and scaling.

[0202] Example 14 Treatment of ZEP3 for Carpometacarpal Osteoarthritis (CMC Arthritis) of the Thumb A 75 - year - old male subject was diagnosed by an orthopedic surgeon with CMC - type arthritis of the thumb. His big toe was swollen, red, and painful, so he could not walk.

[0203] The sodium salt of ZEP3 in 0.5% cream was applied to the swollen area 5 times a day. The subject reported relief within 1 day after treatment and resolution of the condition within 3 - 4 days.

[0204] Example 15 Effects of ZEP3 and ZEP3Na Peptides on TNF - Alpha Secretion in Human Keratinocytes TNF - alpha is a major pro - inflammatory mediator of keratinocytes. The compounds were tested for anti - inflammatory effects by measuring their impact on TNF - alpha levels.

[0205] Method: SCCE020 cells (EpiGRO™ Human Epidermal Keratinocytes) were grown in complete growth medium (EpiGRO™ Human Keratinocyte Complete Medium; Millipore catalog number SCMK001) for 4 passages. At passage 5, the cells were seeded at 3 per well at a volume of 1 ml of complete growth medium per well (3×105 cells / well). Three additional control wells contained only medium without cells. Once attached, the cell medium was replaced with basal medium (starvation medium) containing L-glutamine without supplements, and the cells were starved overnight. The day after 24 hours of seeding, the cells were treated with ZEP3 or ZEP3Na or the appropriate diluent (PBS or DMSO). After 4 hours of incubation, LPS (from E. coli 055:B5; Sigma catalog number L6529-1MG) was added to the wells at a final concentration of 20 or 30 μg / ml. The experimental groups (3 wells for each treatment) are summarized in Table 9.

[0206] The cells were incubated in a tissue culture incubator for 24 hours. After collecting the cell supernatants from all wells, the cells from the treatment groups (1, 2, 3, 8, 9, 10, 13, 14 (without DMSO group)) were pelleted and stored at -80°C for further analysis. The cells were collected as follows: after collecting the supernatant, the wells were rinsed with trypsin and then incubated with trypsin until separated from the plate surface, centrifuged, washed with PBS, and stored as cell pellets. The TNF-α concentration in the supernatant was measured 5 times by ELISA (Quantikine HS ELISA; R&D Systems catalog number HSTA00D).

[0207] Results: ZEP3 and ZEP4 effectively decreased the TNF alpha levels in human keratinocyte cell cultures. The TNF alpha concentration in the samples was calculated according to the equation of the trend line of the standard curve: OD = 0.063x + 0.1718. The percent inhibition of TNF alpha production was calculated for each treatment group compared to the corresponding control group (same conditions, without peptide). The results are summarized in Table 9.

Table 9

[0208] Conclusion: The quantitative assay by ELISA showed a dose-dependent inhibition of TNF-α production in human keratinocytes treated with LPS together with either ZEP3 or ZEP3Na, as compared with the amount secreted by cells simulated with LPS alone.

[0209] Although the invention has been described in connection with its particular embodiments, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended that all such alternatives, modifications, and variations which fall within the spirit and broad scope of the appended claims be included.

[0210] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Further, any reference or identification of a reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not necessarily be construed as limiting.

Claims

1. An isolated sodium salt of a peptide having the sequence shown in SEQ ID NO:

1.

2. The isolated sodium salt according to claim 1, consisting of the sequence shown in SEQ ID NO:

1.

3. The isolated sodium salt according to claim 1 or 2, having a purity of at least 99.7% as measured by HPLC.

4. The isolated sodium salt according to claim 1 or 2, having a sodium content of 3.1%.

5. The isolated sodium salt according to claim 1 or 2, which is in solid phase.

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