Pharmaceutical compositions for inhibiting inflammatory cytokines

Abstract

The present invention provides pharmaceutical compositions comprising specific tetrapeptides for reducing the release of or inhibiting the activity of inflammatory cytokines and mediators, and for treating diseases and disorders associated with these cytokines and mediators.

Description

Technical Field

[0001] The present invention provides pharmaceutical compositions and methods for alleviating symptoms associated with the release of inflammatory cytokines in inflammatory conditions including, but not limited to, inflammatory ocular 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 protective response involving immune cells, blood vessels and molecular mediators. The function of inflammation is to eliminate the initial cause of cell damage, remove necrotic cells and tissues damaged during the original injury and inflammation, and initiate tissue repair.

[0003] Classical symptoms of inflammation are fever, pain, redness, swelling, and loss of function. Inflammation is a universal response and is therefore considered a mechanism of innate immunity.

[0004] Inflammatory diseases include a variety of disorders and conditions characterized by inflammation. Examples include allergies, asthma, transplant rejection, autoimmune diseases, and ocular inflammation, to name just a few.

[0005] Inflammatory eye disorders

[0006] Uveitis is a general term that describes a group of inflammatory eye diseases that can mildly reduce vision or cause severe vision loss. The diseases usually affect the part of the eye called the uvea, but are 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 in the eye, or it can be part of an inflammatory disease that affects other parts of the body. Uveitis can be acute or chronic, and is generally classified as infectious or noninfectious.

[0007] Keratoconjunctivitis sicca, also known as dry eye disease (DED) or dry eye syndrome (DES), is a common eye disease that prompts millions of people to seek ophthalmic care. Regardless of the underlying etiology, dry eye disease has been shown to be associated with abnormalities of the precorneal tear film and subsequent inflammatory changes in the entire ocular surface including the adnexa, conjunctiva, and cornea. Activation of inflammatory cytokines such as IL-6 plays a key role in the pathophysiology of DED.

[0008] Since the recognition of the role of inflammation in dry eye disease, a large number of treatments aimed at inhibiting various inflammatory pathways have been investigated, such as cyclosporine A, corticosteroids, tacrolimus, tetracycline derivatives and autologous serum (Michelle Hessen M. and Karamursel Akpek, EJ Ophthalmic Vis Res. 2014, 9(2), 240–250).

[0009] U.S. Pat. No. 4,619,916 teaches 13 tripeptides of the formula pGlu-X-Trp, wherein pGlu is cyclized glutamic acid (pyroglutamic acid), and X can be Gly, Val, Glu, Asp, Ser, Ala, Asn, Gln, Ile, Leu, Pro, Lys, and Arg. The reference does not describe or suggest the use of the peptides for treating inflammation.

[0010] U.S. Patent No. 7,220,725 and International Patent Publication No. WO200212269 teach novel peptides including pGlu-Asn-Trp-Lys(octanoyl)-OH(ZEP3) and pGlu-Asn-Trp-Thr-OH(ZEP4) and their use for treating pain. The references do not describe or suggest the use of the peptides for treating inflammation.

[0011] US Patent No. 9,012,397 and WO2012 / 131676 teach topical pharmaceutical compositions comprising peptides ZEP3 or ZEP4 and their use for improving symptoms of skin disorders. Said references do not describe or suggest the use of said peptides for treating inflammation.

[0012] Gaynes et al. (Invest. Ophthalmol. Vis. Sci. 54, E-Abstract 5416, 2013) describe the analgesic effect of the peptide ZEP4 in reducing ocular pain and altering nociceptive pathways in a rat model of experimentally induced chemical corneal injury. The reference does not describe or suggest anti-inflammatory activity of the peptide.

[0013] The present invention addresses the ongoing need to improve and develop new safe and effective treatments that ameliorate inflammatory conditions through intervention of the inflammatory cascade. Summary of the invention

[0014] The present invention provides compositions and methods for improving symptoms associated with the release of inflammatory cytokines. These cytokines are known to be part of the etiology and pathology of many inflammatory disorders. Diseases and disorders suitable for treatment with the compositions of the present 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, where administration of the drug may be long-term in order to prevent deterioration.

[0015] The present invention is based in part on the discovery in in vitro and in vivo models that peptides referred to as ZEP3 and ZEP4 unexpectedly exhibit potent activity in attenuating the expression and / or release of inflammatory and pro-inflammatory mediators, including specific cytokines interferon gamma (IFN-γ, IFNγ), interleukin 1 beta (IL-1β, IL-1β), interleukin 10 (IL-10), tumor necrosis factor alpha (TNFα, TNFα) and interleukin 6 (IL-6) and the mediators reactive oxygen species (ROS) and RANTES (a chemokine that regulates normal T cell expression and possible secretion upon activation).

[0016] Surprisingly, the activity of the peptides in inhibiting the inflammatory cascade was found to be exerted even when the peptides were administered before the onset of the inflammatory response. Thus, the peptides may improve or inhibit the symptoms induced in a variety of different inflammatory diseases and disorders, and may be used to eliminate the inflammatory response altogether. It is envisioned that the compositions of the invention may be used to prevent the exacerbation of chronic inflammatory diseases.

[0017] It should be expressly understood that treatment of inflammatory diseases using the compositions of the present invention does not include treatment of the skin disorder or pain itself.

[0018] According to one aspect of the present invention, a pharmaceutical composition is provided, comprising a pharmaceutically acceptable carrier and a pGlu-X 1 -X 2 -X 3 -OH described peptide or its pharmaceutically acceptable salt or derivative as an active ingredient, wherein X 1 is a polar amino acid residue; X 2 is an aromatic or hydrophobic amino acid residue; and X 3 Selected from positively charged amino acid residues and polar amino acid residues; used for reducing the release of inflammatory cytokines or inhibiting their activity.

[0019] According to certain embodiments, X 1 is selected from the group consisting of Asn, Gln, His, Ser, Thr, Tyr and Cys; X 2 is selected from the group consisting of Trp, Phe, Tyr, Ala, Ile, Leu, Met, Val and Gly; and X 3 Selected from Lys, Lys derivatives, Arg, His, Asn, Gln, His, Ser, Thr and Tyr.

[0020] According to other embodiments, X 1 Selected from Asn and Thr; X 2 is selected from Trp, Phe and Tyr; and X 3 Selected from Lys, Lys derivatives and Thr.

[0021] According to certain embodiments, the peptide derivative comprises an alkyl group attached to a free functional group of the peptide sequence.

[0022] According to other embodiments, the alkyl group is linked to the free amino group of the side chain or N-terminus of the peptide via an amide bond or a linking bond.

[0023] According to certain embodiments, the alkyl group is a C4 to C30 alkyl group.

[0024] According to certain specific embodiments, C8 alkyl (octanoyl herein) is connected to the side chain of the Lys residue of the peptide sequence or the terminal amino group of the peptide via an amide bond. According to certain embodiments, the carboxyl end of the peptide is modified to form, for example, an amide, an alcohol or an ester end.

[0025] According to certain specific embodiments, the peptide is selected from 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.

[0026] According to certain embodiments, the inflammatory cytokine is selected from the group consisting of TNFα, IL-1β, IL-6, IL-10 and IFNγ.

[0027] According to another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pGlu-X 1 -X 2 -X 3 -OH described peptide or its pharmaceutically acceptable salt or derivative as an active ingredient, wherein X 1 is a polar amino acid residue; X 2 is an aromatic or hydrophobic amino acid residue; and X 3 Selected from positively charged amino acid residues and polar amino acid residues; used to reduce the release of inflammatory mediators or chemokines.

[0028] According to certain specific embodiments, the peptide is selected from 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.

[0029] According to certain embodiments, the inflammatory mediator or cytokine is selected from reactive oxygen species (ROS) and RANTES (chemokine regulated by normal T cells expressed and possibly secreted upon activation).

[0030] According to another aspect of the present invention, there is provided a method for reducing the release of at least one inflammatory or proinflammatory cytokine or inhibiting its activity, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pGlu-X according to Formula I: 1 -X 2 -X 3 -OH or a pharmaceutically acceptable salt or derivative thereof, wherein X 1 is a polar amino acid residue; X 2 is an aromatic or hydrophobic amino acid residue; and X 3 Selected from positively charged amino acid residues and polar amino acid residues.

[0031] According to another aspect, a method for reducing the release of inflammatory mediators or chemokines is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pGlu-X according to Formula I: 1 -X 2 -X 3 -OH or a pharmaceutically acceptable salt or derivative thereof, wherein X 1 is a polar amino acid residue; X 2 is an aromatic or hydrophobic amino acid residue; and X 3 Selected from positively charged amino acid residues and polar amino acid residues.

[0032] According to certain embodiments, the inflammatory mediator or chemokine is selected from ROS and RANTES.

[0033] According to another aspect, the present invention provides a method for treating an inflammatory disease or disorder. According to a specific embodiment, the inflammatory disease or disorder is selected from an ocular inflammatory disease or disorder, an otic inflammatory disease or disorder, a pulmonary inflammatory disease or disorder, an intestinal inflammatory disease or disorder, or an inflammatory autoimmune disease or disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pGlu-X 1 -X 2 -X 3 -OH or a pharmaceutically acceptable salt or derivative thereof, wherein X 1 is a polar amino acid residue; X 2 is selected from aromatic amino acid residues and hydrophobic amino acid residues; and X 3 Selected from positively charged amino acid residues and polar amino acid residues.

[0034] According to certain embodiments, the present invention provides methods for treating chronic inflammatory diseases. According to specific embodiments, the step of administering a therapeutically effective amount of a peptide according to Formula I to a subject in need is performed even before the symptoms have appeared or occurred. These methods for treating chronic inflammatory diseases effectively prevent the condition from worsening.

[0035] According to another aspect, the present invention provides a method for treating an ocular inflammatory disease or disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to Formula I: pGlu-X 1 -X 2 -X 3 -OH or a pharmaceutically acceptable salt or derivative thereof, wherein X 1 is a polar amino acid residue; X 2 is an aromatic or hydrophobic amino acid residue; and X 3 Selected from positively charged amino acid residues and polar amino acid residues.

