Novel anti-inflammatory peptides and uses thereof

Abstract

The invention relates to a novel immunomodulatory peptide capable of controlling inflammatory response and application of the novel immunomodulatory peptide. The peptides of the present invention can be used to treat inflammation, various immune diseases, or cancers by modulating immune cell activity.

Description

Technical Field

[0001] The present invention relates to a novel peptide, a pharmaceutical composition for preventing or treating immune diseases or cancer, and an anti-inflammatory cosmetic composition containing the same. Background Art

[0002] Resolution of inflammation is a biological mechanism that terminates the innate immune response, restores tissue homeostasis, and induces an appropriate adaptive immune response. Previously, resolution of inflammation was considered a passive phenomenon after the occurrence of an inflammatory response. In recent years, mediators of resolution of inflammation have been discovered, revealing that resolution of inflammation is an active biological mechanism that is carried out step by step according to the inflammatory response and involves multiple factors. When a wound, foreign body contact, or infection occurs, a defense mechanism is generated through the acute inflammatory response of the innate immune system, and then the resolution of inflammation controls the inflammatory response to an appropriate level, inducing healing and recovery of damaged tissues. Resolution of inflammation limits further infiltration of neutrophils into inflamed tissues, induces cell death, and mediates the reduction of lipid inflammatory substances, cytokines, and chemokines that cause inflammatory responses. Subsequently, the clearance of damaged tissues or dead cells is promoted by inducing endocytosis of macrophages. To this end, inflammatory macrophages are reduced and an increase in macrophages with anti-inflammatory properties is induced. Appropriate resolution of inflammation mediates normal adaptive immune responses and enhances the ability to cope with future repeated inflammatory situations. However, excessive / persistent inflammation without sufficient resolution can lead to various chronic inflammatory diseases, and it is an abnormal adaptive immune response that can lead to autoimmune diseases.

[0003] Resolution of inflammation is induced by substances secreted by cells, such as pro-resolving factors, called specialized pro-resolving mediators (SPMs). When inflammation begins to resolve, various pro-resolving factors such as lipids, peptides, and proteins control inflammatory responses, such as the activation of immune cells, through specific receptors such as GPCRs.

[0004] Current inflammatory treatments inhibit the activity of specific enzymes or the occurrence of inflammatory responses by antagonizing specific receptors or their ligands. Existing anti-inflammatory strategies can effectively and strongly inhibit acute inflammatory responses, but they also inhibit normal inflammatory responses, causing various side effects. Of particular concern is the safety of patients with chronic inflammatory / autoimmune diseases who require long-term medication, such as decreased immunity and the risk of secondary infection. Therefore, there is an urgent need to develop treatments with safe inflammatory response control efficacy and safety through new methods. The strategy of inducing inflammatory resolution is an innovative immunotherapy strategy that uses agonists to control excessive / persistent inflammatory responses to enhance the mechanism of inflammatory resolution. In particular, it has attracted attention as an alternative to broad-spectrum immunosuppressants, which have a high risk of side effects when treating chronic inflammatory / autoimmune diseases that require long-term medication due to their limited effects on normal immune responses. The targets of the therapeutic strategy of inducing inflammatory resolution include not only inflammatory diseases that occur due to immune dysfunction in various tissues such as the skin, respiratory system, digestive system, eye, musculoskeletal system, and circulatory system, but also diseases in which the inflammatory response occurring at the onset of the disease has a significant impact on the progression of the disease. Specifically, representative examples of these diseases are Parkinson's disease and Alzheimer's disease, which are well-known neurological diseases in which neuroinflammation greatly affects the deterioration of symptoms. In addition, as the importance of inflammatory responses in the tumor microenvironment on cancer progression has been confirmed, especially in solid cancers, anticancer therapy through immunomodulation by inducing inflammation resolution is also being considered as a new strategy.

[0005] Formyl peptide receptor (FPR)-based receptors are G protein-coupled receptors (GPCRs) that are mainly found in immune cells and mediate various cellular functions through downstream signal transduction systems. FPR1 mainly binds to bacterial-derived peptides to induce acute inflammatory responses and mediate defense functions, while FPR2 has been shown to control inflammatory responses by binding to various types of endogenous ligands (peptides, proteins, and lipids) and has attracted attention as a target for the treatment of inflammatory diseases. FPR2 is a receptor for representative pro-resolving factors (LXA4, AnxA1, Ac2-26, RvD1, etc.), which is expressed in innate immune cells and mediates the induction of inflammatory resolution. Based on the complex characteristics of FPR2 ligands, attempts have been made to develop various substances such as peptides, lipids, proteins, and small molecule compounds as FPR2 targeted therapeutic materials that induce inflammatory resolution. These attempts have mainly focused on developing treatment methods based on the results of research on the therapeutic effects on various inflammatory diseases, such as atopic dermatitis, psoriasis, asthma, lung injury, lung inflammation, conjunctivitis, keratitis, dry eye, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, and systemic sclerosis, which are inflammatory diseases that occur in various tissues such as the skin, respiratory system, digestive system, eye, musculoskeletal system, and circulatory system due to abnormal local or systemic immune responses. In addition, as the importance of neuroinflammation occurring in microglia or neurons involved in immune functions in the brain in degenerative brain diseases such as Parkinson's disease and Alzheimer's disease, and the importance of the interaction between inflammatory responses occurring in immune cells and basal cells of the tumor microenvironment and cancer cells in cancer progression have been confirmed, a large number of research and development results on the effectiveness of therapeutic strategies for neurological diseases and cancers obtained by resolving inflammation have been reported, and these strategies are considered to be new approaches. Among them, clinical trials of peptides and small molecule compounds targeting FPR2 for atopic dermatitis, heart failure, myocardial infarction, cardiovascular thrombosis, Crohn's disease, etc. are underway.

[0006] The present inventors have developed and patented a peptide having antibacterial activity in previous studies (Patent Document 1), and have developed and patented a peptide having excellent antibacterial and immune function regulating abilities (Patent Document 2).

[0007] [Related technical literature]

[0008] [Patent Document]

[0009] Patent Document 1: Korean Patent No. 10-1855170

[0010] Patent Document 2: Korean Patent No. 10-2170236 Summary of the invention

[0011] [Technical issues]

[0012] The present invention aims to provide a novel peptide having immunomodulatory function and producing a therapeutic effect on immune diseases by inducing inflammation regression.

[0013] The present invention also aims to provide various uses of the peptide, such as a pharmaceutical composition for preventing or treating immune diseases or cancer, and an anti-inflammatory cosmetic composition.

