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, etc.

[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 can be caused by problems or diseases occurring in the eye, or can 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) or dry eye syndrome (DES), also known as keratoconjunctivitis sicca, is a common eye condition that affects millions of people seeking ophthalmic treatment. Regardless of the underlying etiology, dry eye has been shown to be associated with abnormalities in the pre-corneal tear film and subsequent inflammatory changes throughout the entire surface of the eye, including the adnexa, conjunctiva, and cornea. Activation of inflammatory cytokines, such as IL-6, plays a crucial role in the pathophysiology of DED.

[0007] Since the role of inflammation in dry eye was recognized, many treatments designed to inhibit various inflammatory pathways have been studied, including cyclosporine A, corticosteroids, tacrolimus, tetracycline derivatives, and autologous serum (Michelle He ssen M., and Karamursel Akpek EJ 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 (wherein 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 to treat inflammation.

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

[0010] U.S. Patent No. 9,012,397 and WO2012 / 131676 teach topical pharmaceutical compositions containing peptides ZE P3 or ZEP4 and their use to improve symptoms of skin diseases. This reference does not describe or suggest the use of peptides to treat inflammation.

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

[0012] This invention addresses the ongoing need to improve and develop new, safe, and effective treatments that improve inflammatory conditions by intervening in the inflammatory cascade. [Overview of the project]

[0013] This 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 pathogenesis of many inflammatory diseases. Diseases and disorders suitable for treatment with the compositions of this invention include, but are not limited to, inflammatory diseases of the eyes, ears, lungs, and intestines. Diseases and disorders suitable for treatment also include autoimmune diseases in which the administration of the medicine may become chronic to prevent exacerbations.

[0014] This invention is partly based on the discovery that peptides shown in ZEP3 and ZEP4 exhibit unexpectedly strong activity in attenuating the expression and / or release of inflammatory and pro-inflammatory 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 (which are regulated upon activation and expressed and possibly secreted in normal T cells).

[0015] Surprisingly, the peptide's activity in inhibiting the inflammatory cascade was found to be exerted even when the peptide was administered before the inflammatory response began. Therefore, the peptide may be useful in improving or suppressing symptoms induced in various inflammatory diseases and disorders, and in completely eliminating the inflammatory response. The composition of the present invention is expected to be useful in preventing exacerbations 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 involve the treatment of skin disorders or pain itself.

[0017] According to one aspect of the present invention, a pharmaceutical composition is provided comprising 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, as an active ingredient for use in reducing the release of inflammatory cytokines or inhibiting their activity; and a pharmaceutically acceptable carrier.

[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 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.

[0019] In further 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 includes an alkyl group bonded to a free functional group of the peptide sequence.

[0021] In yet another embodiment, the alkyl group is bonded to the free amino group at the side chain or N-terminus of the peptide by an amide bond or linkage.

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

[0023] According to some specific embodiments, a C8 alkyl group (octanoyl in this specification) is attached by amide linkage to the side chain of a Lys residue in the peptide sequence or to the terminal amino group of the peptide. According to some embodiments, the carboxyl 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 their pharmaceutically acceptable derivatives and salts.

[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 presumably secreted).

[0029] According to another aspect of the present invention, there is provided a method of reducing the release of at least one inflammatory or inflammation-inducing cytokine or inhibiting its activity, 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 a therapeutically effective amount of a peptide according to formula I to a subject in need thereof is also performed before symptoms appear or develop. These methods of treating chronic inflammatory diseases are effective in preventing exacerbation.

[0034] In yet another aspect, the present invention provides a method for treating an inflammatory disease or disorder of the eye, comprising the step of administering to a subject in need 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] According to 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 further 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] According to some specific embodiments, the Lys derivative is Lys(octanoyl).

[0038] According to some embodiments, the peptide derivative includes an alkyl group bonded to a functional group of the peptide sequence.

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

[0040] According to some embodiments, the alkyl group is a C4-C30 alkyl group.

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

[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 their pharmaceutically acceptable salts or derivatives.

[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 Sequence 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 may be selected from the form of a cream, ointment, paste, lotion, gel, or eye drops.

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

[0051] According to some embodiments, the cream composition contains 0.1 to 5% w / w of peptides. According to other embodiments, the composition contains 0.5 to 2% of peptides. According to yet another embodiment, the composition contains about 1% of peptides.

[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 yet another embodiment, 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, compositions for administration to subjects with eye diseases or injuries are 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 disease, allergic eye disease, keratitis, conjunctivitis, meibomian gland dysfunction, and Sjögren's syndrome.

[0056] In further embodiments, the pharmaceutical composition may be 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 further embodiments, 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] In 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 further embodiments, inflammatory diseases or disorders of the lungs are 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] In a further embodiment, the inflammatory disease or disorder of the ear is selected from the group consisting of inflammatory symptoms associated with ear infections, otitis media, otitis externa, mastoiditis, and otomastalitis.

[0065] According to a further characteristic embodiment, administration is by eye drop administration.

[0066] In a further embodiment, administration is by oral administration.

[0067] In a further embodiment, administration is performed via nasal administration.

[0068] In a further embodiment, administration is parenteral.

[0069] The present invention successfully addresses the shortcomings of currently known compositions by providing pharmaceutical compositions for use in reducing the release or inhibiting the activity of inflammatory cytokines and mediators, and in treating diseases or disorders related to inflammatory cytokines and mediators.

[0070] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which the invention relates. Similar or equivalent methods and materials may be used in the practice or testing of embodiments of the invention, but exemplary methods and / or materials are described below. In case of any conflict, the patent specification, including the definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not necessarily intended to be limiting. [Brief explanation of the drawing]

[0071] Some embodiments of the present invention are described herein by reference only, with reference to the accompanying drawings. It is emphasized hereby that, with particular close reference to the drawings, the details shown are illustrative and intended to aid in the description of embodiments of the present invention. In this regard, the description made in conjunction with the drawings will make it clear to those skilled in the art how embodiments of the present invention may be put into practice.