[0036] According to certain embodiments, X 1 is selected from the group consisting of Asn, Gln, His, Ser, Thr, Tyr and Cys; X 2 is selected from the group consisting of Trp, Phe, Tyr, Ala, Ile, Leu, Met, Val and Gly; and X 3 Selected from Lys, Lys derivatives, Arg, His, Asn, Gln, His, Ser, Thr and Tyr.

[0037] According to other embodiments, X 1 Selected from Asn and Thr; X 2 is selected from Trp, Phe and Tyr; and X 3 Selected from Lys, Lys derivatives and Thr.

[0038] According to certain specific embodiments, the Lys derivative is Lys(octanoyl).

[0039] According to certain embodiments, the peptide derivative comprises an alkyl group attached to a functional group of the peptide sequence.

[0040] According to other embodiments, the alkyl group is linked to the side chain or terminal amino group of the peptide via an amide bond.

[0041] According to certain embodiments, the alkyl group is a C4 to C30 alkyl group.

[0042] According to certain specific embodiments, the C8 alkyl group (octanoyl group herein) is linked to the side chain or terminal amino group of the Lys residue of the peptide sequence via an amide bond.

[0043] According to certain specific embodiments, the peptide is selected from pGlu-Asn-Trp-Lys(octanoyl)-OH (SEQ ID NO: 1), pGlu-Asn-Trp-Thr-OH (SEQ ID NO: 2), and pharmaceutically acceptable salts or derivatives thereof.

[0044] According to certain embodiments, the peptide has the amino acid sequence shown in SEQ ID NO:1.

[0045] According to certain embodiments, the peptide has the amino acid sequence shown in SEQ ID NO:2.

[0046] According to certain embodiments, the pharmaceutical composition comprises a derivative or salt of a peptide having an amino acid sequence shown in a sequence selected from SEQ ID NO: 1 and SEQ ID NO:2.

[0047] According to certain embodiments, the method comprises administering a derivative or salt of a peptide having an amino acid sequence shown in a sequence selected from SEQ ID NO:1 and SEQ ID NO:2.

[0048] According to certain embodiments, the pharmaceutical composition comprises the sodium salt of the peptide set forth in any one of SEQ ID NO: 1 and SEQ ID NO:2.

[0049] According to certain embodiments, the pharmaceutical composition is formulated for administration via a route selected from the group consisting of topical administration, ocular administration, oral administration, nasal administration, and parenteral administration.

[0050] According to various embodiments, the formulation for topical administration may be selected from a cream, an ointment, a paste, a lotion, a gel or take the form of eye drops.

[0051] According to certain specific embodiments, a cream formulation comprising a peptide selected from SEQ ID NOs: 1 and 2 or a pharmaceutically acceptable salt thereof is provided for topical administration.

[0052] According to certain embodiments, the cream composition comprises 0.1-5% w / w of the peptide. According to other embodiments, the composition comprises 0.5-2% of the peptide. According to other embodiments, the composition comprises about 1% of the peptide.

[0053] According to certain embodiments, the composition according to the present invention has a pH of about 4 to about 8. According to certain embodiments, the composition has a pH of about 4.5 to about 6.5. According to other embodiments, the composition has a pH of about 7 to about 9.

[0054] According to certain 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.

[0055] According to other embodiments, the composition for administration to a subject having an ocular disease or injury is formulated in a form selected from the group consisting of liquid solutions or suspensions for use as eye drops, emulsions, creams, ointments, sprays, and gels.

[0056] According to certain embodiments, the ocular 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.

[0057] According to other embodiments, the pharmaceutical composition is formulated as eye drops, eye ointment, eye spray, eye suspension, eye emulsion, eye solution, eye gel or intravitreal injection.

[0058] According to other embodiments, the inflammatory disease or disorder is an autoimmune disease or disorder.

[0059] According to other embodiments, the autoimmune disease or disorder is selected from rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, lupus and Sjögren's syndrome.

[0060] According to other embodiments, the inflammatory disease or disorder is inflammatory bowel disease.

[0061] According to other embodiments, the inflammatory bowel disease is selected from Crohn's disease, ulcerative colitis and celiac disease.

[0062] According to other embodiments, the inflammatory disease or disorder is a pulmonary inflammatory disease or disorder.

[0063] According to other embodiments, the pulmonary inflammatory disease or disorder is selected from asthma, bronchitis, pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis and cystic fibrosis.

[0064] According to other embodiments, the inflammatory disease or disorder is an inflammatory disease or disorder of the ear.

[0065] According to other embodiments, the inflammatory disease or disorder of the ear is selected from inflammatory symptoms associated with ear infection, otitis media, otitis externa, mastoiditis and otomycomastoiditis.

[0066] According to other embodiments, the administration is ocular administration.

[0067] According to other embodiments, the administration is oral administration.

[0068] According to other embodiments, the administration is nasal administration.

[0069] According to other embodiments, the administration is parenteral administration.

[0070] The present invention successfully addresses the shortcomings of currently known arrangements by providing pharmaceutical compositions for reducing the release or inhibiting the activity of inflammatory cytokines and mediators and for treating diseases or conditions associated with inflammatory cytokines and mediators.

[0071] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice and testing of embodiments of the invention, exemplary methods and / or materials are described below. In the event of a conflict, the present patent specification, including definitions, shall prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Some embodiments of the present invention are described herein by way of example only with reference to the accompanying drawings. With specific reference now to the drawings in detail, it should be emphasized that the details shown are merely examples and are for the purpose of illustrative discussion of embodiments of the present invention. In this regard, the description in conjunction with the drawings may make it apparent to those skilled in the art how embodiments of the present invention may be practiced.

[0073] In said drawings:

[0074] Figure 1 is a bar graph illustrating the effect of ZEP4 (Z) at concentrations ranging from 3.125–100 μg / ml on the amount of TNFα produced by inflammation-induced macrophages. Lines marked with **** indicate statistically significant differences at p<0.0001.

[0075] Figure 2 : is a bar graph illustrating the effect of ZEP4 (Z) (50 μg / ml) on the amount of TNFα produced by inflammation-induced macrophages treated with lipopolysaccharide (LPS, 100 ng / ml) and palmitic acid (PA, 100, 200 or 400 μM). The line marked with **** indicates a statistically significant difference at p < 0.0001.

[0076] Figure 3 is a bar graph illustrating the effect of ZEP3 (Z) (concentration range: 3.125-100 μg / ml) on the amount of TNFα produced by inflammation-induced macrophages. Lines marked with ****, ***, **, *, and ns indicate statistically significant differences at p<0.0001, p<0.001, p<0.01, P<0.1, and not significant, respectively.

[0077] Figure 4 is a bar graph illustrating the effect of ZEP4(Z) (50 μg / ml) on the amount of IL-1β produced by inflammatory nigericin-induced macrophages. Lines marked with **** indicate statistically significant differences at p<0.0001.

[0078] Figure 5 is a bar graph illustrating the effect of ZEP3 on the viability of inflammation-induced macrophages.

[0079] Figure 6 is a bar graph illustrating the protective effect of ZEP3 at different concentrations on the viability of corneal epithelial cells exposed to 70 mM NaCl (black bars) or NHE20 (grey bars) induced by inflammation as determined by LDH assay. Lines marked with **** and * indicate statistically significant differences at p<0.0001 and p<0.05, respectively.

[0080] Figure 7 is a bar graph illustrating the effect of ZEP3 at different concentrations on the amount of IL-6 produced by inflammation-induced corneal epithelial cells exposed to 4-hydroxynonenal (4-HNE) for 24 hrs. Lines marked with * indicate statistically significant differences at p<0.05.

[0081] Figure 8 is a bar graph illustrating the protective effect of ZEP3 at different concentrations on the amount of reactive oxygen species (ROS, measured by DCFDA kit) produced by corneal epithelial cells exposed to 4-HNE for 24 hours, induced by inflammation. Lines marked with ****, ***, and ** indicate statistically significant differences at p<0.0001, p<0.001, and p<0.01, respectively.

[0082] Fig. 9 Depicted is IFN-γ production in normal PBMCs from 4 human donors in response to stimulation with different concentrations of ZEP3, as well as medium, stimulation, and dexamethasone controls. Statistically significant differences are indicated as: *p<0.05, **p<0.01, compared to stimulation controls by one-way ANOVA followed by Dunette's multiple comparisons.

[0083] Fig.10 Depicted are IL-10 production in normal PBMCs from 4 human donors in response to stimulation with different concentrations of ZEP3, as well as media, stimulation, and dexamethasone controls. Statistically significant differences are indicated as: *p<0.05, **p<0.01, compared to stimulation controls by one-way ANOVA followed by Dunette's multiple comparisons.

[0084] Fig.11 Depicted is IL-1β production in normal PBMCs from 4 human donors in response to stimulation with different concentrations of ZEP3, as well as medium, stimulation, and dexamethasone controls. Statistically significant differences are indicated as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, compared to stimulation controls by one-way ANOVA followed by Dunette's multiple comparisons.

[0085] Fig.12 RANTES production in normal PBMCs from 4 human donors in response to stimulation with different concentrations of ZEP3, as well as media, stimulation and dexamethasone controls are represented. Statistically significant differences are indicated as: *p<0.05, compared to stimulation control by one-way ANOVA followed by Dunette's multiple comparisons.

[0086] Fig.13A and 13B It is shown that in the oxazolone-induced atopic dermatitis model in mice, ZEP3, ZEP3Na ( Fig.13A ) or ZEP4( Fig. 13B ) Plot of the evolution of dorsal skin thickness over time in mice topically treated with β-lactamase compared to vehicle and betamethasone controls. Statistically significant differences are indicated as: *p<0.05, **p<0.0, ****p<0.001, compared to stimulated controls by one-way ANOVA followed by Dunette's multiple comparisons.

[0087] Fig.14A and 14B Describes the effects of ZEP3, ZEP3Na ( Fig.14A ) or ZEP4( Fig. 14B ) Evolution of baseline-corrected right ear thickness over time in mice topically treated with , compared with vehicle and betamethasone controls.

[0088] Fig.15A and 15B Representative figures show the effects of ZEP3, ZEP3Na( Fig.15A ) or ZEP4( Fig. 15B ) Evolution of erythema scores over time in mice topically treated with β-lactamase compared to vehicle and betamethasone controls. Statistically significant differences are indicated as: *p<0.05, **p<0.001, ****p<0.0001, compared to stimulated controls by one-way ANOVA followed by Dunette's multiple comparisons.