[0014] [Technical solution]

[0015] In order to achieve the above object, one aspect of the present invention provides a peptide consisting of an amino acid sequence represented by X1-X2-X3-X4-X5-X6-m,

[0016] wherein X1 is absent, or X1 is arginine (R), lysine (K), histidine (H) or proline (P); X2 is methionine (M) or tryptophan (W); X3 is a basic amino acid, such as arginine (R) or lysine (K); X4 is tyrosine (Y); X5 is a basic amino acid, such as arginine (R) or lysine (K), or X5 is an aromatic amino acid, such as tryptophan (W) or tyrosine (Y); X6 is proline (P), tyrosine (Y) or valine (V); and m is D-methionine.

[0017] Another aspect of the present invention provides a peptide consisting of an amino acid sequence represented by X2-X3-X4-X5-X6-m,

[0018] wherein X2 is isoleucine (I), proline (P) or histidine (H); X3 is lysine (K); X4 is tyrosine (Y) or phenylalanine (F); X5 is lysine (K) or tryptophan (W); X6 is valine (V), proline (P), tyrosine (Y) or methionine (M); and m is D-methionine.

[0019] Yet another aspect of the present invention provides a peptide consisting of an amino acid sequence represented by X1-X2-X3-X4-X5-X6-m,

[0020] wherein X1 is a basic amino acid, such as arginine (R) or lysine (K); X2 is an aromatic amino acid, such as tryptophan (W); X3 is a basic amino acid, such as arginine (R) or lysine (K); X4 is tyrosine (Y); X5 is arginine (R), lysine (K), leucine (L), asparagine (N), phenylalanine (F) or threonine (T); X6 is isoleucine (I), threonine (T), tyrosine (Y) or valine (V); and m is D-methionine.

[0021] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating immune diseases, comprising the above peptide as an active ingredient.

[0022] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the above peptide as an active ingredient.

[0023] Still another aspect of the present invention provides an anti-inflammatory cosmetic composition comprising the above peptide as an active ingredient.

[0024] [Beneficial Effects]

[0025] Since the novel peptide of the present invention has the function of regulating the activity of immune cells and the function of treating immune diseases or cancer caused by the disappearance of inflammation, it can be used as a pharmaceutical composition for treating immune diseases or cancer containing the above peptide, or an anti-inflammatory cosmetic composition containing the above peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Results of an analysis of the ability of peptides to inhibit the production of inflammatory cytokines at the individual level are shown.

[0027] Figure 2 Results of analysis of the peptides' efficacy in improving collagen-induced arthritis severity scores are shown.

[0028] Figure 3 Shown are the results of analysis of corneal damage (A) and corneal opacity scores (B) according to peptide administration in the BAC-induced dry eye model.

[0029] Figure 4 Shown are the results of analysis of body weight (A) and colon length (B) according to peptide administration in the DSS-induced colitis model.

[0030] Figure 5 Shown are the results of analysis of the ability of peptides to inhibit cytokine expression in the SK-N-MC neuronal cell line (A) and rat DRG neurons (B).

[0031] Figure 6 Shown are the results of analysis of the ability of the peptide to improve the density of dopamine neuronal cells in nerve terminals (A) and the number of neuronal cells (B) in the MPTP-induced Parkinson's disease model.

[0032] Figure 7 Results of analyses of the peptides' efficacy in inhibiting tumor growth are shown. DETAILED DESCRIPTION

[0033] The present invention is described in detail below.

[0034] The present invention provides a novel peptide which binds to FPR2 receptor to exert the function of controlling inflammatory response.

[0035] In one aspect of the present invention, a peptide consisting of an amino acid sequence represented by X1-X2-X3-X4-X5-X6-m is provided, wherein

[0036] X1 is absent, or X1 is arginine (R), lysine (K), histidine (H) or proline (P); X2 is methionine (M) or tryptophan (W); X3 is a basic amino acid, such as arginine (R) or lysine (K); X4 is tyrosine (Y); X5 is a basic amino acid, such as arginine (R) or lysine (K), or X5 is an aromatic amino acid, such as tryptophan (W) or tyrosine (Y); X6 is proline (P), tyrosine (Y) or valine (V); and m is D-methionine.

[0037] The term "peptide" as used herein refers to a polymer composed of one or more amino acids connected by amide bonds (or peptide bonds), including peptides and pharmaceutically acceptable salts of peptides. Typically, the amino terminus (or N-terminus) of the peptide is shown at the left end of the amino acid sequence of the peptide, and the carboxyl terminus (or C-terminus) is shown at the right end of the amino acid sequence of the peptide. The amino acid sequence of the peptide can be written as a single letter symbol to represent the amino acids covalently bonded by peptide bonds. When the D conformation is not specifically indicated, the L conformation can be assumed, and the amino acid residues constituting the peptide can be natural or non-natural amino acid residues.

[0038] The peptides of the present invention can be obtained by various methods known in the art. For example, after extracting the protein from an organism, it can be treated with a proteolytic enzyme to reduce the molecular weight, or it can be produced biologically using gene recombination or a protein expression system, or it can be produced by in vitro synthesis using chemical synthesis methods such as peptide synthesis and cell-free protein synthesis.

[0039] In one embodiment of the present invention, X1 may be absent, or may be arginine (R), lysine (K), histidine (H) or proline (P); X2 may be methionine (M) or tryptophan (W); X3 may be a basic amino acid, such as arginine (R) or lysine (K); X4 may be tyrosine (Y); X5 may be a basic amino acid, such as arginine (R) or lysine (K); X6 may be proline (P), tyrosine (Y) or valine (V); and m may be D-methionine.

[0040] In one embodiment of the present invention, X1 may be absent, or may be arginine (R), lysine (K); X2 may be methionine (M) or tryptophan (W); X3 may be a basic amino acid, such as arginine (R) or lysine (K); X4 may be tyrosine (Y); X5 may be an aromatic amino acid, such as tryptophan (W); X6 may be proline (P), tyrosine (Y) or valine (V); and m may be D-methionine.

[0041] In one embodiment of the present invention, X1 may be absent, or may be arginine (R), lysine (K); X2 may be methionine (M) or tryptophan (W); X3 may be a basic amino acid, such as arginine (R); X4 may be tyrosine (Y); X5 may be an aromatic amino acid, such as tyrosine (Y); X6 may be proline (P), tyrosine (Y) or valine (V); and m may be D-methionine.

[0042] In one embodiment of the present invention, X1 may be absent, or may be arginine (R), lysine (K), histidine (H) or proline (P); X2 may be methionine (M) or tryptophan (W); X3 may be a basic amino acid, such as arginine (R) or lysine (K); X4 may be tyrosine (Y); X5 may be a basic amino acid, such as lysine (K); X6 may be proline (P), tyrosine (Y) or valine (V); and m may be D-methionine.