[0072] [Figure 1] This bar graph shows the effect of ZEP4(Z) at concentrations ranging from 3.125 to 100 μg / ml on the amount of TNFα produced by pro-inflammatory macrophage cells. Lines marked with **** represent statistically significant differences at p<0.0001. [Figure 2] This bar graph shows the effect of ZEP4(Z) (50 μg / ml) on the amount of TNFα produced by pro-inflammatory macrophage cells treated with lipopolysaccharide (LPS, 100 ng / ml) and palmitic acid (PA, 100, 200, or 400 μM). Lines marked with **** represent statistically significant differences at p<0.0001. [Figure 3] This bar graph shows the effect of ZE P3(Z) (concentration range: 3.125~100 μg / ml) on the amount of TNFα produced by pro-inflammatory macrophage cells. Lines marked with ****, ***, **, *, and ns represent statistically significant differences at p< 0.0001, p<0.001, p<0.01, p<0.01, and p<0.1, respectively, and non-significant differences. [Figure 4] This bar graph shows the effect of ZEP4(Z) (50 μg / ml) on the amount of IL-1 beta produced by inflammatory nigericin-induced macrophage cells. Lines marked with ** represent statistically significant differences at p<0.0001. [Figure 5] This bar graph shows the effect of ZEP3 on the viability of pro-inflammatory macrophage cells. [Figure 6] This bar graph shows the protective effect of ZEP3 at various concentrations on the viability of inflammatory corneal epithelial cells exposed to 70 mM NaCl (black bars) or NHE20 (gray bars), as determined by an LDH assay. Lines marked with **** and * represent statistically significant differences at p<0.0001 and p<0.05, respectively. [Figure 7] This bar graph shows the effect of various concentrations of ZEP3 on the amount of IL-6 produced by inflammatory corneal epithelial cells exposed to 4-hydroxynonenal (4-HNE) for 24 hours. Lines marked with * represent statistically significant differences at p<0.05. [Figure 8] This bar graph shows the protective effect of ZEP3 at various concentrations on the amount of reactive oxygen species (ROS, measured by the DCFDA kit) produced by inflammatory corneal epithelial cells exposed to 4-HNE for 24 hours. Lines marked with ****, ***, and ** represent statistically significant differences at p<0.0001, p<0.001, and p<0.01, respectively. [Figure 9]This figure illustrates IFN-γ production in normal PBMCs from four human donors in response to ZEP3 at different concentrations, as well as stimulation with culture medium, stimulus, and dexamethasone control. Statistically significant differences are represented by *p<0.05 and **p<0.01, and are compared to the stimulus control by Dunette's multiple comparisons following one-way ANOVA. [Figure 10] This figure illustrates IL-10 production in normal PBMCs from four human donors in response to ZEP3 at different concentrations, as well as stimulation with culture medium, stimulus, and dexamethasone control. Statistically significant differences are represented by *p<0.05 and **p<0.01, and are compared to the stimulus control by Dunette's multiple comparisons following one-way ANOVA. [Figure 11] This figure illustrates IL-1B production in normal PBMCs from four human donors in response to ZEP3 at different concentrations, as well as stimulation with culture medium, stimulus, and dexamethasone control. Statistically significant differences are expressed as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, and are compared to the stimulus control by Dunette's multiple comparisons following one-way variance analysis. [Figure 12] This figure shows RANTE S production in normal PBMCs from four human donors in response to ZEP3 at different concentrations, as well as stimulation with culture medium, stimulation, and dexamethasone control. Statistically significant differences are expressed as *p<0.05 and compared to the stimulation control by Dunette's multiple comparisons following one-way ANOVA. [Figure 13A] This graph shows the time course of dorsal skin thickness in mice topically treated with ZEP3, ZEP3Na (Figure 13A), or ZEP4 (Figure 13B) compared to excipients and betamethasone controls in a mouse oxazolone-induced atopic dermatitis model. Statistically significant differences are expressed as *p<0.05, **p<0.01, and ****p<0.001, and are compared to the stimulation control by Dunette's multiple comparisons following one-way ANOVA. [Figure 13B]This graph shows the time course of dorsal skin thickness in mice topically treated with ZEP3, ZEP3Na (Figure 13A), or ZEP4 (Figure 13B) compared to excipients and betamethasone controls in a mouse oxazolone-induced atopic dermatitis model. Statistically significant differences are expressed as *p<0.05, **p<0.01, and ****p<0.001, and are compared to the stimulation control by Dunette's multiple comparisons following one-way ANOVA. [Figure 14A] This figure illustrates the time course of baseline-adjusted right ear thickness in mice topically treated with ZEP3, ZEP3Na (Figure 14A), or ZEP4 (Figure 14B) compared to excipients and betamethasone controls in a mouse oxazolone-induced atopic dermatitis model. [Figure 14B] This figure illustrates the time course of baseline-adjusted right ear thickness in mice topically treated with ZEP3, ZEP3Na (Figure 14A), or ZEP4 (Figure 14B) compared to excipients and betamethasone controls in a mouse oxazolone-induced atopic dermatitis model. [Figure 15A] This graph shows the time course of erythema scores in mice topically treated with ZEP3, ZEP3Na (Figure 15A), or ZEP4 (Figure 15B) compared to excipients and betamethasone controls in a mouse oxazolone-induced atopic dermatitis model. Statistically significant differences are expressed as *p<0.05, **p<0.001, and ****p<0.0001, and are compared to the stimulus control by Dunette's multiple comparison following one-way ANOVA. [Figure 15B] This graph shows the time course of erythema scores in mice topically treated with ZEP3, ZEP3Na (Figure 15A), or ZEP4 (Figure 15B) compared to excipients and betamethasone controls in a mouse oxazolone-induced atopic dermatitis model. Statistically significant differences are expressed as *p<0.05, **p<0.001, and ****p<0.0001, and are compared to the stimulus control by Dunette's multiple comparison following one-way ANOVA. [Figure 16A]This figure shows the changes in scale scores over time in mice topically treated with ZEP3, ZEP3Na (Figure 16A), or ZEP4 (Figure 16B) compared to excipients and betamethasone controls in a mouse oxazolone-induced atopic dermatitis model. Statistically significant differences are expressed as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, and are compared to the stimulation control by Dunette's multiple comparisons following one-way ANOVA. [Figure 16B] This figure shows the changes in scale scores over time in mice topically treated with ZEP3, ZEP3Na (Figure 16A), or ZEP4 (Figure 16B) compared to excipients and betamethasone controls in a mouse oxazolone-induced atopic dermatitis model. Statistically significant differences are expressed as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, and are compared to the stimulation control by Dunette's multiple comparisons following one-way ANOVA. [Figure 17A] This figure shows the relative changes in animal body weight from day 8 to day 28 in mice topically treated with ZEP3, ZEP3Na (Figure 17A), or ZEP4 (Figure 17B) compared to excipients and betamethasone controls in a mouse oxazolone-induced atopic dermatitis model. [Figure 17B] This figure shows the relative changes in animal body weight from day 8 to day 28 in mice topically treated with ZEP3, ZEP3Na (Figure 17A), or ZEP4 (Figure 17B) compared to excipients and betamethasone controls in a mouse oxazolone-induced atopic dermatitis model. [Modes for carrying out the invention]

[0073] The present invention relates to pharmaceutical compositions comprising specific peptides, as well as salts and derivatives thereof, for use in inhibiting inflammatory cascades. 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 illustrated herein, inhibitory effects on inflammatory cytokine release have 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 a mouse model of inflammatory atopic dermatitis (AD).