[0089] Fig.16A and 16B Represents the results of the study on the effect of ZEP3, ZEP3Na( Fig.16A ) or ZEP4( Fig. 16B ) Evolution of scaling scores over time in mice topically treated with , compared to vehicle and betamethasone controls. Statistically significant differences are indicated as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, compared to stimulated controls by one-way ANOVA followed by Dunette's multiple comparisons.

[0090] Fig.17A and 17B It is shown that in the oxazolone-induced atopic dermatitis model in mice, ZEP3, ZEP3Na ( Fig.17A ) or ZEP4( Fig. 17B Relative change in animal body weight from day 8 to day 28 in mice topically treated with ) compared to vehicle and betamethasone controls. DETAILED DESCRIPTION

[0091] The present invention relates to pharmaceutical compositions comprising specific peptides and their salts and derivatives for inhibiting the inflammatory cascade. The inhibition of inflammatory mediators is beneficial in the prevention and treatment of several inflammatory diseases and disorders, such as ocular inflammatory diseases and disorders.

[0092] As exemplified herein, the inhibitory effect on inflammatory cytokine release was demonstrated in vitro in several models including a mouse macrophage cell line, a human monocyte cell line, a human keratinocyte cell line, human peripheral blood mononuclear cells (human PBMCs), and human corneal epithelial cells (HCECs), and in vivo in an inflammatory atopic dermatitis (AD) mouse model.

[0093] The beneficial effects of the peptides on experimental corneal inflammation were confirmed in a human corneal epithelial cell culture model.

[0094] The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

[0095] Before explaining at least one embodiment of the present invention in detail, it should be understood that the application of the present invention is not necessarily limited to the details set forth in the following description or illustrated by the examples. The present invention can have other embodiments or be practiced or implemented in various ways. In addition, it should be understood that the phrases and terms used herein are for the purpose of description and should not be considered as limiting.

[0096] While putting the present invention into practice, the inventors surprisingly found that peptides ZEP3 and ZEP4 and their sodium salt forms reduced the expression of inflammatory cytokines TNFα, IL-1β and IL-6 in macrophages and corneal epithelial cells induced to cause inflammation (Examples 1-7 below), and reduced the expression of IFNγ, IL-10, IL-1β and RANTES in inflammatory stimulated human PBMCs (Example 12 below). In addition, in a mouse inflammation model of atopic dermatitis, ZEP4, ZEP3 and their sodium salt form ZEP3Na significantly reduced clinical lesions in vivo (Example 13 below). The results show that the peptides of the present invention are uniquely capable of inhibiting or preventing inflammation.

[0097] Therefore, according to one aspect of the present invention, there is provided a pharmaceutical composition for reducing the release of or inhibiting the activity of at least one cytokine or mediator involved in the etiology or 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 an amino acid sequence as shown in any one of SEQ ID NOs: 1 and 2, or a pharmaceutically acceptable salt thereof.

[0098] Thus, the present invention provides methods for ameliorating and treating inflammatory diseases associated with excessive release or activity of specific cytokines and mediators.

[0099] According to certain embodiments, the inflammatory disease is a disease or disorder associated with excessive release or activity of at least one cytokine or mediator, wherein the at least one cytokine or mediator is selected from IFN-γ, IL-1β, IL-10, TNFα, IL-6, ROS and RANTES.

[0100] As used herein, the phrase "inflammatory disease or disorder" refers to a disease, condition or disorder associated with inflammation. As used herein, the term "inflammation" refers to the process by which a subject's immune system coordinates a response to tissue injury, infection, antigenic challenge, and the like. Inflammation may be associated with increased blood supply to tissues, increased capillary permeability in tissues, and / or increased migration of leukocytes into tissues.

[0101] Proinflammatory cytokines are mainly produced by activated macrophages and are involved in the upregulation of inflammatory responses.

[0102] Inflammation can be diagnosed by elevated levels of inflammatory cytokines such as, but not limited to, TNFα, IL-1-α, IL-1β, IFN-γ, and IL-6 in a biological sample obtained from the subject. The biological sample can be, for example, tears, blood, serum, plasma, urine, sputum, saliva, feces, semen, cerebrospinal fluid, bone marrow, lymph or CSF, external secretions of the skin, respiratory, intestinal and urogenital tracts, milk, any human organ or tissue, any sample obtained by lavage, pleural effusion, samples of in vitro or ex vivo cell culture, and cell culture components.

[0103] Reactive oxygen species (ROS) are key signaling molecules that play an important role in the development of inflammatory disorders. Increased ROS production by polymorphonuclear neutrophils (PMNs) at sites of inflammation can cause endothelial dysfunction and tissue damage. Under inflammatory conditions, oxidative stress generated by PMNs leads to the opening of interendothelial junctions and promotes the migration of inflammatory cells across the endothelial barrier.

[0104] According to certain embodiments of the present invention, the inflammatory disease or disorder is an ocular inflammatory disease or disorder. The ocular inflammatory disease or disorder may be, but is not limited to, uveitis, dry eye syndrome, inflammatory symptoms associated with viral, bacterial or fungal ocular infections, allergic eye disease, keratitis, conjunctivitis, meibomian gland dysfunction and Sjögren's syndrome.

[0105] In one embodiment, the ocular inflammatory disease or disorder is uveitis. As used herein, the term "uveitis" refers to inflammation of the uvea, which is the layer between the sclera and the retina and includes the iris, ciliary body, and choroid. Uveitis is also commonly referred to as iritis, pars planitis, choroiditis, chorioretinitis, anterior uveitis, and posterior uveitis. The most common form of uveitis is anterior uveitis, which involves inflammation of the front part of the eye that is usually isolated from the iris. This condition is commonly referred to as iritis.

[0106] In one embodiment, the ocular inflammatory disease or condition is dry eye syndrome. The phrase "dry eye syndrome (DES)" used herein refers to a condition in which the tear composition is altered by a diseased or dysfunctional lacrimal gland functional unit. There is evidence that inflammation causes structural changes in the tear glands. Changes in tear composition caused by lacrimal gland dysfunction, increased evaporation and / or poor clearance have a pro-inflammatory effect on the ocular surface. This inflammation is partly responsible for irritation symptoms, ocular surface epithelial diseases, and changes in corneal epithelial barrier function in dry eye. Anti-inflammatory therapies for DES target one or more inflammatory mediators / pathways identified in dry eye. DES is also referred to as dry eye disease (DED), keratoconjunctivitis sicca, and keratitis sicca by those skilled in the art.

[0107] In one embodiment, the ocular inflammatory disease or condition is associated with Sjögren's syndrome. When used herein, the phrase "Sjögren's syndrome" refers to a systemic autoimmune inflammatory disorder characterized by reduced tearing, dry mouth and other mucous membrane dryness, and is usually accompanied by autoimmune rheumatic disorders such as rheumatoid arthritis. Dryness of the eyes and mouth is the most common symptom of this syndrome. The symptoms may occur alone, or with symptoms associated with rheumatoid arthritis or other connective tissue diseases. There may be associated enlargement of the salivary glands. Other organs may be affected. The syndrome may be associated with rheumatoid arthritis, systemic lupus erythematosus (SLE), scleroderma, polymyositis and other diseases.

[0108] In certain embodiments, the ocular inflammatory disease or condition results from surgery involving the eye, such as corneal transplant / keratoplasty, corneal repair surgery, lamellar transplant, or selective endothelial transplant.

[0109] In certain embodiments, the ocular inflammatory disease or disorder affects the ocular surface, such as, but not limited to, corneal ocular surface inflammatory disorders, corneal neovascularization, keratitis including peripheral ulcerative keratitis and microbial keratitis, conjunctivitis, or pemphigoid syndrome.

[0110] In certain embodiments, the ocular inflammatory disease or condition is an autoimmune disorder affecting the eye, such as, but not limited to, sympathetic ophthalmopathy, Vogt-Koyanagi Harada (VKH) syndrome, birdshot chorioretinopathy, ocular cicatricial pemphigoid (OCP), Sjögren's syndrome, or Fuchs' metachronous iridocyclitis.

[0111] According to certain embodiments of the present invention, the inflammatory disease or disorder is an autoimmune disease or disorder. The autoimmune disease or disorder can be, but is not limited to, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, pemphigus vulgaris, ankylosing spondylitis, juvenile idiopathic arthritis, lupus and Sjögren's syndrome.

[0112] In one embodiment, the autoimmune disease or disorder is rheumatoid arthritis. As used herein, the phrase "rheumatoid arthritis" refers to a chronic autoimmune disorder in which multiple joints are inflamed. Inflammation of the synovial joint lining is accompanied by joint pain and stiffness, and often leads to bone and joint destruction, deformity, disability, and even death.

[0113] In one embodiment, the autoimmune disease or disorder is lupus. When used in this article, the term "lupus" refers to a chronic inflammatory autoimmune disorder known as lupus erythematosus, which may affect many organ systems, including skin, joints and internal organs. Lupus is a general term, including many specific types of lupus, including systemic lupus, lupus nephritis and lupus encephalitis. In systemic lupus erythematosus (SLE), the natural defense of the human body turns to the human body, and the deteriorated immune cells attack human tissues. Antibodies that can react with the blood cells, organs and tissues of the human body may be produced. This reaction causes immune cells to attack the affected system, thereby producing a chronic disease. Lupus nephritis, also known as lupus glomerular disease, is a renal disorder, which is usually a complication of SLE, and is characterized by the damage of glomeruli and the progressive loss of renal function. Lupus encephalitis refers to another complication of SLE, which is an inflammation of the brain and / or central nervous system.

[0114] In one embodiment of the present invention, the inflammatory disease or disorder is osteoarthritis (OA). OA is also known as hypertrophic osteoarthritis, osteoarthrosis and degenerative joint disease. OA is a chronic degenerative disease of skeletal joints that affects specific joints in adults of all ages, typically the knee, hip, hand joints and spine. OA is characterized by the following manifestations, including degeneration and thinning of articular cartilage and the occurrence of accompanying "ulcers" or depressions, osteophyte formation, marginal bone hypertrophy, synovial changes and enlargement of affected joints. In addition, osteoarthritis is accompanied by pain and stiffness, especially after long-term activities. The peptides of the present invention, their salts and derivatives, and pharmaceutical compositions comprising them can be used to treat osteoarthritis. The characteristic imaging features of osteoarthritis include narrowing of the joint space, subchondral sclerosis, osteophytes, subchondral cyst formation, and loose bone bodies (or "joint loose bodies").