[0043] In one embodiment of the present invention, X1 may be absent or may be arginine (R); X2 may be methionine (M) or tryptophan (W); X3 may be a basic amino acid, such as arginine (R) or lysine (K); X4 may be tyrosine (Y); X5 may be an aromatic amino acid, such as or tryptophan (W); X6 may be proline (P), tyrosine (Y) or valine (V); and m may be D-methionine.

[0044] In one embodiment of the present invention, X1 may be absent or may be arginine (R); X2 may be methionine (M) or tryptophan (W); X3 may be a basic amino acid, such as arginine (R); X4 may be tyrosine (Y); X5 may be an aromatic amino acid, such as tyrosine (Y); X6 may be proline (P), tyrosine (Y) or valine (V); and m may be D-methionine.

[0045] In the present invention, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorption, efficacy, efficacy, etc.), changed specificity (e.g., wide spectrum biological activity) and reduced antigenicity, one or more amino acids of the peptide can be chemically modified. "Stability" used herein includes not only in vivo stability (which protects the peptide of the present invention from attack by proteolytic enzymes in the body), but also storage stability (e.g., room temperature storage stability). For example, a protecting group can be added to the N-terminus or C-terminus of the peptide of the present invention. Preferably, the protecting group can be acetyl, butyryl, hexanoyl, octanoyl, fluorenylmethoxycarbonyl, palmitoyl, myristoyl, stearoyl, butoxycarbonyl, acryloxycarbonyl or polyethylene glycol (PEG), but can be any component that can change the properties of the peptide, especially improve the stability of the peptide, without limitation.

[0046] In the present invention, in the N-terminal amino acid of the peptide, the terminal amino group (H2N-) can be alkylated or acylated. For example, the N-terminal amino group of the peptide can be acylated to include aliphatic acyl groups (i.e., including acetyl, myristoyl or butyryl), carboxylic acid, benzoic acid, trifluoroacetic acid or sulfonic acid. In another example, the N-terminal amino acid of the peptide can be alkylated with aliphatic alkyl, halides or aliphatic alkyne sulfonates.

[0047] In the present invention, the C-terminal carboxyl group (-COOH) of the peptide can be amidated or esterified. For example, the C-terminal carboxyl group can be chemically modified by forming an amide with an amine. In another example, the C-terminal carboxyl group can be chemically modified by forming an ester with an alcohol.

[0048] In addition, in the present invention, the side chains in the amino acids of the peptide can be chemically modified. For example, the phenyl group in tyrosine can be substituted by an aliphatic alkyl group, an aliphatic carboxyl group or an alkoxy group, but the present invention is not limited thereto. The epsilon amino group in lysine can be chemically modified by forming an amide with, for example, an aliphatic carboxyl group, a benzoic acid group or an amino acid group. In addition, the epsilon amino group in lysine can be chemically modified by alkylation of one or two C1 to C4 aliphatic alkyl groups, but the present invention is not limited thereto.

[0049] In one embodiment of the present invention, any one selected from the group consisting of an acetyl group, a butyryl group, a hexanoyl group, and an octanoyl group may be added to the N-terminus of the peptide.

[0050] In one embodiment of the present invention, the C-terminus of the peptide may be amidated.

[0051] In one embodiment of the present invention, the peptide may consist of any one of the amino acid sequences of SEQ ID NOs: 1-127.

[0052] In one aspect of the present invention, a peptide consisting of an amino acid sequence represented by X2-X3-X4-X5-X6-m is provided, wherein

[0053] X2 can be isoleucine (I), proline (P) or histidine (H); X3 can be lysine (K); X4 can be tyrosine (Y) or phenylalanine (F); X5 can be lysine (K) or tryptophan (W); X6 can be valine (V), proline (P), tyrosine (Y) or methionine (M); and m can be D-methionine.

[0054] In one embodiment of the present invention, X2 can be isoleucine (I), proline (P) or histidine (H); X3 can be lysine (K); X4 can be tyrosine (Y) or phenylalanine (F); X5 can be lysine (K) or tryptophan (W); X6 can be valine (V) or proline (P); and m can be D-methionine.

[0055] In one embodiment of the present invention, X2 can be isoleucine (I) or histidine (H); X3 can be lysine (K); X4 can be tyrosine (Y) or phenylalanine (F); X5 can be lysine (K) or tryptophan (W); X6 can be tyrosine (Y) or methionine (M); and m can be D-methionine.

[0056] In one embodiment of the present invention, X2 can be isoleucine (I) or proline (P); X3 can be lysine (K); X4 can be tyrosine (Y) or phenylalanine (F); X5 can be lysine (K) or tryptophan (W); X6 can be valine (V); and m can be D-methionine.

[0057] In one embodiment of the present invention, X2 can be isoleucine (I) or histidine (H); X3 can be lysine (K); X4 can be tyrosine (Y) or phenylalanine (F); X5 can be lysine (K) or tryptophan (W); X6 can be proline (P); and m can be D-methionine.

[0058] In one embodiment of the present invention, X2 can be isoleucine (I); X3 can be lysine (K); X4 can be phenylalanine (F); X5 can be lysine (K) or tryptophan (W); X6 can be tyrosine (Y) or methionine (M); and m can be D-methionine.

[0059] In one embodiment of the present invention, X2 can be isoleucine (I) or proline (P); X3 can be lysine (K); X4 can be tyrosine (Y) or phenylalanine (F); X5 can be lysine (K); X6 can be valine (V); and m can be D-methionine.

[0060] In one embodiment of the present invention, X2 can be isoleucine (I) or histidine (H); X3 can be lysine (K); X4 can be tyrosine (Y) or phenylalanine (F); X5 can be lysine (K) or tryptophan (W); X6 can be proline (P); and m can be D-methionine.

[0061] In one embodiment of the present invention, X2 may be isoleucine (I); X3 may be lysine (K); X4 may be phenylalanine (F); X5 may be lysine (K) or tryptophan (W); X6 may be tyrosine (Y) or methionine (M); and m may be D-methionine.

[0062] In one embodiment of the present invention, any one selected from the group consisting of an acetyl group, a butyryl group, a hexanoyl group, and an octanoyl group may be added to the N-terminus of the peptide.

[0063] In one embodiment of the present invention, the C-terminus of the peptide may be amidated.

[0064] In one embodiment of the present invention, the peptide may consist of any one of the amino acid sequences in SEQ ID NOs: 128-151.

[0065] In one aspect of the present invention, a peptide consisting of an amino acid sequence represented by X1-X2-X3-X4-X5-X6-m is provided.

[0066] wherein X1 is a basic amino acid, such as arginine (R) or lysine (K); X2 is an aromatic amino acid, such as or tryptophan (W); X3 is a basic amino acid, such as arginine (R) or lysine (K); X4 is tyrosine (Y); X5 is arginine (R), lysine (K), leucine (L), asparagine (N), phenylalanine (F) or threonine (T); X6 is isoleucine (I), threonine (T), tyrosine (Y) or valine (V); and m is D-methionine.