[0075] The beneficial effects of peptides on experimental corneal inflammation were demonstrated in a human corneal epithelial cell culture model.

[0076] The principles and operation of this invention can be better understood by referring to the drawings and accompanying description.

[0077] Before describing in detail at least one embodiment of the present invention, it should be understood that the present invention is not necessarily limited in its application to the details described below or illustrated by the examples. Other embodiments of the present invention are possible, or can be practiced or carried out in various ways. It should also be understood that any expressions or terms used herein are for illustrative purposes only and should not be construed as limiting.

[0078] While putting the present invention into practice, the inventors surprisingly discovered that peptides ZEP3 and ZEP4, as well as their sodium salt forms, reduced the expression of inflammatory cytokines TNF-alpha, IL-1 beta, and IL-6 by inflammation-induced macrophages and corneal epithelial cells (Examples 1-7 below), and reduced the expression of IFNγ, IL-10, IL-1β, and RANTES in inflammation-stimulated human PBMCs (Example 12 below). Furthermore, in a mouse inflammatory model of atopic dermatitis, ZEP4, ZEP3, and their sodium salts formed ZEP3Na, which significantly reduced clinical lesions in vivo (Example 13 below). The results demonstrate that the peptides of the present invention can independently inhibit or prevent inflammation.

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

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

[0081] According to some embodiments, an inflammatory condition is a disease or disorder associated with the overrelease or activity of at least one cytokine or mediator, the at least one cytokine or mediator being 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 term “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 in question modulates its response to tissue damage, infection, antigen load, etc. Inflammation may be associated with increased blood supply to tissue, increased capillary permeability of tissue, and / or increased migration of white blood cells into tissue.

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

[0084] Inflammation can be diagnosed by elevated levels of inflammatory cytokines such as TNFα, IL-1-alpha, IL-1-beta, IFN-gamma, and IL-6, etc., in biological samples obtained from subjects. Biological samples may include, for example, tears, blood, serum, plasma, urine, sputum, saliva, feces, semen, cerebrospinal fluid, bone marrow, lymph, or CSF, exocrine secretions from the skin, the respiratory tract, the intestinal tract, and the genitourinary system, milk, human organs or tissues, samples obtained by washing, multiple exudates, samples from in vitro or ex vivo cell cultures, and cell culture components.

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

[0086] According to some embodiments of the present invention, an inflammatory disease or disorder is an inflammatory disease or disorder of the eye. The inflammatory eye disease or disorder may 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 Sjögren's syndrome.

[0087] In one embodiment, an 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, which includes the iris, ciliary body, and choroid. Uveitis is also commonly called iritis, squamous inflammation, choroiditis, chorioretinitis, anterior uveitis, and posterior uveitis. The most common form of uveitis is anterior uveitis, which involves inflammation of the anterior part of the eye, usually isolated by 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 term “dry eye syndrome (DES)” refers to a condition of altered tear composition resulting from a diseased or dysfunctional lacrimal gland functional unit. Evidence suggests that inflammation causes structural changes in the lacrimal glands. Altered tear composition resulting from lacrimal gland dysfunction, increased evaporation, and / or poor clearance exerts an inflammatory effect on the surface of the eye. This inflammation is partly responsible for irritation symptoms, ocular surface epithelial disease, and alterations in the corneal epithelial barrier function of dry eye. Anti-inflammatory therapies for DES target one or more 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, an inflammatory disease or condition of the eye is associated with Sjögren's syndrome. As used herein, the term “Sjögren's syndrome” refers to a systemic autoimmune inflammatory disorder characterized by decreased tear production, dry mouth, and other mucosal dryness, often associated with autoimmune rheumatic disorders such as rheumatoid arthritis. Dryness of the eyes and mouth are the most common symptoms of this syndrome. Symptoms may occur alone or in conjunction with symptoms associated with rheumatoid arthritis or other connective tissue diseases. Associated enlargement of the salivary glands may also occur. Other organs may also be affected. This syndrome may also be associated with rheumatoid arthritis, systemic lupus erythematosus (SLE), scleroderma, polymyositis, and other diseases.

[0090] In some embodiments, inflammatory diseases or conditions of the eye result from eye-related procedures, such as corneal transplantation / corneal reconstruction, corneal prosthesis surgery, lamellar transplantation, or selective endothelial transplantation.

[0091] In some embodiments, inflammatory diseases or conditions of the eye affect the surface of the eye, including but not limited to corneal ocular surface inflammation, corneal neovascularization, keratitis including peripheral ulcerative keratitis and microbial keratitis, conjunctivitis, or bullous pemphigoid syndrome.

[0092] In some embodiments, the inflammatory disease or condition of the eye is an autoimmune disorder affecting the eye, including but not limited to sympathetic ptosis, Vogt-Koyanagi-Harada (VKH) syndrome, Birdshott retinomyelopathy, ocular scarring pemphigoid (OCP), Sjögren's syndrome, or Fuchs' metachronous 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 is not limited to, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, pemphigus vulgaris, ankylosing spondylitis, juvenile idiopathic arthritis, lupus, and Sjögren'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 leads to 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 lupus erythematosus, which 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 (SLE), the body’s natural defenses go against the body, and malformed immune cells attack the body’s tissues. Antibodies that can react to the body’s blood cells, organs, and tissues may be produced. This reaction leads to immune cells attacking the affected systems, causing a chronic disease. Lupus nephritis, also known as lupus glomerulositis, 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, which 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 called hypertrophic osteoarthritis, osteoarthritis, and degenerative joint disease. OA is a chronic degenerative disease of the skeletal joints that affects specific joints in adults of all ages, usually the knees, hips, wrists, and spine. OA is characterized by several symptoms, including degeneration and thinning of articular cartilage with associated development of “ulcers” or craters, osteophyte formation, bone hypertrophy at the margins, and synovial changes and enlargement of the affected joint. Furthermore, osteoarthritis is accompanied by pain and stiffness, especially after prolonged activity. The peptides of the present invention, their salts and derivatives, and pharmaceutical compositions containing them can be used to treat osteoarthritis. Characteristic radiographic features of osteoarthritis include joint cavity narrowing, subchondral sclerosis, osteohypoplasia, subchondral cyst formation, and loose bone bodies (or “joint mice”).