[0115] According to some embodiments of the present invention, the inflammatory disease or disorder is inflammatory bowel disease. The inflammatory bowel disease may be, but is not limited to, Crohn's disease, ulcerative colitis and celiac disease.

[0116] According to certain embodiments of the present invention, the inflammatory disease or disorder is a pulmonary inflammatory disease or disorder. The pulmonary inflammatory disease or disorder 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.

[0117] According to some embodiments of the present invention, the inflammatory disease or disorder is an inflammatory disease or disorder of the ear. The inflammatory disease or disorder of the ear may be, but is not limited to, inflammatory symptoms associated with ear infection, otitis media, otitis externa, mastoiditis and otomytomastoiditis.

[0118] The present invention also provides a pharmaceutical composition for treating diseases or disorders associated with excessive release of inflammatory mediators and chemokines, including but not limited to ROS, RANTES and monocyte chemoattractant protein-1 (MCP-1). Inflammatory diseases associated with the release of ROS that can be treated using the composition of the present invention include but are not limited to pulmonary diseases, cardiovascular diseases, vascular calcification associated with kidney diseases and inflammatory metabolic diseases.

[0119] The chemokines RANTES and MCP-1 have been implicated in allergic lung inflammation, lung leukocyte infiltration, bronchial hyperresponsiveness, and recruitment of eosinophils in asthma pathogenesis.

[0120] The term "treat" refers to inhibiting, preventing or arresting the development of a disease or disorder and / or causing the reduction, remission or regression of a disease or disorder.

[0121] The peptides, derivatives and salts used in the compositions and methods of the invention can be synthesized using any method known in the art, including but not limited to solid phase and liquid phase peptide synthesis. Certain peptides used in the compositions of the invention can be produced using recombinant methods or a combination of recombinant and synthetic methods.

[0122] In one embodiment of the present invention, the peptide is pGlu-Asn-Trp-Lys(octanoyl)-OH (SEQ ID NO: 1; hereinafter referred to as "ZEP3"), wherein pGlu is pyroglutamic acid. The peptide ZEP3 can be produced, for example, by the procedure described in US Pat. No. 7,220,725.

[0123] In one embodiment of the present invention, the peptide is pGlu-Asn-Trp-Thr-OH (SEQ ID NO: 2; hereinafter referred to as "ZEP4"). The peptide ZEP4 can be produced, for example, by the following procedure:

[0124] The synthesis of peptide ZEP4 was performed by sequential synthesis of 9-fluorenylmethoxycarbonyl (Fmoc) amino acids on a solid support of chlorotrityl chloride resin (CTC). CTC resin (125 gr) was loaded with Fmoc-threonine (tert-butyl; 79 gr) and diisopropylamine (DIPEA; 160 gr) acting as a coupling agent between the amino acid and the solid support. The Fmoc protecting group was removed by a mixture of 25% piperidine, the resin-peptide was filtered and washed with dimethylformamide (DMF). The second amino acid Fmoc-Trp (85 gr) was activated with a mixture of 3-[bis(dimethylamino)methylureido]-3H-benzotriazole-1-oxide, hexafluorophosphate (HBTU) / hydroxybenzothiazole (OHBT) and coupled to the first amino acid by adding DIEA. The Fmoc group was removed as described above, the resin-peptide was filtered and washed with dimethylformamide (DMF). The third amino acid Fmoc-Asn(trt) (119 gr) was activated with HBTU / HOBT and coupled by adding DIEA. The Fmoc group was removed as described above, and the resin-peptide was filtered and washed with dimethylformamide (DMF). The fourth amino acid pGlu (26 gr) was activated with HBTU / HOBT and coupled by adding DIEA.

[0125] The peptide-resin was washed thoroughly with DMF, then IPA and dried under reduced pressure. The peptide was cleaved from the resin and the protecting groups of Thr and Asn were also cleaved by TFA (95%) and triisopropylsilane (TIS) (5%) at room temperature for 2 hr. The peptide was precipitated by adding methyl tert-butyl ether (MTBE), filtered and dried (46 gr obtained).

[0126] The crude product (46 gr) was dissolved in a mixture of acetonitrile (ACN) / water, loaded on a preparative HPLC system (4", RP C-18 100-120A pore size) and purified using the following gradient system: phase A - 0.1% TFA in water; phase B - ACN. Elution was performed by gradually increasing phase B (3% to 33%) over 45 min. Fractions with a purity higher than 97% were collected. The combined fractions were eluted on the same HPLC system using the following gradient: phase A: 0.2% acetic acid; phase B: ACN. Elution was performed by gradually increasing phase B (10% to 40%) over 30 min. Fractions with a purity higher than 97% were collected, combined and freeze-dried (yielding 29 gr). The final product had a MW (MS) of 530.5 and a purity of 97.3% (HPLC).

[0127] Also included within the scope of the present invention are salts and derivatives of the peptides used in the disclosed compositions and methods.

[0128] When used in this article, the term "salt" refers to both the salt of the carboxyl group of the peptide molecule and the acid addition salt of its amino or guanidinium group. The salt of the carboxyl group can be formed by means known in the art, and includes inorganic salts such as sodium, calcium, ammonium, iron or zinc salts, etc., and salts with organic bases such as salts formed with amines such as triethanolamine, piperidine, procaine, etc. Acid addition salts include, for example, salts with inorganic acids such as acetic acid or oxalic acid. The salts described here also include ionic components added to the peptide solution to enhance hydrogel formation and / or calcium salt mineralization.

[0129] As used herein, "derivatives" of the peptides of the invention encompass derivatives that can be prepared from functional groups present as side chains on the residues or the N- or C-terminal groups by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the peptide, do not provide toxic properties to the composition containing it, and do not adversely affect its immunogenic properties.

[0130] These derivatives may include, for example, aliphatic esters of carboxyl groups, amides of carboxyl groups produced by reaction with ammonia or with primary or tertiary amines, N-acyl derivatives of the free amino groups of amino acid residues formed by reaction with acyl moieties (e.g., alkanoyl or aroyl groups), or O-acyl derivatives of the free hydroxyl groups (e.g., the free hydroxyl groups of seryl or threonyl groups) formed by reaction with acyl moieties.

[0131] 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).

[0132] ZEP3 sodium salt can be produced, for example, by the following procedure:

[0133] ZEP3 (3.1 g) was dissolved in NaHCO 3 The solution was injected into an HPLC ion exchange column (2.5 x 22 cm Luna C18, 100A, 15 microns) and eluted by the following gradient: Mobile phase A: 2 mM NaHCO 3 Aqueous solution; Mobile phase B: CH 3 CN / H 2 2 mM NaHCO in O(8 / 2) 3 ; and mobile phase: C: 100 mM NaHCO 3Aqueous solution. Loading per run: up to 5% (W / W% peptide / stationary phase). Flow rate: 4.8 cm / min (24 ml / min). Gradient program is as follows: 20 min phase C; 5 min phase A; 18 min phase B; and 7 min phase C. Fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile (110 g / l) and then freeze-dried [yield 2.2 g (71%)]. The final product has a purity of 99.7% (HPLC), a sodium content of 3.1% and a solubility in water of 50 mg / ml.

[0134] In one embodiment of the present invention, the peptide is the sodium salt of the peptide shown in SEQ 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:

[0135] Dissolve ZEP4 (5 g) in NaHCO 3 The solution was injected into an HPLC ion exchange column (2.5 x 22 cm Luna C18, 100A, 15 microns) and eluted by the following gradient: Mobile phase A: 2 mM NaHCO 3 Aqueous solution; Mobile phase B: CH 3 CN / H 2 2 mM NaHCO in O(8 / 2) 3 ; and mobile phase: C: 100 mM NaHCO 3 Aqueous solution. Loading per run: up to 5% (W / W% peptide / stationary phase). Flow rate: 4.8 cm / min (24 ml / min). Gradient program is as follows: 20 min phase C, then 5 min phase A, then 20 min phase B and 10 min phase C. Fractions containing the product are collected and concentrated under reduced pressure to remove acetonitrile (110 g / l) and then freeze-dried [4 g (80%) are obtained]. The final product has a purity of 97.5% (HPLC), a sodium content of 2.5% and a solubility in water of 50 mg / ml.

[0136] The peptides of the invention can be used in therapy per se or as part of a pharmaceutical composition (active ingredient).

[0137] As used herein, a "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0138] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably to refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Adjuvants are included under these phrases.

[0139] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and starch types, polysaccharides, cyclodextrins, suspending agents such as polyvinyl pyrrolidone, 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.

[0140] Techniques for the formulation and administration of drugs may be found in the most recent edition of Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0141] Suitable routes of administration may, for example, include ocular, topical, oral, rectal, transmucosal, nasal, enteral or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injections.

[0142] The pharmaceutical composition of the present invention can be manufactured by methods known in the art, such as conventional mixing, dissolving, suspending, solubilizing, granulating, sugar-coating, grinding, emulsifying, encapsulating, embedding, spray-drying or freeze-drying methods.

[0143] Thus, pharmaceutical compositions for use according to the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising pharmaceutically acceptable excipients and auxiliaries which facilitate processing of the active ingredients into preparations. Suitable formulations depend on the chosen route of administration.

[0144] The pharmaceutical compositions described herein can be formulated for ocular administration. The phrase "ocular administration" used herein refers to administration of the peptide or pharmaceutical composition of the present invention in the external eye (e.g., conjunctival sac) or in the vitreous. The dosage form suitable for ocular administration can be, but is not limited to, eye drops, eye ointments, eye sprays, eye suspensions, eye emulsions, eye solutions, eye gels, or intravitreal injections.

[0145] The pharmaceutical compositions described herein can be formulated for topical administration. Dosage forms suitable for topical administration can be, but are not limited to, drops, lotions, gels, ointments, emulsions, suspensions, lotions, sprays, atomized liquids, suppositories, creams, powders, foams, crystals and liposomes.