[0067] In one embodiment of the present invention, X1 can be a basic amino acid, such as arginine (R) or lysine (K); X2 can be an aromatic amino acid, such as or tryptophan (W); X3 can be a basic amino acid, such as arginine (R) or lysine (K); X4 can be tyrosine (Y); X5 can be arginine (R) or lysine (K); X6 can be isoleucine (I) or threonine (T); and m can be D-methionine.

[0068] In one embodiment of the present invention, X1 can be a basic amino acid, such as arginine (R) or lysine (K); X2 can be an aromatic amino acid, such as or tryptophan (W); X3 can be a basic amino acid, such as arginine (R) or lysine (K); X4 can be tyrosine (Y); X5 can be leucine (L), asparagine (N), phenylalanine (F) or threonine (T); X6 can be tyrosine (Y) or valine (V); and m can be D-methionine.

[0069] In one embodiment of the present invention, X1 can be a basic amino acid, such as arginine (R); X2 can be an aromatic amino acid, such as or tryptophan (W); X3 can be a basic amino acid, such as arginine (R); X4 can be tyrosine (Y); X5 can be arginine (R) or lysine (K); X6 can be isoleucine (I) or threonine (T); and m can be D-methionine.

[0070] In one embodiment of the present invention, X1 can be a basic amino acid, such as arginine (R); X2 can be an aromatic amino acid, such as or tryptophan (W); X3 can be a basic amino acid, such as arginine (R); X4 can be tyrosine (Y); X5 can be leucine (L), asparagine (N), phenylalanine (F) or threonine (T); X6 can be tyrosine (Y) or valine (V); and m can be D-methionine.

[0071] In one embodiment of the present invention, any one selected from the group consisting of an acetyl group, a butyryl group, a hexanoyl group, and an octanoyl group may be added to the N-terminus of the peptide.

[0072] In one embodiment of the present invention, the C-terminus of the peptide may be amidated.

[0073] In one embodiment of the present invention, the peptide may consist of any one of the amino acid sequences in SEQ ID NOs: 152-160.

[0074] In this specification, the peptide can be used directly or indirectly as an immunomodulatory peptide to exert an immunomodulatory effect, such as regulating the inflammatory response of immune cells, regulating the migration of immune cells, regulating the inflammatory response of epidermal or endothelial cells. In the present invention, the immunomodulatory peptide can induce inflammation resolution by activating phagocytes such as neutrophils and monocytes, antigen presenting cells such as dendritic cells, immune cells such as eosinophils, basophils, mast cells and microglia, neuronal cells, epidermal cells and endothelial cells expressed in formyl peptide receptors (FPR2), thereby exhibiting immunomodulatory function.

[0075] As confirmed in the experimental examples described later, the peptide of the present invention showed high FPR2 activation efficacy even at low concentrations, and was also confirmed to have high selectivity compared to FPR1. In addition, the present inventors confirmed that inflammatory cytokines (TNFα, IL-6, IL-1β) were lower than those in the control group by measuring changes in intracellular cytokine gene expression during inflammation, and demonstrated the efficacy of the peptide of the present invention in inhibiting inflammatory responses at the individual level in an animal model.

[0076] Therefore, the present invention provides a pharmaceutical composition for preventing or treating an immune disease comprising the above-mentioned peptide as an active ingredient, use of the peptide for preventing or treating an immune disease, and a method for preventing or treating an immune disease, the method comprising administering the peptide to a subject.

[0077] In one embodiment of the present invention, the immune disease can be atopic dermatitis, psoriasis, infantile eczema, sepsis, conjunctivitis, keratitis, dry eyes, asthma, lung injury, lung inflammation, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, peritonitis, systemic sclerosis, neuroinflammation, Parkinson's disease or Alzheimer's disease, but the present invention is not limited thereto.

[0078] In the present invention, the peptide can be used as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for preventing or treating atopic dermatitis. For more details, see Baxendell et al. J Immunol May 1, 2018, 200 (1 Supplement) 105.4.

[0079] In the present invention, the peptide can be used as a ligand that binds to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating psoriasis. For more information, see Liu et al. Sci Rep. 2017 Aug 2; 7(1): 7100. doi: 10.1038 / s41598-017-07485-1.

[0080] In the present invention, the peptide can be used as a ligand binding to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating infantile eczema. For more details, see Wu et al. Br JDermatol. 2013 Jan; 168(1): 172-8. doi: 10.1111 / j.1365-2133.2012.11177.x.

[0081] In the present invention, the peptide can be used as a ligand binding to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating sepsis. For more details, see Walker et al. SHOCK, Vol. 36, No. 4, pp. 410Y416, 2011.

[0082] In the present invention, the peptide can be used as a ligand binding to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating conjunctivitis. For more details, see Hodges et al. Mucosal Immunol. 2017 Jan; 10(1): 46-57. doi: 10.1038 / mi.2016.33. Epub 2016 Apr 13.

[0083] In the present invention, the peptide can be used as a ligand that binds to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating keratitis. For more details, see Zhu et al. Int Immunopharmacol. 2021 Jul; 96: 107785. doi: 10.1016 / j.intimp.2021.107785. Epub 2021 May 24.

[0084] In the present invention, the peptide can be used as a ligand binding to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating dry eye. For more details, see Gao et al. J Immunol. 2015 Oct 1; 195(7): 3086-99. doi: 10.4049 / jimmunol.1500610. Epub 2015 Aug 31.

[0085] In the present invention, the peptide can be used as a ligand binding to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating asthma. For more details, see Barnig et al. Eur Respir Rev. 2015 Mar; 24(135): 141-53. doi: 10.1183 / 09059180.00012514.

[0086] In the present invention, the peptide can be used as a ligand that binds to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating lung injury. For more details, see Ba ​​et al. J Thorac Dis. 2019 Aug; 11(8): 3599-3608. doi: 10.21037 / jtd.2019.08.86.

[0087] In the present invention, the peptide can be used as a ligand that binds to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating pulmonary inflammation. For more details, see Sekheri et al. Proc Natl Acad Sci US A. 2020 Apr 7; 117(14): 7971-7980. doi: 10.1073 / pnas.1920193117. Epub 2020 Mar 23.

[0088] In the present invention, the peptide can be used as a ligand binding to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating rheumatoid arthritis. For more details, see Kao et al. Br J Pharmacol. 2014 Sep; 171(17): 4087-96. doi: 10.1111 / bph.12768.

[0089] In the present invention, the peptide can be used as a ligand binding to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating ankylosing spondylitis. For more details, see Kao et al. Br J Pharmacol. 2014 Sep; 171(17): 4087-96. doi: 10.1111 / bph.12768.