[0097] According to some embodiments of the present invention, the inflammatory disease or disorder is inflammatory bowel disease. 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, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, 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, an infection of the ear, otitis media, otitis externa, mastoiditis, and otomastitis.

[0100] The present invention also provides pharmaceutical compositions for use in treating diseases or disorders associated with the excessive release of inflammatory mediators and chemokines, including but not limited to ROS, RANTES, and monocyte chemotactic protein-1 (MCP-1). Inflammatory diseases associated with ROS release that can be treated 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 eosinophil recruitment in pulmonary allergic inflammation, pulmonary leukocyte infiltration, bronchial hypersensitivity, and the pathogenesis of asthma.

[0102] The terms “treatment” or “to treat” may be used interchangeably herein and mean inhibiting, preventing or halting the onset of a disease or disorder, and / or causing 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 may 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 may be produced using recombinant methods or a combination of recombinant and synthetic methods.

[0104] In one embodiment of the present invention, the peptide is the pGlu-Asn-Trp-Lys(octanoyl)-OH (SEQ ID NO: 1; hereinafter referred to as "ZEP3") peptide, where pGlu is pyroglutamic acid. Peptide ZEP3 can be produced, 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"). Peptide ZEP4 can be produced, for example, by the following procedure.

[0106] The synthesis of peptide ZEP4 is carried out by the sequential synthesis of 9-fluoromethoxycarbonyl (Fmoc) amino acids on a chlorotrityl chloride resin (CTC) solid support. Fmoc-threonine (t-butyl; 79g) is loaded onto CTC resin (125g), and diisopropylamine (DIPEA; 160g) is used as a coupling agent for the amino acids to the solid support. The Fmoc protecting group is removed with a 25% piperidine mixture, and the resin-peptide is filtered and washed with dimethylformamide (DMF). The second amino acid, Fmoc-Trp (85g), is activated with a mixture of 3-[bis(dimethylamino)methyliminyl]-3H-benzotriazole-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) (119g), 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 (26g), is activated with HBTU / HOBT and coupled with DIEA.

[0107] The peptide-resin is thoroughly washed with DMF, then washed with IPA and dried under reduced pressure. The peptide is cleaved from the resin and the protecting groups of Thr and Asn with 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 46g).

[0108] The crude product (46g) is dissolved in a mixture of acetonitrile (ACN) and water, loaded into 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 performed by gradually increasing phase B over 45 minutes (from 3% to 33%). Fractions with a purity of over 97% are collected. The combined fractions are eluted using the same HPLC system with a gradient consisting of phase A: 0.2% acetic acid and phase B: ACN. Elution is performed by gradually increasing phase B over 30 minutes (from 10% to 40%). Fractions with a purity of over 97% are collected, combined, and lyophilized (yield 29g). The final product has a MW (MS) of 530.5 and a purity of 97.3% (HPLC).

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

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

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

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

[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 (wherein n is 1 or 2), hereinafter referred to as "ZEP3 sodium salt" or ZEP3Na).

[0114] The sodium salt of ZEP3 can be produced, for example, by the following procedure: Dissolve ZEP3 (3.1 g) in NaHCO3 (100 mM) (50 g / l) in water. Inject the solution into an HPLC ion exchange column (2.5 × 22 cm Luna C18, 100A, 15 micron) and elute using 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 fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile (110 g / l), and then freeze-dried [yield 2.2 g (71%)]. The final product had a purity of 99.7% (HPLC), a sodium content of 3.1%, and solubility in 50 mg / ml of water.

[0115] In one embodiment of the present invention, the peptide is the sodium salt of the peptide described in Sequence ID No. 2 (pGlu-Asn-Trp-Thr-OH.nNa (wherein n is 1 or 2); hereinafter referred to as "ZEP4 sodium salt" or ZEP4Na). ZEP4 sodium salt can be produced, for example, by the following procedure: Dissolve ZEP4 (5 g) in NaHCO3 (100 mM) (50 g / l) in water. Inject the solution into an HPLC ion exchange column (2.5 × 22 cm Luna C18, 100A, 15 micron) and elute by a gradient consisting of mobile phase A: NaHCO3 2 mM in H2O; mobile phase B: NaHCO3 2 mM in CH3CN / H2O(8 / 2); and mobile phase C: NaHCO3 100 mM 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 then phase C for 10 minutes. The fraction containing the product is 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 solubility in 50 mg / ml of water.

[0116] The peptides of the present invention can be used therapeutically, either on their own 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, comprising 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 organism.

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

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

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

[0121] Preferred routes of administration include, for example, intraocular, topical, oral, rectal, transmucosal, transnasal, intestinal, or parenteral delivery (including intramuscular, subcutaneous, and intrathecal injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections).

[0122] The pharmaceutical compositions of the present invention can be produced by processes well known in the art, for example, by conventional mixing, dissolution, suspension, solubilization, granulation, sugar-coated tablet production, polishing, emulsification, encapsulation, encapsulation, 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 comprising excipients and adjuvants that facilitate the treatment of the active ingredient into a preparation that can be used as a pharmaceutical. The appropriate formulation depends on the selected route of administration.

[0124] The pharmaceutical compositions described herein may be formulated for ophthalmic administration. As used herein, the term “ophthalmic administration” refers to the administration of the peptides or pharmaceutical compositions of the present invention into the external eye (e.g., the conjunctival sac) or intravitreous cavity. Suitable dosage forms 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 intravitreous injections.

[0125] The pharmaceutical compositions described herein may be formulated for topical administration. Suitable dosage forms for topical administration may include, but are not limited to, droplets, liquid cleansers, gels, ointments, emulsions, suspensions, lotions, sprays, spray liquids, suppositories, creams, powders, foams, crystals, and liposomes.