[0146] For injection, the active ingredients of the pharmaceutical composition can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution or physiological saline buffer. One route of administration suitable for the pharmaceutical composition of the present invention is subperiosteal injection as described in U.S. Patent No. 6,525,030 to Erikkson. For transmucosal administration, penetrants suitable for the barrier to be penetrated are used in the formulation. Such penetrants are well known in the art.

[0147] For oral administration, the pharmaceutical composition can be easily formulated by combining the active compound with a pharmaceutically acceptable carrier known in the art. Such a carrier can prepare the pharmaceutical composition into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, emulsions, etc., for oral ingestion by patients. Pharmaceutical preparations for oral use can be manufactured as follows: using solid excipients, and optionally grinding the resulting mixture and processing the mixture of particles after adding suitable adjuvants when necessary to obtain tablets or dragee cores. Suitable excipients are specifically fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose; and / or physiologically acceptable polymers such as polyvinyl pyrrolidone (PVP). If necessary, disintegrants such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or its salts such as sodium alginate can be added. As used herein, the term "oral administration" includes administration of the drug compound to any oral surface, including the tongue, gums, palate, sublingual or other buccal surfaces.

[0148] The dragee cores are provided with a suitable coating. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talcum powder, polyvinyl pyrrolidone, carbomer gel, polyethylene glycol, titanium dioxide, varnish solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablet or dragee coatings for identification or characterization of different combinations of active compound agents.

[0149] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin and sealed soft capsules made of gelatin and plasticizers such as glycerol or sorbitol. The push-fit capsules can contain a mixture of the active ingredient with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and an optional stabilizer. In the soft capsule, the active ingredient can be dissolved or suspended in a suitable liquid such as a fatty oil, liquid paraffin or liquid polyethylene glycol. In addition, a stabilizer can be added. All preparations for oral administration should be in a dosage suitable for the selected route of administration.

[0150] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0151] For administration by nasal inhalation, the active component used according to the present invention is presented in the form of an aerosol spray, using suitable propellants such as dichlorodifluoromethane, trichloromonofluoromethane, dichlorotetrafluoroethane or carbon dioxide, and is conveniently delivered from a pressurized package or aerosol. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver the metered amount. Capsules and cartridges such as gelatin used in a dispenser can be prepared, containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0152] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example by bolus injection or continuous infusion. The injection preparations can be present in unit dosage form, for example in ampoules or multi-dose containers, and optionally preservatives are added. The compositions can be suspensions, solutions or emulsions in oily or aqueous media, and can contain preparatons such as solubilizers, suspending agents, stabilizers and / or dispersants.

[0153] Pharmaceutical compositions for parenteral administration include aqueous solutions of active agents in water-soluble form. In addition, the suspension of the active ingredient can be prepared into suitable oily or water-based injection suspensions. Suitable lipophilic solvents or media include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglyceride emulsions or liposomes. Aqueous injection suspensions can include materials that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextrose. Optionally, the suspension can also include a suitable stabilizer or an agent that improves the solubility of the active ingredient to allow the preparation of a high concentration solution.

[0154] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, a sterile, pyrogen-free, water based solution, before use.

[0155] The pharmaceutical compositions of this invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, eg, conventional suppository bases such as cocoa butter or other glycerides.

[0156] Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredient is contained in an amount effective to achieve the desired purpose. More specifically, a therapeutically effective amount means an amount of the active ingredient that is effective to prevent, alleviate or ameliorate the symptoms of a disease or disorder in the subject to be treated.

[0157] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0158] For any formulation used in the methods of the invention, the therapeutically effective amount or dosage can be estimated first from in vitro and cell culture assays. For example, a dosage can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful dosages in humans.

[0159] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined in cell culture or experimental animals by standard in vitro pharmacology procedures. The data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a dosage range for human use. The dosage can be changed according to the dosage form used and the route of administration adopted. Each physician can select the exact formulation, route of administration and dosage according to the patient's condition. (See, for example, Fingl et al., 1975, in The Pharmacological Basis of Therapeutics Ch. 1p. 1).

[0160] Dosage amount and interval can be adjusted individually to achieve a level of active ingredient sufficient to achieve the minimum effective concentration (MEC). The MEC varies for each preparation but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma or tissue concentrations.

[0161] Depending on the severity and responsiveness of the condition to be treated, administration may be single or multiple administrations, and the course of treatment may last from several days to several weeks or until a diminution of the disease state is achieved.

[0162] The amount of composition to be administered will, of course, be dependent on the subject to be treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like.

[0163] Therefore, if necessary, the composition and / or article of some embodiments of the present invention can be present in a package or a dispenser device such as a FDA-approved medicine box, which can contain one or more unit dosage forms containing active ingredients. The package can, for example, include metal or plastic foil, such as a blister package. The package or dispenser device can be accompanied by instructions for administration. The package or dispenser can also include a notice accompanying the container, which takes the form specified by the government agency that regulates drug production, use or sales, and the notice reflects the approval of the form of the composition or human or veterinary administration by the agency. For example, such notices can be prescription drug labels approved by the U.S. Food and Drug Administration or product instructions for approval. As further described above, the composition comprising the preparation of the present invention prepared in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container (e.g., lyophilized vial), and added with a label for treating a specified disease.

[0164] The peptides or pharmaceutical compositions of the present invention can be used to inhibit the release or activity of inflammatory cytokines and mediators and treat inflammatory diseases or disorders by providing a therapeutically effective amount of the peptides or pharmaceutical compositions of the present invention to a subject in need thereof.

[0165] As used herein, the phrase "subject in need thereof" refers to a male or female mammalian subject (e.g., human) diagnosed with an inflammatory disease or disorder. In certain embodiments, the term encompasses individuals at risk of developing an inflammatory disease or disorder. The subject may be of any gender or age, including neonates, infants, young children, teenagers, young adults, adults, and the elderly.

[0166] As used herein, the term "about" refers to ± 10%.

[0167] The terms "including", "comprising", "having" and variations thereof mean "including but not limited to".

[0168] The term "consisting of" means "including and limited to."

[0169] The term "consisting essentially of" means that a composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially change the basic and novel characteristics of the claimed composition, method or structure.

[0170] As used herein, the singular includes plural referents unless the context clearly states otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0171] Throughout this application, various embodiments of the present invention may be presented in the format of a range. It should be understood that the description of the range format is only for the purpose of convenience and simplicity, and should not be interpreted as a rigid limitation on the scope of the invention. Therefore, the description of the range should be considered as specifically disclosing all possible sub-ranges and each numerical value within the range. For example, the description of the range from 1 to 6 should be considered as specifically disclosing sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and each number within the range such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, this applies.

[0172] Whenever a numerical range is indicated herein, it is intended to include any cited value (fractional or integer) within the indicated range. The phrases "range between a first indicated number and a second indicated number" and "range from a first indicated number to a second indicated number" are used interchangeably herein and are intended to include the first and second indicated numbers and all fractional and integer values ​​therebetween.

[0173] As used herein, the term "method" refers to manners, means, techniques, and procedures for accomplishing a given task, including but not limited to those manners, means, techniques, and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine, or readily developed from known manners, means, techniques, and procedures.

[0174] It should be understood that certain features of the invention described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for the sake of brevity may also be provided in any other described implementation of the invention separately or in any suitable subcombination or where appropriate. Certain features described in the context of various different embodiments should not be considered essential features of those embodiments unless the embodiments are inoperable without those elements.

[0175]

[00136] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section herein find experimental support in the following examples.

[0176] Example

[0177] Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

[0178] Example 1 - Effect of ZEP4 on TNFα expression in inflammation-induced macrophages

[0179] Materials and methods

[0180] The peptide pGlu-Asn-Trp-Thr-OH (hereinafter referred to as "ZEP4", SEQ ID NO: 2) was synthesized as described above.

[0181] TNFα expression in B6 macrophages

[0182] 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 at 37°C and 5% CO. 2 Incubate overnight at 4 °C. After incubation, the culture medium in some experimental groups was replaced with fresh culture medium containing 100 ng / mL LPS, and then incubated for 3 hours. Then, the culture medium in some experimental groups (containing LPS or not containing LPS) was replaced with fresh culture medium containing ZEP4 with a concentration in the range of 3.125 to 100 μg / ml, and then incubated for 1 hour. Next, palmitic acid (PA, 200 μM) was added to the suitable wells, and the plate was continued to be incubated for 24 hours, and the amount of TNFα in each well was then measured. Table 1 lists different treatments and control groups. The reduction of TNFα production in the treatment group was calculated relative to the cells treated with LPS and PA (CLP200).

[0183] result

[0184] Figure 1 As shown in Table 1 below, ZEP4 significantly reduced the amount of TNFα produced by inflammation-induced macrophages treated with LPS and palmitic acid for 24 hrs. A ZEP4 concentration of 3.125 μg / ml resulted in a 70.6% reduction in TNFα expression. The most effective ZEP4 concentration was 50 μg / ml, which resulted in a 77.0% reduction in TNFα expression (p<0.0001).

[0185] Table 1

[0186] Effect of ZEP4 concentration on TNFα expression in macrophages treated with LPS and palmitic acid

[0187]

[0188] Example 2 - Effect of ZEP4 on TNFα expression in inflammation-induced macrophages

[0189] Materials and methods

[0190] Peptide ZEP4 (SEQ ID NO: 2) was synthesized as described above.

[0191] TNFα expression in B6 macrophages

[0192] 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 at 37°C and 5% CO. 2 Incubate overnight at 4 °C. After incubation, the culture medium was replaced with a fresh culture medium containing 100 ng / mL LPS, and then incubated for 3 hours. The LPS-containing culture medium in the treatment group was then replaced with a fresh culture medium containing 50 μg / ml ZEP4, and then incubated for 1 hour. Then, aliquots (100, 200 or 400 μM) of palmitic acid were added to suitable wells. The plate was continued to be incubated for 24 hours, and the amount of TNFα in each well was measured. Table 2 lists different treatments and control groups. The reduction of TNFα production in the treatment group was calculated relative to the cells treated with LPS and PA, according to the PA concentration.

[0193] result

[0194] Figure 2 As shown in Table 2 below, palmitic acid concentrations of 100, 200 and 400 μM induced macrophage TNFα expression of 416, 462 and 593 pg / mL, respectively. ZEP4 at a concentration of 50 μg / ml significantly reduced macrophage TNFα expression at all palmitic acid concentrations (****p<0 / 0001).