[0090] In the present invention, the peptide can be used as a ligand binding to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating ulcerative colitis or Crohn's disease. For more details, see Kim et al. Exp Mol Med. 2013 Sep 13; 45(9): e40. doi: 10.1038 / emm.2013.77.

[0091] In the present invention, the peptide can be used as a ligand that binds to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating systemic sclerosis. For more details, see Park et al. Front Immunol. 2019 Sep 3; 10: 2095. doi: 10.3389 / fimmu.2019.02095. eCollection 2019.

[0092] In the present invention, the peptide can be used as a ligand that binds to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating neuroinflammation. For more details, see Ponce et al. Front Aging Neurosci. 2022 Feb 17; 14: 780811. doi: 10.3389 / fnagi.2022.780811. eCollection 2022.

[0093] In the present invention, the peptide can be used as a ligand that binds to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating Parkinson's disease. For more details, see Krashia et al. Nat Commun. 2019 Sep 2; 10 (1): 3945. doi: 10.1038 / s41467-019-11928-w.

[0094] In the present invention, the peptide can be used as a ligand that binds to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating Alzheimer's disease. For more details, see Pamplona et al. Transl Psychiatry. 2022 Oct 10; 12(1): 439. doi: 10.1038 / s41398-022-02208-1.

[0095] In the present invention, the peptide can act as a pro-resolving factor that binds to the FPR2 receptor.

[0096] Furthermore, the present inventors have also demonstrated that when an animal model is treated with the peptide of the present invention, tumor growth is inhibited. Thus, it has been demonstrated that the peptide that induces resolution of inflammation can inhibit tumor growth through immunomodulation.

[0097] Therefore, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the peptide as an active ingredient, use of the peptide for preventing or treating cancer, and a method for preventing or treating cancer, which comprises administering the peptide to a subject.

[0098] In one embodiment of the present invention, the cancer can be a solid cancer or a non-solid cancer, and can be gastric cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, colorectal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumors, urethral cancer, prostate cancer, bronchial cancer, bone marrow tumors or lymphoma.

[0099] Preferably, the cancer may be breast cancer, colon cancer, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, melanoma or lymphoma, but the present invention is not limited thereto.

[0100] In the present invention, the peptide can serve as a ligand binding to the FPR2 receptor and can be used in a pharmaceutical composition for preventing or treating cancer. For more details, see Kim et al. PLoS One. 2012;7(1):e30522.doi:10.1371 / journal.pone.0030522.Epub 2012Jan 25; Wang et al. Antioxidants (Basel). 2021Sep14;10(9):1459.doi:10.3390 / antiox10091459.; Liu et al. Cancer Res. 2013Jan 15;73(2):550-60.doi:10.1158 / 0008-5472.CAN-12-2290.Epub 2012Nov 8; Gilligan et al. Proc Natl Acad Sci US A. 2019Mar 26;116(13):6292-6297.doi:10.1073 / pnas.1804000116.Epub 2019Mar 12;Du et al.AnnTransl Med.2021May;9(9):802.doi:10.21037 / atm-21-1873.Erratumin:Ann TranslMed.2022Apr;10(7):428;Sulciner et al.J Exp Med.2018Jan 2;215(1):115-140.doi:10.1084 / jem.20170681.Epub 2017Nov 30;Zhang et al.Front Immunol.2017Feb 2;8:71.doi:10.3389 / fimmu.2017.00071; Liotti et al. Cancers(Basel).2022Jul 8;14(14):3333.doi:10.3390 / cancers14143333.

[0101] The term "prevention" as used herein refers to all behaviors of suppressing or delaying immune diseases or cancers by administering the pharmaceutical composition of the present invention, and the term "treatment" as used herein refers to all behaviors of alleviating or beneficially changing immune diseases or cancers by administering the pharmaceutical composition of the present invention. "Subject" as used herein refers to an individual who needs to prevent or treat a disease, more specifically, any mammal, for example, including not only humans or other primates, but also including but not limited to livestock such as cattle, pigs, sheep, horses, dogs and cats, but preferably humans.

[0102] The route of administration of the pharmaceutical composition comprising peptide as an active ingredient can be any general route capable of delivering the drug to the target tissue, and the pharmaceutical composition can be applied to the object by oral or parenteral routes. For example, the pharmaceutical composition can be provided by abdominal cavity, intravenous, intraarterial, intramuscular, subcutaneous, intradermal, oral, local (including skin application, instillation and inhalation), transdermal, intrathecal and epidural, intranasal, intraocular, intrapulmonary, rectal or intravaginal administration, but the present invention is not limited thereto. In addition, the pharmaceutical composition comprising peptide as an active ingredient can be applied in any convenient form of pharmaceutical product, such as tablets, powders, granules, capsules, oral liquids, solutions, dispersants, suspensions, syrups, sprays, suppositories, gels, emulsions or patches. However, since the peptide can be digested during oral administration, the oral composition is preferably formulated as a package active agent or protected from decomposition in the stomach.

[0103] The pharmaceutical composition comprising the peptide of the present invention can also include pharmaceutically or physiologically acceptable carriers, excipients and diluents. The term "pharmaceutically acceptable carriers, excipients and diluents" used herein refers to carriers, excipients and diluents that do not stimulate the organism and do not inhibit the biological activity and properties of the administered compound. Examples of suitable carriers, excipients and diluents that can be included in the composition can include saline, sterile water, Ringer's solution, buffered saline, albumin injection, glycerol, ethanol, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When the composition is formulated for pharmaceutical use, it may also include conventional dispersants, fillers, extenders, binders, disintegrants, surfactants, anticoagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives or lyophilizers.

[0104] In the present invention, the pharmaceutical composition can be administered in the form of an injection. The injection can be formulated as an intravenous injection or a subcutaneous injection. In the case of a parenteral injection, the ingredients contained in a general injection composition can be included. For example, the injection composition includes liquid carriers such as sterile water, water for injection, and physiological saline. In addition, the injection composition may also contain amino acids, sugars, lipids, vitamins, electrolytes, pH regulators, stabilizers, osmotic pressure regulators, or solubilizers.

[0105] In the present invention, when applied topically, the pharmaceutical composition can be formulated into the form of, for example, an ointment, a gel, a cream or a lotion. The type of topical application can be, but is not limited to, for example, application to the skin, dropping into the eyes, using microneedle transdermal penetration or intradermal injection. For example, it is preferred to apply the composition or attach a patch containing the composition. The composition may include, for example, a base material, an excipient, a lubricant and a preservative. For example, when the pharmaceutical composition is applied in the form of eye drops, a buffer, a viscosity agent, an isotonic agent, a pH adjusting agent or a solvent may also be included. In addition, when the pharmaceutical composition is applied in the form of an ointment for the skin, a gelling agent, a stabilizer, an emulsifier or a suspending agent may also be included.