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

[0127] For oral administration, pharmaceutical compositions can be readily formulated by combining the active compound with a pharmaceutically acceptable carrier known in the art. Such carriers allow the pharmaceutical composition to be formulated for oral intake by patients as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, emulsions, etc. Pharmacological preparations for oral use can be prepared using solid excipients, and the resulting mixture can be optionally ground, and after adding suitable adjuvants as desired, the granular mixture can be processed to obtain tablets or sugar-coated tablet cores. Suitable excipients include sugars, particularly lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carbomethylcellulose, etc.; and / or fillers such as physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, may be added. As used herein, the term “oral administration” includes administration of the pharmaceutical compound to any oral surface, including the tongue, gingiva, palate, sublingual area, or other buccal surface.

[0128] The core of the sugar-coated tablet is coated with a suitable coating. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopole gel, polyethylene glycol, titanium dioxide, lacquer solution, and a suitable organic solvent or solvent mixture. Dyes or pigments may be added to the tablet or sugar-coated tablet coating for identification or to characterize different combinations of active compound doses.

[0129] Pharmaceutical compositions that can be used orally include push-type capsules made of gelatin, and soft-seal capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-type 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. Further stabilizers may be added. All formulations for oral administration must be in a dosage suitable for the selected route of administration.

[0130] For oral administration, the composition may take the form of tablets or lozenges formulated using 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 a pressurized pack or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve for delivering a measured amount. Capsules and cartridges, for example, gelatin capsules and cartridges for use in dispensers, 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 example, for parenteral administration by bolus injection or continuous infusion. Formulations for injection may be provided in unit dosage forms, for example, in ampoules or multi-dose containers, with the optional addition of preservatives. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may also contain formulation agents such as solubilizers, suspending agents, stabilizers and / or dispersants.

[0133] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active formulation in a water-soluble form. Furthermore, suspensions of the active ingredient can be prepared as suitable oily or water-based injectable 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 injectable suspensions may also 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 high-concentration solutions.

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

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

[0136] Suitable pharmaceutical compositions for use in connection with the present invention include compositions containing an active ingredient 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 improve the symptoms of the disease or disorder being treated.

[0137] Determining the therapeutically effective dose is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosures provided herein.

[0138] For any preparation used in the method of the present invention, the therapeutically effective amount or dose can first 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 potency. Using such information, a useful dose in humans can be determined more accurately.

[0139] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard in vitro pharmaceutical procedures in cell culture or experimental animals. Data obtained from these in vitro and cell culture assays, as well as animal studies, can be used when formulating various dosages for human use. Dosages may vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage may be selected by individual physicians considering the patient's condition. (See, for example, Fingl, E. et al. (1975), "The Pharmacological Basis of Therapeutics," Ch.1, p.1.)

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

[0141] Depending on the severity and responsiveness of the condition being treated, the medication may be administered as a single dose or multiple doses, the course of treatment may last from several days to several weeks, or a reduction in the condition may be achieved.

[0142] The amount of the composition administered will naturally depend on the patient being treated, the severity of their suffering, the method of administration, and the judgment of the prescribing physician.

[0143] Accordingly, compositions and / or articles of some embodiments of the present invention may be presented in packs or dispenser devices, such as FDA-approved kits, which may optionally contain one or more unit dosage forms comprising the active ingredient. Packs may include, for example, metal or plastic foil, such as blister packs. Packs or dispenser devices may be accompanied by instructions for administration. Packs or dispensers may also correspond to notices relating to the form of container prescribed by government agencies that regulate the manufacture, use, or sale of pharmaceuticals, which reflect the agency's approval of the composition or the form of administration in humans or veterinary medicine. Such notices may be, for example, labels approved by the U.S. Food and Drug Administration for prescription drugs or package inserts for approved products. Compositions comprising preparations of the present invention formulated on a suitable pharmaceutical carrier may also be prepared for the treatment of an indication, as further detailed above, placed in appropriate containers (e.g., lyophilized vials), and labeled.

[0144] The peptides or pharmaceutical compositions of the present invention may be used to treat inflammatory diseases or disorders by inhibiting the release or activity of inflammatory cytokines and mediators, and by providing a therapeutically effective amount of the peptide or pharmaceutical composition to a target that requires such inhibition.

[0145] As used herein, the term “subjects requiring it” refers to male or female mammalian subjects (e.g., humans) diagnosed with an inflammatory disease or disorder. In certain embodiments, this term encompasses individuals at risk of developing an inflammatory disease or disorder. Subjects may be of any sex or age and include neonates, infants, boys, adolescents, adults, and the elderly.

[0146] As used herein, the term “approximately” refers to ±10%.

[0147] The terms "comprises," "comprising," "includes," "including," and "have," as well as their variations, all mean "to include, but not limited to."

[0148] The phrase "~consists of" means "to include and be limited to ~".

[0149] The term “essentially derived from” means that the composition, method, or structure may include additional components, steps, and / or parts, provided that the additional components, steps, and / or parts do not substantially alter the basic and novel properties of the claimed composition, method, or structure.

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

[0151] Throughout this application, various embodiments of the present invention may be presented in range form. It should be understood that the range form is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the present invention. Therefore, the range description should be considered to specifically disclose all possible subranges and the individual numbers within those ranges. For example, a range description such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, 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] Where a numerical range is indicated herein, it is always intended to include any cited digit (fraction or integer) within that range. The expressions “ranging / ranges” between a first number and a second number, and “ranging / ranges from” the first number to the second number, are used interchangeably herein and mean including the first and second numbers, as well as all fractions and integers between them.

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

[0154] For clarity, certain features of the present invention described in relation to separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the present invention described in relation to a single embodiment for brevity may be provided separately, in any suitable partial combination, or in any other described embodiment of the present invention. Certain features described in relation to various embodiments should not be considered essential features of those embodiments unless the embodiments would not function without those elements.

[0155] Various embodiments and aspects of the present invention, as described above and claimed in the following claims, are experimentally supported in the following examples. [Examples]

[0156] Herein, the following embodiments will be used to illustrate the present invention in a non-limiting manner, in conjunction with the above description.