[0195] Table 2

[0196] Effect of ZEP4 (50 μg / ml) on TNFα expression in macrophages treated with LPS (100 ng / ml) and palmitic acid (100, 200, and 400 μM)

[0197] Example 3 - Effect of ZEP3 on TNFα expression in inflammation-induced macrophages

[0198] Materials and methods

[0199] The peptide pGlu-Asn-Trp-Lys(octanoyl)-OH (hereinafter referred to as "ZEP3", SEQ ID NO: 1) was synthesized as described in US Pat. No. 7,220,725.

[0200] TNFα expression in B6 macrophages

[0201] 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 at 37°C and 5% CO. 2Incubate overnight at 4 °C. After incubation, the culture medium in some experimental groups was replaced with fresh culture medium containing 100 ng / mL LPS, and then incubated for 3 hours. Then, the culture medium in some experimental groups (containing LPS or not containing LPS) was replaced with fresh culture medium containing ZEP3 with a concentration in the range of 3.125 to 100 μg / ml, and then incubated for 1 hour. Next, palmitic acid (200 μM) aliquots were added to suitable wells, and the plate was continued to be incubated for 24 hours, and the amount of TNFα in each well was then measured. Table 3 lists different treatments and control groups. The reduction of TNFα production in the treatment group was calculated relative to the cells treated with LPS and PA.

[0202] result

[0203] Figure 3 As shown in Table 3 below, ZEP3 significantly reduced TNFα expression in macrophages treated with LPS and palmitic acid. The most effective ZEP3 concentration under the experimental conditions was 3.125 μg / ml (TNFα expression was reduced by 82.9%; p<0.0001). ZEP3ED 50 It was estimated to be 51.94 μg / mL.

[0204] Table 3

[0205] Effect of ZEP3 concentration on TNFα expression in macrophages treated with LPS and palmitic acid

[0206]

[0207] Example 4 - Effect of ZEP4 on IL-1β expression in nigericin-activated macrophages

[0208] Materials and methods

[0209] Peptide ZEP4 (SEQ ID NO: 2) was synthesized as described above.

[0210] IL-1β expression in nigericin-activated macrophages

[0211] 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 at 37°C and 5% CO. 2The plates were incubated overnight at 4 °C. After overnight incubation, the culture medium was replaced with fresh culture medium containing 100 ng / mL LPS. After 3 hours, for the treated wells, the LPS-containing culture medium was replaced with fresh culture medium containing 50 μg / ml ZEP4. After 1 hour of incubation, aliquots of nigericin (20 mM) were added to the ZEP4-treated wells and the LPS control wells. The plates were incubated for a further 24 hours and the amount of IL-1β in each well was measured. Table 4 lists the different controls and treatment groups. The reduction in TNFα production in the treatment groups was calculated relative to cells treated with LPS and nigericin (LPS+N).

[0212] result

[0213] Figure 4 As shown in Table 4 below, ZEP4 at a concentration of 50 μg / ml reduced IL-1β release from nigericin-activated macrophages by 53.8% (****p<0.0001).

[0214] Table 4

[0215] Effect of ZEP4 on IL-1β release from nigericin-activated macrophages

[0216]

[0217] Example 5 - Effect of ZEP3 on the viability of inflammation-induced macrophages

[0218] Materials and methods

[0219] 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 with LPS and palmitic acid

[0220] 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 at 37°C and 5% CO. 2 Incubate overnight at 4 °C. After overnight incubation, the culture medium was replaced with a fresh culture medium containing 100 ng / mL LPS, and then incubated for 3 hours. The LPS culture medium in the treatment group was then replaced with a fresh culture medium containing ZEP3 at a concentration of 50 μg / ml, and then incubated for 1 hour. Next, palmitic acid (100, 200 or 400 μM) was added to the appropriate wells. The plate was continued to be incubated for 24 hours, and the viability of the cells was estimated using an LDH cytotoxicity assay kit. Table 5 lists different treatments and control groups.

[0221] result

[0222] Figure 5As shown in Table 5 below, ZEP3 had no significant effect on the viability of inflammation-induced macrophages under the experimental conditions.

[0223] Table 5

[0224] Effect of ZEP3 on the viability of B6 macrophages activated with LPS and palmitic acid

[0225] Example 6 - Effect of ZEP3 pretreatment on TNFα expression in inflammation-induced macrophages

[0226] Materials and methods

[0227] Peptide ZEP3 (SEQ ID NO: 1) was synthesized as described in US Pat. No. 7,220,725.

[0228] TNFα expression in B6 macrophages

[0229] 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 at 37°C and 5% CO. 2 After overnight incubation, the medium was replaced with fresh medium containing 50 μg / ml ZEP3. After 1 hour of incubation, 100 ng / mL LPS was added to the appropriate wells, the plates were incubated for an additional 24 hours, and then the amount of TNFα in each well was measured.

[0230] result

[0231] Table 6 below shows that pre-treatment of macrophages with ZEP3 prior to LPS induction resulted in a 46.7% reduction in TNFα production by the cells (**p<0.05).

[0232] Table 6

[0233] Effect of ZEP3 pretreatment on TNFα expression in LPS-treated macrophages

[0234]

[0235] Example 7 - Anti-inflammatory effect of ZEP3 in corneal epithelial cell culture dry eye model

[0236] The anti-inflammatory effect of the peptides in experimental corneal inflammation was tested in a cell culture model using various inflammasome inducers such as LPS, 4-HNE, nigericin, etc., to establish a dry eye disease (DED) model. Using a dose-response model, human primary corneal epithelial cells were pretreated with the peptides and then exposed to DED inducers. The expression of IL-6, cell viability, and ROS production were measured.

[0237] Materials and methods

[0238] Human primary corneal epithelial cells (PCS-700-010; HCEC) were treated with ZEP3 at concentrations ranging from 3 to 12 μg / ml for 2 hours and then exposed to LPS, 4-HNE or nigericin for 24 hours. After incubation, the levels of the proinflammatory cytokine IL-6, cell viability and reactive oxygen species (ROS) production in cell cultures were measured.

[0239] result

[0240] like Figure 7 As shown in , IL-6 levels in cell cultures decreased from 52.45±0.9121 pg / mL in untreated cell cultures to 38.08±1.368 pg / mL in ZEP3-treated cells (27.4% decrease; P<0.05).

[0241] like Figure 8 As shown in , ROS levels in cell cultures (reported as relative fluorescence units; RFU) decreased from 27043±952 RFU in untreated cell cultures to 13000±800 RFU in cell cultures treated with ZEP3 (51.9% decrease; P<0.0001).

[0242] like Figure 6 As shown in , the cell death density decreased from 21.125±.942% in untreated cell cultures to 3.625±1.092% in cell cultures treated with ZEP3 (82.8% decrease; P<0.0001).

[0243] In summary, the ZEP3 peptide effectively reduced corneal inflammation, restored HCEC viability and reduced ROS production, factors that are important in the severity of DEA. The results provide a basis for the use of this and similar peptides to prevent and treat DED caused by different pathologies.

[0244] Examples 1-7 above show that peptides ZEP3 (SEQ ID NO: 1) and ZEP4 (SEQ ID NO: 2) reduce the expression of inflammatory cytokines TNFα, IL-1β and IL-6 in inflammation-induced macrophages and corneal epithelial cells. In addition, the peptides are able to restore the viability of cells exposed to inflammatory stress. The results show that the peptides of the present invention are able to effectively inhibit or prevent inflammation in macrophages and corneal epithelial cells.

[0245] Example 8 - Effects of ZEP3, ZEP3 sodium salt and ZEP4 on endotoxin-induced uveitis (EIU) in rats

[0246] The model was established according to De Vos et al., Exp Eye Res 61:667-675, 1995. In male Lewis rats weighing 180 to 200 g, EIU was induced by footpad injection of 200 μg LPS diluted in 0.1 mL sterile water. The animals were then randomly divided into (4 peptides) x (three concentrations) = 12 groups. An untreated induced group was used as an induced control.

[0247] Animals were examined using a slit lamp biomicroscope 24 hours after, corresponding to the peak severity of EIU. Clinical ocular inflammation in each eye was scored using a scale of 0 to 7 as described below: iris hyperemia and cells in the anterior chamber 0-2 (0 = no sign; 1 = mild; 2 = severe), and redness, miosis, and hypopyon were scored, with 0 being no sign and 1 being present. The highest possible score was 7 (the sum of the scores for the 5 parameters).

[0248] Example 9 - Effects of ZEP3, ZEP3 sodium salt and ZEP4 in a controlled environment chamber (CEC) dry eye mouse model

[0249] The model establishment procedure is described in Barabino et al. (IOVS 46:2766–2771, 2005). Briefly, 8- to 12-week-old BALB / c mice were placed in a controlled environment chamber (CEC) where relative humidity (RH), temperature (T), and air flow (AF) were regulated and monitored. The mice were randomized into groups and treated with ZEP3, ZEP sodium salt, and ZEP4 (test peptide). An untreated induced group was used as a control. The mice were then placed in a CEC and exposed to specific environmental controlled conditions (RH = 18.5% ± 5.1%, AF = 15 L / min, T = 21–23°C) for 3, 7, 14, and 28 days. Control mice were kept in a normal environment (RH = 50%–80%, no AF, T = 21–23°C) for the same length of time. Tear production using a cotton thread test, corneal fluorescein staining (score 0–15), and goblet cell density in the upper and lower conjunctiva were measured by an observer wearing a mask.

[0250] Example 10 - Effects of ZEP3, ZEP3 sodium salt and ZEP4 in a scopolamine-induced dry eye rat model

[0251] Male Sprague-Dawley rats weighing between 300 g and 350 g were used. Dry eye was induced using scopolamine (Sigma-Aldrich, St. Louis, Mo.) via an osmotic pump (2ML4) filled with scopolamine and implanted subcutaneously in the dorsal midline region between the shoulder blades. The animals were anesthetized in a 99.9% isoflurane USP (Abraxis Bioscience, Richmond Hill, Ontario) chamber prior to the pump implantation surgery and prior to all endpoint testing. Scopolamine was delivered at a rate of 12.5 mg / day and data were evaluated on day 14.