[0106] In addition, the pharmaceutical composition of the present invention can be used differently according to the purpose of application and the disease. The amount of the active ingredient actually applied can be appropriately selected by a person of ordinary skill in the art according to various relevant factors such as the disease to be treated, the severity of the disease, whether it is co-administered with other drugs, the activity of the drug, the sensitivity to the drug, the age, sex, weight, diet, administration time, administration route, and the administration ratio of the composition. The composition can be administered once a day or in 1 to 3 times, but the dosage and administration route can be adjusted according to the type and severity of the disease.

[0107] The pharmaceutical composition comprising the peptide of the present invention as an active ingredient can be administered at a dose of 0.001 mg / kg to 1 g / kg, more preferably 0.1 mg / kg to 100 mg / kg. However, the dose can be appropriately adjusted according to the age, sex and condition of the patient.

[0108] In addition, the present invention also provides an anti-inflammatory cosmetic composition comprising the peptide as an active ingredient, use of the peptide for inhibiting inflammation, and a method for inhibiting inflammation, which comprises applying the peptide to a subject.

[0109] The anti-inflammatory cosmetic composition comprising the peptide of the present invention can be made into a general emulsion preparation or a solubilized preparation. For example, the anti-inflammatory cosmetic composition can be formulated into the following forms: solution, external ointment, toner (e.g., skin softener or nourishing water), emulsion (e.g., facial lotion or body lotion), cream (e.g., nourishing cream, moisturizing cream or eye cream), essence, cosmetic cream, spray, gel, mask, sunscreen, primer, liquid, solid or spray foundation, loose powder, makeup removal product (e.g., cleansing cream, makeup removal liquid or makeup removal oil), cleansing foam, cleanser (e.g., soap or shower gel), oil or patch, but the present invention is not limited thereto.

[0110] In the present invention, the cosmetic composition may further include one or more supplements and carriers conventionally used in the cosmetic field, such as pearlescent agents, thickeners, viscosity modifiers, pH regulators, lipid substances, organic solvents, solubilizers, concentrates, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, chelating agents, preservatives, vitamins, occlusive agents, moisturizers, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators, lipid vesicles or any other ingredients commonly used in cosmetics.

[0111] Below, by referring to the embodiments and drawings described in detail below, the advantages and features of the present invention and the methods for implementing the present invention will become apparent. However, the present invention is not limited to the exemplary embodiments disclosed below and can be embodied in many different forms. These exemplary embodiments are only used to make the disclosure of the present invention complete and fully convey the scope of the present invention to those of ordinary skill in the art, and the present invention should only be limited by the appended claims.

[0112] Example 1: Preparation of immunomodulatory peptides

[0113] In the present invention, in order to synthesize novel peptides having excellent immunomodulatory function compared with conventional peptides, conventional amino acid synthesis method (Umbarger, HE, Ann Rev Biochem., 47:533-606, 1978) is adopted, and the synthesized peptides are referred to as immunomodulatory candidate peptides. In addition, the inventors have chemically modified the N-terminal and / or C-terminal of the synthetic peptides. The synthesized peptide sequence is shown in Table 1.

[0114] [Table 1]

[0115]

[0116]

[0117]

[0118]

[0119] Experimental Example 1: Measuring changes in calcium activity caused by peptides

[0120] The present inventors confirmed whether the immunomodulatory peptide candidate prepared according to one embodiment of the present invention activated the immune function of the organism by observing the change of calcium ion permeability. Specifically, whether the peptide activated FPR2 was confirmed by measuring the calcium ion concentration in the cell.

[0121] 1) Preparation of cells and peptides

[0122] The present inventors used RBL cells that do not express FPR2 (RBL-WT) and RBL cells that overexpress FPR2 (RBL-FPR2), and used Fura-2 / AM, a staining substance that has a strong binding ability to calcium, as a method for sensitively measuring intracellular calcium ion release.

[0123] Peptides dissolved in water or 50% DMSO at a concentration of 0.5 mM were diluted to the appropriate concentration in HBSS (Sigma, #55037C) + 20 mM HEPES buffer.

[0124] 2) Culture conditions

[0125] In this experiment, cells were cultured in DMEM (Lonza, #BE12-604F) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0126] 3) Experimental methods

[0127] The day before measuring calcium activity, 3 × 10 5 Cells were prepared in a 96-well transparent flat-bottom black culture plate (Costar, #3603) at a density of 10 cells / well. The prepared cells were treated with the same volume of Fura-2 QBT calcium kit (component A; Molecular Devices, #8197) per well. After incubation at 37°C for 1 hour, the cells were incubated in a dark room at room temperature for 30 minutes. Fura-2 treated cells, prepared peptides and special pipette tips were placed on various parts of the FlexStation 3 multifunctional microplate reader (Molecular Devices, #Flex3), and then the changes in calcium activity caused by the peptides were measured using SoftMax Pro 7 software (Molecular Devices, #FLEX3-INSTALL-OS).

[0128] 4) Experimental results

[0129] The results showed that most peptides exhibited FPR2 activation, indicating that the peptides of the present invention can regulate the immune function of individuals by binding to and activating FPR2. The analysis results of the FPR2 activation effect of the peptides were graded according to the numerical values ​​and summarized in the following Table 2. The standards for each grade are shown in Table 3.

[0130] [Table 2]

[0131] Analysis of FPR2 and FPR1 activation effects of peptides

[0132]

[0133]

[0134]

[0135] [Table 3]

[0136]

[0137] As shown in Table 2, it was confirmed that the immunomodulatory peptides of the present invention have an EC 50 The values ​​are usually 100 nM or lower, but the EC for FPR1 50 The value was 10,000 nM or higher. Thus, it was confirmed that the immunomodulatory peptide specifically binds to FPR2.

[0138] Experimental Example 2: Measuring the Cytokine Inhibitory Ability of Peptides

[0139] The inventors observed the cytokine inhibitory ability of the immunomodulatory peptide candidate prepared according to one embodiment of the present invention. Specifically, in order to confirm whether the peptide can reduce the inflammatory response of cells, the changes in cytokine gene expression in cells during inflammation were measured.

[0140] 1) Preparation of cells and peptides

[0141] The present inventors used RAW264.7 cell line, i.e., mouse macrophages, and lipopolysaccharide (LPS; Sigma-Aldrich, #L2630), a major component of the cell wall of Gram-negative bacteria, as a substance that induces an inflammatory response. A peptide dissolved in water at a concentration of 100 μM was prepared.