[0157] Example 1 - Effect of ZEP4 on TNF-alpha expression by pro-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] TNF-alpha Expression by B6 Macrophage Cells: Macrophages derived from C57BL / 6 mice were cultured at a density of 200,000 cells / mL in 24-well plates (500 μl wells) 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, followed by incubation for 3 hours. Then, the medium (with or without LPS) of a specific experimental group was replaced with fresh medium containing ZEP4 at concentrations ranging from 3.125 to 100 μg / mL, followed by incubation for 1 hour. Palmitic acid (PA, 200 μM) was then added to the appropriate wells, and the plates were incubated for a further 24 hours, after which the amount of TNF-alpha in each well was measured. Table 1 shows the various treatment and control groups. The reduction in TNF-alpha production in the treatment groups was calculated compared to cells treated with LPS and PA (CLP200).

[0159] Results Figure 1 and Table 1 below show that ZEP4 significantly reduced the amount of TNF-alpha produced by pro-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 pro-inflammatory macrophage cells Materials and Methods 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 at a density of 200,000 cells / mL in 24-well plates (500 μl wells) 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. Next, the LPS-containing medium of the treatment group was replaced with fresh medium containing 50 μg / mL of ZEP4 and incubated for 1 hour. Then, aliquots of palmitic acid (100, 200 or 400 μM) were added to the appropriate wells. The plates were incubated for a further 24 hours and the amount of TNF-alpha in each well was measured. Table 2 shows the various treatment and control groups. The reduction in TNF-alpha production in the treatment group was calculated based on PA concentration, compared to cells treated with LPS and PA.

[0161] Results Figure 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 macrophage TNF-alpha expression at all palmitic acid concentrations (****p<0 / 0001). [Table 2]

[0162] Example 3 - Effect of ZEP3 on TNF-alpha expression by pro-inflammatory 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] TNF-alpha expression by B6 macrophage cells Macrophage cells derived from C57BL / 6 mice were cultured at a density of 200,000 cells / mL in 24-well plates (500 μl wells) 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, followed by incubation for 3 hours. Then, 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, followed by incubation for 1 hour. Subsequently, palmitic acid (200 μM) aliquots were added to the appropriate wells, and the plates were incubated for a further 24 hours, after which the amount of TNF-alpha in each well was measured. Table 3 shows the various treatment and control groups. The reduction in TNF-alpha production in the treatment groups was calculated compared to cells treated with LPS and PA.

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

[0165] Example 4 - Effect of ZEP4 on IL-1 beta expression 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 at a density of 200,000 cells / mL in 24-well plates (500 μl wells) 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, aliquots of nigericin (20 mM) were added to the ZEP4-treated and LPS control wells. The plates were incubated for a further 24 hours, and the amount of IL-1 beta in each well was measured. Table 4 shows various control and treatment groups. The reduction in TNF-alpha production in the treatment group was calculated compared to cells treated with LPS and nigericin (LPS+N).

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

[0167] Example 5 - Effect of ZEP3 on the viability of pro-inflammatory macrophage cells Materials and Methods 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 at a density of 200,000 cells / mL in 24-well plates (500 μl wells) 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 incubated for 3 hours. The LPS-containing medium of the treatment group was then replaced with fresh medium containing ZEP3 at a concentration of 50 μg / mL and incubated for 1 hour. Palmitic acid (100, 200 or 400 μM) was then added to the appropriate wells. The plates were incubated for a further 24 hours, and cell viability was estimated using an LDH cytotoxicity assay kit. Table 5 shows various treatment groups and control groups.

[0168] Results Figure 5 and Table 5 below show that ZEP3 did not have a significant effect on the viability of pro-inflammatory macrophage cells under experimental conditions. [Table 5]

[0169] Example 6 - Effect of ZEP3 pretreatment on TNF-alpha expression by pro-inflammatory 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 at a density of 200,000 cells / mL in 24-well plates (500 μl wells) and placed overnight in an incubator at 37°C and 5% CO2. 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 a further 24 hours, after which the amount of TNF-alpha in each well was measured.

[0170] Results: Table 6 below shows that pretreatment of macrophage cells with ZEP3 before LPS induction reduced TNF-alpha production 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 peptides in experimental corneal inflammation was tested in a cell culture model that simulates dry eye disease (DED) using various inflammasome inducers such as LPS, 4-HNE, and nigericin. Using a dose-response model, human primary corneal epithelial cells were pretreated with peptides and subsequently exposed to DED inducers. IL-6 expression, cell viability, and ROS production were measured.

[0172] material and method 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, followed by exposure to LPS, 4-HNE, or nigericin for 24 hours. After incubation, 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, IL-6 levels in cell culture decreased from 52.45 ± 0.9121 pg / mL in untreated cell cultures to 38.08 ± 1.368 pg / mL in cells treated with ZEP3 (a 27.4% reduction; P < 0.05).

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

[0175] As shown in Figure 6, the cell death density decreased from 21.125 ± 0.942% in untreated cell cultures to 3.625 ± 1.092% in cultured cells treated with ZEP3 (an 82.8% reduction; P < 0.0001). The ZEP3 peptide is effective in reducing corneal inflammation, restoring HCEC viability, and decreasing ROS production, and is concluded to be a key 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 demonstrate 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 pro-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. A model was performed according to De Vos et al., Exp Eye Res 61:667-675, 1995. Male Lewis rats weighing 180-200 g were given EIU by injecting 200 μg of LPS diluted in 0.1 mL of sterile water into the paw. The animals were then randomized into (4 peptides) × (3 concentrations) = 12 groups. The untreated induction group was used as a control for induction.

[0178] Animals are examined under a slit-lamp biomicroscope 24 hours after the peak severity of the EIU. Clinical ocular inflammation is scored in each eye using a scale of 0 to 7 as follows: iris hyperemia and intra-anterior chamber cells are rated 0 to 2 (0 = no signs; 1 = mild; 2 = severe); flare, miosis, and anterior chamber hypopyon are rated 0 if no signs are present and 1 if present. The maximum possible score is 7 (sum of the five parameter scores).

[0179] Example 9 - Effects of ZEP3, ZEP3 sodium salt, and ZEP4 in a controlled environment chamber (CEC) mouse model of dry eye. The model procedure is described in Barabino et al. (IOVS 46:2766-2771, 2005). Briefly, 8-12 week old BALB / c mice are used in a controlled environment chamber (CEC) where relative humidity (RH), temperature (T), and airflow (AF) are controlled and monitored. Mice are randomized and treated with ZEP3, ZEP sodium salt, and ZEP4 (test peptides). An untreated induction group is used as a control. Mice are then placed in the CEC and exposed to specific controlled environmental 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 in a normal environment (RH=50%~80%, no AF, T=21~23°C) for the same period. Aqueous tear production is measured by a masked observer using a cotton thread test, corneal fluorescein staining (score, 0-15), and goblet cell density in the upper and lower conjunctiva.