[0252] A sterile solution of 0.175 g / mL scopolamine hydrobromide (Sigma-Aldrich, St. Louis Mo.) was prepared in saline (0.9%) and filtered through a 0.22 um syringe tip filter (Millex-GC, Millipore Corp., Bedford, Mass.). 2 mL of 4% hyoscine was added according to the manufacturer's instructions. The pump was loaded with 2 mL of 0.175 g / mL scopolamine solution.

[0253] The groups of rat eyes tested were as follows: Group 1: control rats (n=12 eyes from 6 rats). Group 2: rats (n=12 eyes from 6 rats) with dry eye induced by continuous systemic administration of scopolamine, and measurements of fluorescein staining were taken on day 14. Group 3: rats (n=14 eyes from 7 rats) with dry eye induced by continuous systemic administration of scopolamine, and topically treated once with saline on day 8. Group 4: rats (n=14 eyes from 7 rats) with dry eye induced by continuous systemic administration of scopolamine, and topically treated once with the test peptide on day 8.

[0254] Endpoints were: (i) reduction in corneal fluorescein staining measured at day 13 compared to saline-treated controls; and (ii) tear production and tear flux measured at day 13 compared to untreated or scopolamine-treated controls.

[0255] Example 11 - Effects of ZEP3, ZEP3 sodium salt and ZEP4 in a rabbit model of keratoconjunctivitis sicca

[0256] Keratoconjunctivitis sicca (KCS) was produced in the right eyes of 8 New Zealand white rabbits by surgical closure of the lacrimal drainage duct and removal of the nictitating membrane, nictitans glands and Harderian glands. All rabbits were kept untreated for 8 weeks and KCS was confirmed by measuring the increase in tear film osmotic pressure by collecting 0.1-0.4 μL tear samples as described by J.Gilbard et al. (Ophthalmol. 96: 677, 1978). A 3.0 mmol solution of UTP or an analog was prepared in a preserved isotonic buffer solution. The rabbits were randomly divided into groups and treated with ZEP3, ZEP sodium salt and ZEP4 (test peptide). The untreated group was used as a control. After the start of treatment, 0.1-0.4 μL tear samples were obtained from all rabbits before the first dose for molar osmotic pressure concentration measurement. The animals were sacrificed at week 20, and goblet cell density was measured by staining with Alcian blue and periodic acid-Schiff's reagent (D. Dartt et al., Exp. Eye Res. 67:27, 1996).

[0257] Example 12 - Effects of ZEP3 and its sodium salt (ZEP3Na) on cytokine production in an in vitro inflammation model in human PBMCs

[0258] 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 with lipopolysaccharide (LPS) to produce cytokines.

[0259] Methods: Briefly, cryopreserved human PBMCs from four healthy non-smokers not taking any corticosteroids or other inflammatory drugs were grown in RPMI 1640 (complete production medium, CGM) containing 10% heat-inactivated fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / mL streptomycin.

[0260] For all groups (1-6, Table 7), human PBMCs were thawed and plated and left to stand for 1 hour before treatment. The cell groups were treated with the test substance ZEP3 (SEQ ID NO: 1) or dexamethasone at concentrations of 40, 100 and 150 μg / ml for 1 hour and then stimulated with LPS for 24 hours. Alamar blue was added 4 hours before supernatant collection. 24 hours after adding LPS, Alamar blue fluorescence at 585nm (related to cell viability) was measured and supernatant was collected for cytokine determination. Three parallel samples were tested for each group.

[0261] Table 7

[0262] Experimental groups used in the study

[0263] Group Stimulation (100pg / ml) deal with Processing time schedule 1 NA NA (medium control) NA 2 LPS NA (stimulation control) NA 3 LPS 1uM dexamethasone 1 hour before treatment 4 LPS ZEP3 (40 μg / ml) 1 hour before treatment 5 LPS ZEP3 (100 μg / ml) 1 hour before treatment 6 LPS ZEP3 (150 μg / ml) 1 hour before treatment

[0264] Detailed Methods: Cells were thawed according to the manufacturer's instructions, washed in CGM and viability assessed using trypan blue staining. A stock solution of 2x10^6 cells / mL was prepared in CGM. 100 μL of the stock solution was added to the appropriate wells of a 96-well black-walled plate (one plate per donor) and 2x10^6 cells / mL were seeded per well. 5 The cells were cultured at 37°C and 5% CO 2 Incubate for 1 hour at 37°C and 5% CO. Add 100 μL of 2X test article, dexamethasone, or 1X CGM to the appropriate wells to a final volume of 200 μL / well. Incubate the plate at 37°C and 5% CO. 2 Incubate for 1 hour at 37°C and 5% CO. After 1 hour, add 20 μL of LPS to groups 2-9 to a final volume of 220 μL. Add 20 μL of IM to group 1. Incubate the cells at 37°C and 5% CO. 2 Incubate for 20 hours at 37°C and 5% CO. At the 20 hour mark, 20 μL of Alamar Blue was added to all wells. Plates were incubated at 37°C and 5% CO. 2 Continue incubation for 4 hours at 40 ℃. 24 hours after LPS stimulation, read the plate to estimate cell viability, harvest the cell culture supernatant and store it at -80 ℃ for cytokine analysis. The remaining cells are washed once with sterile PBS. The cells are incubated in a trypsin solution to detach from the plate, centrifuged and deposited and the trypsin solution is removed. The cells are then stored at -80 ℃ for optional further analysis. Luminex assays are used to determine the concentrations of GM-CSF, IFN-g, IL-10, IL-12p40, IL-17a, IL-1β, IL-4, IL-6, RANTES and TNFα present in the supernatant samples collected after the Alamar Blue assay. The manufacturer's process is followed in using the cytokine assay. The cytokine production and percentage of the sample stimulation are calculated for the mean value of the stimulation control.

[0265] Result: As Figure 9-12 As indicated in the literature, the major mediators of inflammation induced by ZEP3 include interferon γ (IFNγ, Fig. 9 )、IL-10( Fig.10 ), IL-1β( Fig.11 ) and RANTES( Fig.12 ), supporting the concept of its anti-inflammatory effect. In some of the cytokines measured, a dose-dependent response was observed. After treatment with all concentrations of the peptide tested, no significant changes in cell viability were observed in all donors, nor any trend, indicating that the peptide is not cytotoxic to PBMC. In all groups tested, IL-4 and IL-17a levels were below the detection limit.

[0266] The reduction in cytokine production was directly associated with ZEP3, confirming the anti-inflammatory effects of ZEP3 in human blood cells.

[0267] Example 13 - In vivo atopic dermatitis model

[0268] The effect of topical administration of ZEP3 (SEQ ID NO: 1) and its sodium salt form (ZEP3Na) and ZEP4 (SEQ ID NO: 2) was evaluated in an oxazolone-induced atopic dermatitis (AD) mouse model. This model is widely used and well described in the literature as an animal model for evaluating the acceptance of drugs for AD therapy in humans (Avci et al., 2013 Expert Opin Drug Discov. 8, 331-355; Petersen TK 2006, Basic Clin. Pharmacol. Toxicol. 99, 104-115).

[0269] Method: On day 0, after the animals were shaved and depilated, the animals were sensitized by topical administration of a 2% oxazolone solution in ethanol on the back (sensitization phase). Then, after one week, a challenge was performed by topical administration of a 1% oxazolone solution in ethanol on the back and right ear every two days for a total of 10 times, i.e., a total of 3 weeks. During the challenge phase, the test compound was applied as a ready-to-use cream formulation by a topical route every two days. The surface applied was the same as that used for the challenge (back + ear). 100 μL was applied on the back and the back was gently massaged. Then, the remaining formulation on the finger cuff was applied to the inner side of the ear. The thickness of the back skin and ear was measured every other day using a caliper, and the following clinical scores were recorded from the back skin: erythema and scaling. The animals were also weighed every other day (the approximate weight of the mice at the beginning was about 20 g). Calibrated digital images of the back skin area were obtained on the 8th, 18th and 28th days. At the end of the experiment on the 28th day, the animals were bled and plasma samples were separated. Right ear and dorsal skin samples were collected on euthanized animals and frozen at -80°C (specimens from dorsal skin and plasma) or kept in formalin (dorsal skin and right ear samples) for further analysis. Table 8 describes the dosing groups.

[0270] Table 8

[0271] Experimental groups tested in AD models

[0272]

[0273] *In a volume of 100 μl for each animal.

[0274] The formulations used for topical administration of the test compounds were:

[0275] ZEP3A 1% cream, having a pH of 7.1, contains: propylene glycol, polysorbate 80, disodium edetate, syloid, methylparaben, white petrolatum, isopropyl myristate, cetyl alcohol and glyceryl monostearate.

[0276] ZEP3Na A 1% cream, having a pH of 8.0, contains: propylene glycol, polysorbate 80, disodium edetate, syloid, methylparaben, white petrolatum, isopropyl myristate, cetyl alcohol and glyceryl monostearate.

[0277] ZEP4 A 1% cream, has a pH of 4.5 and contains: propylene glycol, glycerin, polysorbate 80, disodium EDTA, xanthan gum, silica (syloid), methylparaben, white petrolatum, isopropyl myristate, cetyl alcohol, stearyl alcohol and glyceryl monostearate (kolliwax).

[0278] Results: The test compounds ZEP3, ZEP3Na and ZEP4 were well tolerated when administered at 50 mg / kg to atopic dermatitis-induced mice. All peptides significantly reduced clinical lesions of atopic dermatitis, back skin thickness ( Fig.13A and 13B )、Ear thickness( Fig.14A and 14B ) and erythema ( Fig.15A and 15B In mice treated with ZEP3 ( Fig.16A ) from day 18 to 24 and with ZEP3Na ( Fig.16A ) or ZEP4( Fig. 16B ) treated mice showed a decrease in scaling from days 14 to 24, indicating a delayed effect. Body weights of mice treated with ZEP3, ZEP3Na, or ZEP4 were normal compared to vehicle ( Fig.17A and 17B ).

[0279] In detail, body weight was fairly stable in the first few days of the study for ZEP3, ZEP3Na, and ZEP4, and showed an increase of approximately 3-4% on day 28 ( Fig.17A and 17B ). For the group treated with betamethasone, as expected, a weight loss of approximately 8-10% was observed.