[0142] 2) Culture conditions

[0143] In this experiment, cells were cultured in DMEM (Lonza, #BE12-604F) containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0144] 3) Experimental methods

[0145] The cells were cultured at 2.5 × 10 5 The cells were seeded at a density of 10 cells / well in a 24-well plate (Costar, #3603). After 24 hours, the prepared cells were treated with 100 nM of peptide. After incubation at 37°C for 30 minutes, they were treated with 200 ng / mL of LPS to induce inflammation. After incubation at 37°C for 6 hours, the cells without culture medium were treated with TRIzol TM (Invitrogen, #15596018) was used to lyse the cells. RNA was obtained by a given experimental method and cDNA was generated using reverse transcriptase (Promega, #M1705). Subsequently, SYBR Green (Enzynomics, RT530) and mouse primers for each inflammatory cytokine gene (TNFα, IL-6 and IL-1β) were used to measure cytokine expression levels.

[0146] 4) Experimental results

[0147] The results confirmed that in most peptides, inflammatory cytokines (TNFα, IL-6 and IL-1β) were reduced compared to the control. This indicates that the peptides of the present invention can reduce the inflammatory immune response of individuals. The analysis results of the changes in cytokine gene expression of the peptides were graded according to the numerical values ​​and summarized in Table 4 below. The standards for each grade are shown in Table 5.

[0148] [Table 4]

[0149] Analysis of the ability of peptides to inhibit cytokine expression

[0150]

[0151]

[0152] [Table 5]

[0153] Reduction rate (%) grade 81％-100％ A 61％-80％ B 41％-60％ C 21％-40％ D 0％-20％ E <0％ F

[0154] In the following experimental examples, peptides with excellent effects were selected from candidate immunomodulatory peptides, and the efficacy of the peptides was confirmed in animal models.

[0155] Experimental Example 3: Confirmation of the efficacy of peptides in inhibiting inflammatory responses at the individual level

[0156] The inventors observed the inflammatory response inhibition efficacy at the individual level using the immunomodulatory peptide candidate prepared according to one embodiment of the present invention. Specifically, in order to confirm whether the peptide controls the inflammatory response at the individual level, the changes in cytokines were observed in the LPS (Sigma-Aldrich, #L2630)-induced acute peritonitis model.

[0157] 1) Methods of inducing acute peritonitis model and administering peptides

[0158] The present inventors used C57BL / 6 mice of about 6 weeks old and used LPS mixed with 5% mannitol (D-Mannitol; Sigma-Aldrich, #M8429) as a substance to induce peritoneal inflammatory disease. The inflammatory substance was intraperitoneally injected into the mice to induce peritoneal inflammatory disease, and the peptide was intraperitoneally injected once immediately after the injection of the inflammatory substance.

[0159] 2) Disease assessment methods

[0160] Blood and abdominal lavage fluid were collected 6 or 24 hours after the end of peptide administration and analyzed for inflammatory factors such as cytokines using enzyme-linked immunosorbent assay (ELISA) technology.

[0161] 3) Experimental results

[0162] The results confirmed that in most subjects, when the peptide was administered, inflammatory cytokines (TNFα, IL-6, IL-1β and IL-17a) were reduced compared with the control group. This indicates that the peptide of the present invention can inhibit inflammatory immune responses at the individual level. Figure 1 The results of analysis of cytokine changes after peptide administration are shown.

[0163] Experimental Example 4: Confirmation of the efficacy of peptides in improving rheumatoid arthritis

[0164] The inventors observed the efficacy of the immunomodulatory peptide candidate prepared according to one embodiment of the present invention on improving rheumatoid arthritis. Specifically, in order to confirm whether the peptide can reduce the inflammatory response in cells, the changes in clinical scores and arthritis symptoms such as plantar edema were measured in the collagen-induced arthritis (CIA) model.

[0165] 1) Method of inducing collagen-induced arthritis model and administering peptide

[0166] The inventors used DBA1 / J mice of about 8 to 12 weeks of age, mixed complete Freund's adjuvant (CFA) and bovine type II collagen, which are substances that cause inflammatory reactions, as arthritis-inducing substances. The inflammatory substance was injected into the tail of the mouse (the first injection), and the inflammatory substance was injected again about 2-3 weeks later (the second boost) to induce arthritis. The peptide was injected intraperitoneally once every two days for a total of 10 times over a few days.

[0167] 2) Methods for evaluating the improvement of rheumatoid arthritis

[0168] Body weight and visual changes were observed during the experiment to confirm the overall health of the animals. Severity scores of arthritis and changes in thickness due to edema of the sole and ankle were tracked and evaluated during the experiment. After the last administration of the substance, blood was collected and analyzed for inflammatory factors such as cytokines, and tissue from the ankle area was removed to form paraffin blocks for tissue analysis using hematoxylin and eosin (H&E) staining or immunohistochemistry.

[0169] 3) Experimental results

[0170] The results confirmed that the severity score of collagen-induced arthritis (CIA) was improved when the peptide was administered. This suggests that the peptide of the present invention can improve arthritis as an inflammatory disease at the individual level. The analysis results of the changes in the severity score of the peptide are shown in FIG. Figure 2 The evaluation criteria for severity scores are shown in Table 6.

[0171] [Table 6]

[0172]

[0173] Experimental Example 5: Confirmation of the efficacy of peptides in improving dry eye syndrome

[0174] The inventors observed the improvement effect of dry eye using the immunomodulatory peptide candidate prepared according to one embodiment of the present invention. Specifically, in order to confirm whether the peptide can improve dry eye, changes in dry eye symptoms such as corneal damage and corneal opacity were measured in a benzalkonium chloride (BAC)-induced dry eye (DED) model.

[0175] 1) Method of inducing BAC-induced dry eye model and administering peptide

[0176] The inventors used BAC as a dry eye inducer to treat C57BL / 6 mice of about 12 to 16 weeks of age. Topical administration was performed by instilling BAC into the right eye twice a day for a total of 16 days. Only BAC was administered for 4 consecutive days (8 times in total), and starting from the 5th day, the peptide was also topically administered by instillation in the same manner 30 minutes after BAC administration.

[0177] 2) Evaluation methods for dry eye syndrome

[0178] During the experiment, visual changes were observed every day, and corneal damage was confirmed under a microscope using a fluorescent indicator 0.2% fluorescein on days 4, 8, 11, and 16 after the start of the application. Since the area stained with fluorescein indicates corneal damage, the extent of corneal damage was evaluated by calculating the stained area. At the same time, the eyes were observed under a microscope and the degree of corneal opacity was scored.

[0179] 3) Experimental results

[0180] The results confirmed that the administration of the peptide improved corneal damage and corneal opacity caused by BAC. This indicates that the peptide of the present invention can improve the symptoms of dry eye in individuals. The analysis results of the degree of corneal damage and corneal opacity of the peptide are as follows Figure 3 shown.