[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 300g-350g were used. Dry eye was induced using scopolamine (Sigma-Aldrich, St. Louis, Mo.), which was continuously and systemically delivered to the animals via an osmotic pump filled with scopolamine (2ML4 Alzet®; CedarLane, Burlington, Ontario) and subcutaneously implanted in the middorsal region between the scapulae. The wound was closed with 2-3 wound clips. Postoperatively and again the following day, the animals were subcutaneously injected with carprofen (0.5 mg / 100g), a nonsteroidal anti-inflammatory drug, and a potent, long-acting analgesic for rodents. Animals will be anesthetized before surgical pump implantation and before all endpoint studies in an isofluorane 99.9% USP (Abraxis Bioscience, Richmond Hill, Ontario) chamber. Scopolamine will be delivered at 12.5 mg / day, and data will be evaluated on day 14.

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

[0182] The rat eyes tested were divided into the following groups: Group 1: Control rats (n=12 eyes from 6 rats). Group 2: Rats (n=12 eyes from 6 rats) were given systemic scopolamine to induce dry eye, and fluorescein staining was performed on day 14. Group 3: Rats (n=14 eyes from 7 rats) were given systemic scopolamine to induce dry eye, and treated topically with physiological saline once on day 8. Group 4: Rats (n=14 eyes from 7 rats) were given systemic scopolamine to induce dry eye, and treated topically with the test peptide once on day 8.

[0183] The endpoints are as follows: (i) reduction of corneal fluorescein staining as measured on day 13 compared to saline-treated controls; and (ii) aqueous tear production and aqueous tear turnover as measured on day 13 compared to untreated or scopolamine-treated controls.

[0184] Example 11 - Effects of ZEP3, ZEP3 sodium salt, and ZEP4 in a rabbit model of keratoconjunctivitis sicca. Eight New Zealand white rabbits were given keratoconjunctivitis sicca (KCS) in the right eye by surgically closing the lacrimal duct and removing the nictitating membrane, nictitating membrane gland, and Harderian gland. All rabbits were left untreated for 8 weeks, and KCS was confirmed by taking 0.1–0.4 μL tear samples and measuring the elevation of the tear film osmotic pressure, as described by J. Gilbard et al. (Ophthalmol. 96:677, 1978). 3.0 mmol solutions of UTP or analogues were prepared in stored isotonic buffer. The rabbits were randomized and treated with ZEP3, ZEP3 sodium salt, and ZEP4 (test peptide). The untreated group was used as a control. After the start of treatment, 0.1–0.4 μL tear samples were taken from all rabbits for osmotic measurement before the first dose. At 20 weeks, the animals were sacrificed, and goblet cell density was measured by staining with Alcian blue and Schiff periodate 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 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 using lipopolysaccharide (LPS) to produce cytokines.

[0186] Methods: Briefly, cryopreserved human PBMCs from four healthy, non-smoking individuals who had not received any corticosteroids or other inflammatory drugs were grown in RPMI1640 with 10% thermo-inactivated fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / mL streptomycin (Full Growth Medium, CGM).

[0187] Human PBMCs were thawed and seeded in all groups (1-6, Table 7), and allowed to stand for 1 hour before treatment. The cell populations were treated with ZEP3 (SEQ ID NO: 1), or dexamethasone, at concentrations of 40, 100, and 150 μg / ml for 1 hour, followed by stimulation with LPS for 24 hours. Almar Blue was added 4 hours before supernatant collection. 24 hours after LPS addition, Almar Blue fluorescence at 585 nm (correlated with cell viability) was measured, and the supernatant was collected for cytokine measurement. Each group was treated 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 stock solution was added to appropriate wells of a 96-well black wall plate (1 plate per donor), and 2 × 10^5 cells were seeded per well. Cells were incubated at 37°C and 5% CO2 for 1 hour. 100 μL of 2X test substance, dexamethasone, or 1X CGM was added to appropriate wells to a final volume of 200 μL / well. Plates were incubated at 37°C and 5% CO2 for a further 1 hour. After 1 hour, 20 μL of LPS was added to groups 2-9 to a final volume of 220 μL. 20 μL of IM was added to group 1. Cells were incubated at 37°C and 5% CO2 for 20 hours. At 20 hours, 20 μL of Alamar Blue was added to all wells. The plates were incubated at 37°C and 5% CO2 for a further 4 hours. 24 hours after LPS stimulation, the plates were read to assess cell viability, the cell culture supernatant was collected and stored at -80°C for cytokine analysis. The remaining cells were washed once with sterile PBS. Cells were incubated in trypsin solution to separate them from the plates, centrifuged to form a pellet, and the trypsin solution was removed. The cells were then stored at -80°C for further analysis of any selection. The concentrations of GM-CSF, IFN-γ, IL-10, IL-12p40, IL-17α, IL-1β, IL-4, IL-6, RANTES, and TNF-α in the supernatant samples collected after the Alamar Blue assay were determined using the Luminex assay. The manufacturer's protocol was followed in the use of the cytokine assay. The mean percentages of cytokine production and sample stimulation versus stimulation control were calculated.

[0189] Results: As shown in Figures 9–12, ZEP3 was shown to induce a significant reduction in major inflammatory mediators, including interferon-gamma (IFNγ, Figure 9), IL-10 (Figure 10), IL-1β (Figure 11), and RANTES (Figure 12), supporting the concept of its anti-inflammatory effect. Dose-dependent responses were observed for some of the measured cytokines. No significant changes in cell viability or any donor tendencies were observed after treatment with all tested concentrations of the peptide, indicating that the peptide is not cytotoxic to PBMCs. Levels of IL-4 and IL-17a were below the detection limit in all test groups.

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

[0191] Example 13 - In vivo atopic dermatitis model: The effects of topical administration of ZEP3 (SEQ ID NO: 1), 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 described in detail 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 TK 2006, Basic Clin. Pharmacol. Toxicol. 99, 104-115).