[0280] With the exception of betamethasone-treated mice, dorsal skin thickness increased during the first few days of the study and then remained fairly stable until the end of the study ( Fig.13A and 13B). On day 28, an increase of about 110% was noted for the negative control group dosed with vehicle, a 90% increase for group 3 dosed with ZEP3 1%, a 105% increase for group 4 dosed with ZEP3Na 1%, and an increase of about 90% for group 5 dosed with ZEP4 1%, while a 40% decrease was noted for the second positive control group dosed with betamethasone. All treatment groups (groups 3, 4, and 5) showed a trend showing that the back skin thickness was less than the first negative control group. For ZEP3, a significant difference in back skin thickness was seen on days 20 (-25.5%) and 24 (-16.6%) compared to control group 1. For ZEP4, a significant difference in back skin thickness was seen on day 14 compared to control group 1 (-13.5%).

[0281] Right ear thickness: As is usually observed with this model, all groups showed significant thickening from the first few days until the end of the study, with the exception of the positive control group 2, which showed only a slight increase of approximately 15-20% over the course of the study ( Fig.14A and 14B Significant differences were observed for ZEP3 at days 20, 22, and 28 (-10.1 to -18.0% compared to control), for ZEP3 Na at days 18, 20, and 22 (-18.4 to -22.7% compared to control), and for ZEP4 at days 20, 22, and 28 (-12.3 to -16.0% compared to control).

[0282] For the negative control group (Group 1), the erythema score was maximum on day 16 and then decreased slightly until it was 0 on days 26-28. The erythema scores recorded for test groups 3, 4, and 5 were similar to those of negative control group 1 ( Fig.15A and 15B ). Significant differences were shown at days 22 and 24 for ZEP3, day 24 for ZEP3Na, and days 12, 16, 22, and 24 for ZEP4.

[0283] Scaling scores gradually improved over the first few days, then decreased slightly and remained fairly stable during the second part of the study ( Fig.16A and 16B ). All treatment groups 3, 4 and 5 showed lower scaling scores than the negative control group 1 from day 14 to day 28.

[0284] For treatment groups 3, 4 and 5, the total score (sum of erythema and scaling scores) from day 14 to day 28 was also lower than that of negative control group 1. Significant differences in the total score were shown on days 20, 24 and 28 for ZEP3, on days 14, 18, 20 and 22 for ZEP3Na, and on days 12-20, 24 and 28 for ZEP4.

[0285] 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 clinical lesions of atopic dermatitis as indicated by a reduction in dorsal skin thickness, ear thickness, erythema and scaling.

[0286] Example 14. Treatment of Carpometacarpal Osteoarthritis (CMC Arthritis) with ZEP3

[0287] A 75 year old male subject was diagnosed by an orthopedic surgeon with CMC type arthritis in his big toe. His toe was swollen, red, and so painful that it prevented him from walking.

[0288] 0.5% ZEP3 sodium salt cream was applied 5 times daily to the swollen areas. The subject reported relief within one day of treatment and symptoms resolved within 3-4 days.

[0289] Example 15. Effects of ZEP3 and ZEP3Na peptide on TNFα secretion in human keratinocytes

[0290] TNFα is the major pro-inflammatory mediator in keratinocytes. The anti-inflammatory effects of the compounds were tested by measuring their effects on TNFα levels.

[0291] Methods: SCCE020 cells (EpiGRO TM Human epidermal keratinocytes) in complete growth medium (EpiGRO TM The cells were grown in complete human keratinocyte medium (Millipore catalog number SCMK001) for a total of 4 passages. At passage 5, the cells were plated in triplicate (3*10 5 The cells were grown in 1 ml of complete growth medium (100 cells / well) per well. The other 3 control wells contained only medium without cells. After attachment, the cell culture medium was replaced with a base medium (starvation medium) containing L-glutamine without supplements, and the cells were starved overnight. The next day, 24 hours after inoculation, the cells were treated with ZEP3 or ZEP3Na or a suitable diluent (PBS or DMSO). After incubation for 4 hours, LPS (from E. coli 055:B5; Sigma catalog number L6529-1MG) was added to the wells at a final concentration of 20 or 30 μg / ml. The experimental groups (3 wells for each treatment) are summarized in Table 9.

[0292] Cells were incubated in a tissue culture incubator for 24 hours. After collecting cell supernatants from all wells, the cells from treatment groups 1, 2, 3, 8, 9, 10, 13, 14 (excluding DMSO groups) were centrifuged and deposited, and stored at -80 ° C for further analysis. Cells were collected as described below: after collecting supernatants, wells were rinsed with trypsin, then incubated with trypsin until they were detached from the plate surface, centrifuged, cleaned with PBS, and stored as cell sediments. The TNF-α concentration in the supernatant was measured in quintuplicate by ELISA (Quantikine HS ELISA; R&D Systems catalog number HSTA00D).

[0293] Results: ZEP3 and ZEP4 effectively reduce TNFα levels in human keratinocyte cultures. TNFα concentrations in samples were calculated according to the standard curve trend line equation: OD = 0.063x + 0.1718. The percentage inhibition of TNFα production in each treatment group was calculated compared to the corresponding control group (same conditions, without peptide). The results are summarized in Table 9.

[0294] Table 9

[0295] Experimental conditions, results, and analysis

[0296]

[0297] & Cell-free

[0298] *The fold change for each sample is relative to the TNF-α concentration in sample #2.

[0299] **For each sample (array 1), a t-test was performed using the Microsoft EXCEL function for the control (array 2) for that sample. Parameters for the t-test: two-tailed distribution, unpaired t-test, unequal variance. The same control reference sample was used to calculate the percentage inhibition of TNF-α secretion.

[0300] Conclusions: Quantitative testing by ELISA confirmed a dose-dependent inhibition of TNF-α production by ZEP3 or ZEP3Na in LPS-treated human keratinocytes compared to the amount secreted by cells stimulated with LPS alone.

[0301] Although the present invention has been described in conjunction with its specific embodiments, it is evident that many substitutions, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to cover all such substitutions, modifications and variations that fall within the spirit and broad scope of the claims.

[0302] All publications, patents and patent applications mentioned in this specification are incorporated by reference in their entirety into this specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference herein. In addition, the citation or identification of any reference in this application should not be construed as an admission that the reference can be used as prior art for the present invention. Where section headings are used, they should not be construed as necessary limitations. Sequence Listing <110> SIS Shurov Innovation Sciences Ltd. <120> Pharmaceutical composition for inhibiting inflammatory cytokines <130> SIS001PCT <150> US 62649940 <151> 2018-03-29 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (1) <223> pyroglutamic acid (pGlu) <220> <221> MOD_RES <222> (4) <223> C8 alkyl attached to the epsilon amine of the Lysine side chain to form Lys(Octanoyl) <400> 1 Glu Asn Trp Lys 1 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (1)..(1) <223> pyroglutamic acid (pGlu) <400> 2 Glu Asn Trp Thr 1

Claims

1. Use of a peptide selected from the group consisting of the peptide of SEQ ID NO: 1 and the peptide of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, for preparing a pharmaceutical composition for treating an inflammatory disease or disorder selected from the group consisting of an ocular inflammatory disease or disorder, an otic inflammatory disease or disorder, a pulmonary inflammatory disease or disorder, an intestinal inflammatory disease or disorder, and an inflammatory autoimmune disease or disorder.

2. The use according to claim 1, wherein the ocular inflammatory disease or disorder is selected from uveitis, dry eye syndrome, inflammatory symptoms associated with infectious eye diseases, allergic eye diseases, keratitis, conjunctivitis, meibomian gland dysfunction and Sjögren's syndrome.

3. The use according to claim 1, wherein the inflammatory autoimmune disease or disorder is selected from rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, Sjögren's syndrome and lupus.

4. The use according to claim 1, wherein the intestinal inflammatory disease or disorder is selected from Crohn's disease and ulcerative colitis.

5. The use according to claim 1, wherein the pulmonary inflammatory disease or disorder is selected from asthma, bronchitis, pleurisy, vasculitis, pneumonia, bronchiectasis, idiopathic pulmonary fibrosis and cystic fibrosis.

6. The use according to claim 1, wherein the pulmonary inflammatory disease or disorder is chronic bronchitis or diffuse panbronchiolitis.

7. The use according to claim 1, wherein the pulmonary inflammatory disease or disorder is hypersensitivity pneumonitis or alveolitis.

8. The use according to claim 1, wherein the inflammatory disease or disorder of the ear is selected from inflammatory symptoms associated with ear infection, otitis media, otitis externa, mastoiditis.

9. The use according to claim 1, wherein the inflammatory disease or disorder of the ear is otomastoiditis.

10. The use according to claim 1, wherein the pharmaceutically acceptable salt of the peptide is a sodium salt of the peptide of SEQ ID NO: 1 or the peptide of SEQ ID NO:

2.

11. The use according to any one of claims 1 to 10, wherein the pharmaceutical composition has a pH of 4 to 8.

12. The use according to any one of claims 1 to 11, wherein the pharmaceutical composition is formulated for administration by a route selected from the group consisting of ocular administration, oral administration, nasal administration and parenteral administration.

13. The use according to any one of claims 1 to 11, wherein the pharmaceutical composition is formulated for topical administration.

14. The use according to claim 13, wherein the pharmaceutical composition is formulated as a cream, an ointment, a paste, a lotion, a gel or takes the form of eye drops.

15. The use according to claim 14, wherein the pharmaceutical composition is formulated as a cream comprising 0.1-5% w / w of the peptide or a pharmaceutically acceptable salt thereof.

16. The use of any one of claims 1 to 15, wherein treating an inflammatory disease or disorder comprises reducing the release of or inhibiting the activity of at least one inflammatory cytokine.

17. The use of claim 16, wherein the at least one inflammatory cytokine is selected from interferon gamma (IFNγ), interleukin 1β (IL-1β), interleukin 10 (IL-10), tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6).

18. The use of claim 17, wherein the at least one inflammatory cytokine is selected from TNFα, IL-1-β and IL-6.

19. The use of any one of claims 1 to 15, wherein treating an inflammatory disease or disorder comprises reducing the release of at least one inflammatory mediator or chemokine.

20. The use of claim 19, wherein the at least one inflammatory mediator or chemokine is selected from reactive oxygen species (ROS) and RANTES (chemokine expressed and secreted by normal activated T cells).

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