[0192] Methods: Animals were sensitized by topical administration of 2% oxazolone in ethanol to the back on day 0 after shaving and depilation (sensitization period). Then, one week later, a challenge was performed by topical administration of 1% oxazolone in ethanol to the back and right ear every two days for 10 times, i.e., for a total of 3 weeks. The test compound was applied via a topical route every two days during the challenge phase as a ready-to-use cream formulation. The application surface 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 on a finger glove was applied to the inner surface of the ear. Every other day, the thickness of the skin on the back and ear was measured with calipers, and the following clinical scores were recorded from the skin on the back: erythema and scaling. The weight of the animals was also measured every other day (the approximate weight of the mice at the start was about 20 g). Corrected digital photographs of the skin area on the back were taken on days 8, 18, and 28. The experiment was concluded on day 28, blood was collected from the animals, and plasma samples were separated. For euthanized animals, skin samples from the right ear and back were collected and frozen at -80°C (samples from back skin and plasma), or preserved in formalin for further analysis (samples from back skin and right ear). Table 8 describes the treatment groups. [Table 8]

[0193] The formulations used for topical administration of the test compounds are as follows: ZEP3 is 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 is 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 is 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, were well-tolerated. All peptides significantly reduced clinical lesions of atopic dermatitis, including 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), indicating a delayed effect. Body weight in mice treated with ZEP3, ZEP3Na, or ZEP4 behaved normally compared to the excipients (Figures 17A and 17B).

[0195] In detail, the body weight of ZEP3, ZEP3Na, and ZEP4 remained very stable during the first few days of the study, showing an increase of approximately 3–4% on day 28 (Figures 17A and 17B). As predicted, a weight loss of approximately 8–10% was observed in the betamethasone-treated group.

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

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

[0198] Erythema scoring peaked on day 16 in the negative control group (group 1) and decreased slightly to 0 between days 26 and 28. Erythema scoring recorded in test groups 3, 4, and 5 was similar to that of negative control group 1 (Figures 15A and 15B). Significant differences were observed on days 22 and 24 in ZEP3, on day 24 in ZEP3Na, and on days 12, 16, 22, and 24 in ZEP4.

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

[0200] The total score (sum of 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. Significant differences in total score were observed in ZEP3 on days 20, 24, and 28, in ZEP3Na on days 14, 18, 20, and 22, and in ZEP4 on days 12-20, 24, and 28.

[0201] Conclusion: Compounds ZEP3 and ZEP4 and their sodium salts, administered at 50 mg / kg to atopic dermatitis-induced mice, were well-tolerated and significantly reduced the clinical lesions of atopic dermatitis, as indicated by reductions in dorsal skin thickness, ear thickness, erythema, and scaling.

[0202] Example 14: Treatment of carpometacarpal osteoarthritis (CMC arthritis) with ZEP3. A 75-year-old male subject was diagnosed by an orthopedic surgeon with CMC arthritis of the thumb. His toes were swollen, red, and painful, making it impossible for him to walk.

[0203] A 0.5% ZEP3 sodium salt cream was applied to the swollen area five times a day. Participants reported relief within one day of treatment and complete resolution within three to four days.

[0204] Example 15 Effects of ZEP3 and ZEP3Na peptides on TNF-alpha secretion in human keratinocytes. TNF-alpha is a major inflammatory mediator in keratinocytes. Compounds were tested for their anti-inflammatory effects by measuring their effect on TNF-alpha levels.

[0205] Methods: SCCE020 cells (EpiGRO® human epidermal keratinocytes) were grown in complete growth medium (EpiGRO® human keratinocyte complete medium; Millipore catalog no. SCMK001) for 4 passages. In passage 5, cells were seeded three times per well with a volume of 1 ml of complete growth medium (3 × 10⁵ cells / well). Three additional control wells contained only medium without cells. Once attached, the cell medium was replaced with a basal medium containing L-glutamine without supplements (starvation medium), and the cells were starved overnight. The following day, 24 hours after seeding, the cells were treated with ZEP3 or ZEP3Na or an appropriate diluent (PBS or DMSO). After 4 hours of incubation, LPS (from E.Coli 055:B5; Sigma catalog no. L6529-1MG) was added to the wells at a final concentration of 20 or 30 μg / ml. The experimental groups (three wells for each treatment) are summarized in Table 9.

[0206] Cells were incubated in a tissue culture incubator for 24 hours. After collecting the cell supernatant from all wells, cells from the treatment groups (1, 2, 3, 8, 9, 10, 13, 14 (no DMSO group)) were pelleted and stored at -80°C for further analysis. Cells were collected as follows: After collecting the supernatant, the wells were rinsed with trypsin, then incubated with trypsin until separated from the plate surface, centrifuged, washed with PBS, and stored as a cell pellet. The TNF-α concentration in the supernatant was measured five times by ELISA (Quantikine HS ELISA; R&D Systems catalog number HSTA00D).

[0207] Results: ZEP3 and ZEP4 effectively reduced TNF-alpha levels in human keratinocyte cell cultures. TNF-alpha concentrations in the samples were calculated according to the equation of the trend line of the standard curve: OD = 0.063x + 0.1718. The percentage of inhibition of TNF-alpha production was calculated for each treatment group compared to the corresponding control group (same conditions, no peptide). The results are summarized in Table 9. [Table 9]

[0208] Conclusion: Quantitative ELISA testing showed dose-dependent inhibition of TNF-α production in human keratinocytes treated with LPS along with either ZEP3 or ZEP3Na, compared to the amount secreted by cells simulated with LPS alone.

[0209] While the present invention has been described in relation to its particular embodiments, it is obvious that many alternatives, modifications, and variations are apparent to those skilled in the art. Therefore, it is intended that all such alternatives, modifications, and variations, which fall within the spirit and broad scope of the appended claims, are encompassed.

[0210] All publications, patents, and patent applications referenced herein are incorporated herein by reference in whole to the same extent as when individual publications, patents, or patent applications are specifically and individually indicated as being incorporated herein by reference. Furthermore, any citation or specification of references in this application should not be construed as an acknowledgment that such references are available as prior art of the present invention. Section headings, insofar as they are used, should not necessarily be construed as restrictive.

Claims

1. An isolated sodium salt of a peptide comprising the sequence shown in Sequence ID No.

1.

2. The isolated sodium salt according to claim 1, wherein the purity measured by HPLC is at least 99.7%.

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

4. The isolated sodium salt according to claim 1, which is a solid phase.

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