[0181] Experimental Example 6: Confirmation of the efficacy of peptides in improving colitis

[0182] The inventors observed the improvement effect of colitis using the immunomodulatory peptide candidate prepared according to one embodiment of the present invention. Specifically, in order to confirm whether the peptide can reduce the inflammatory response occurring in the large intestine, the changes in body weight and intestinal length of individuals were measured in the dextran sulfate sodium (DSS)-induced colitis (inflammatory bowel disease (IBD)) model.

[0183] 1) Method of inducing DSS-induced colitis model and administering peptide

[0184] The present inventors used C57BL / 6 mice of about 7 to 8 weeks old, dissolved 2.5% DSS as a colitis-inducing substance in water, and allowed them to eat freely. After 3 days, the original water was poured out, and 2.5% DSS water was re-prepared and provided for drinking for another two days. After a total of 5 days, it was replaced with clean water. From the second day of providing 2.5% DSS, 100 μL of the peptide was intraperitoneally administered once a day.

[0185] 2) Colitis assessment method

[0186] Body weight was measured every day during the experiment, and after 7 days, the mice were euthanized with CO2 and the intestinal length was measured.

[0187] 3) Experimental results

[0188] The results confirmed that weight loss was improved after administration of the peptide, and the intestinal length shortened by inflammation was restored. This indicates that the peptide of the present invention can improve the symptoms of colitis in individuals. The weight and intestinal length measurements of colitis mice are shown in Figure 2. Figure 4 shown.

[0189] Experimental Example 7: Neuroinflammation Inhibitory Effect of Peptides

[0190] The inventors observed the neuroinflammation inhibition efficacy using the immunomodulatory peptide candidate prepared according to one embodiment of the present invention. Specifically, in order to determine whether the peptide can reduce the inflammatory response of neuronal cells, the changes in cytokine gene expression in neuronal cells during inflammation were measured.

[0191] 1) Preparation of cells and peptides

[0192] The inventors used SK-N-MC cell line, i.e., human neuronal cells, and dorsal root ganglion (DRG) neurons, i.e., rat neuronal cells. In this experiment, the cells were cultured in neuronal cell culture medium at 37°C and 5% CO2. Lipopolysaccharide (LPS; Sigma-Aldrich, #L2630), a major component of the cell wall of Gram-negative bacteria, was used as a substance that causes an inflammatory response. Peptides dissolved in water at a concentration of 1 mM were prepared.

[0193] 2) Experimental methods

[0194] The cells were cultured at 2.5 × 10 5 The cells were seeded at a density of 10 cells / well in a 24-well plate (Costar, #3603). After 24 hours, the prepared cells were treated with 1 μM of peptide. After incubation at 37°C for 30 minutes, 500 ng / mL of LPS was used to induce inflammation. After incubation at 37°C for 6 hours, the cells without culture medium were treated with TRIzol TM (Invitrogen, #15596018) was used to lyse the cells. RNA was obtained by a given experimental method and cDNA was generated using reverse transcriptase (Promega, #M1705). Subsequently, SYBR Green (Enzynomics, RT530) and mouse primers for each inflammatory cytokine gene (IL-1β, IL-6) were used to measure cytokine expression levels.

[0195] 3) Experimental results

[0196] The results confirmed that in most of the peptides, inflammatory cytokines (IL-1β and IL-6) were reduced compared to the control. This indicates that the peptides of the present invention can reduce the neuroinflammatory response of individuals. The analysis results of the changes in cytokine gene expression after peptide treatment are shown in Table 5.

[0197] Experimental Example 8: The improving effect of peptides on neuroinflammation-induced degenerative brain disease

[0198] The inventors observed the efficacy of the immunomodulatory peptide candidate prepared according to one embodiment of the present invention on improving neuroinflammatory encephalopathy. Specifically, in order to confirm whether the peptide can alleviate Parkinson's disease based on the inhibitory effect of the inflammatory response of neuronal cells, the changes in the number of dopaminergic neurons and the density of terminal fibers in brain tissue were analyzed in an MPTP-induced Parkinson's disease animal model.

[0199] 1) Methods for inducing MPTP-induced Parkinson's disease model and administering peptides

[0200] The inventors used C57BL / 6 mice of about 7 to 8 weeks old and used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride, which induces neuroinflammation, as a brain lesion-inducing substance. 15 mg / kg MPTP was intraperitoneally administered four times to induce the disease, and then the peptide was intraperitoneally administered once a day for 7 days, for a total of seven times.

[0201] 2) Methods for evaluating the improvement of Parkinson's disease

[0202] On the second day after the last administration, mice were anesthetized, perfused with phosphate buffer solution and fixative (4% paraformaldehyde) to remove blood and fix tissues, and then brains were extracted. Using the extracted brains, frozen blocks were made, slice slides were made, and then dopaminergic neurons were stained using TH staining. The stained tissues were then photographed under a microscope, and the recovery of dopaminergic neuron number and nerve terminal fiber density was analyzed.

[0203] 3) Experimental results

[0204] The results confirmed that when the immunomodulatory peptide was administered, the nerve terminal fiber density of dopaminergic neurons that was reduced by MPTP was restored. This suggests that the peptide of the present invention can improve Parkinson's disease by resolving inflammation and restoring dopaminergic neurons affected by neuroinflammation at the individual level. The analysis results of the severity score changes of the peptide are shown in Figure 6 shown.

[0205] Experimental Example 9: Confirmation of the ability of peptides to inhibit tumor growth

[0206] The inventors observed the tumor growth inhibition ability using the immunomodulatory peptide candidate prepared according to one embodiment of the present invention. Specifically, in order to confirm whether the peptide inhibits tumor growth, the change of tumor size was measured in a cancer cell implantation animal model.

[0207] 1) Cancer cell implantation model and peptide administration method

[0208] The present inventors used C57BL / 6 mice of about 8 weeks old and CT-26 cell line as colon cancer cells. 2.5×10 5 cells, and induced tumor growth for about 2 weeks. Animals whose tumors had grown to uniform size were selected and randomly assigned to drug administration groups. Subsequently, the peptide was injected intraperitoneally five times a week, and 5-fluorouracil (5-FU) was injected twice a week as a positive control drug.

[0209] 2) Evaluation methods for tumor growth inhibition

[0210] During the experiment, body weight and visual changes were observed to confirm the overall health of the animals. The length and width of the tumor were measured every 3 to 4 days, assuming that the tumor was an ellipsoid, and the tumor volume of the ellipsoid was calculated. Changes in tumor volume were tracked during the experiment to evaluate the efficacy of tumor growth inhibition. After the last substance administration, blood was collected for analysis of inflammatory factors or tumor-related factors, and paraffin blocks were made from the extracted tumor tissue, and tissue analysis was performed using hematoxylin and eosin (H&E) staining or immunohistochemistry.

[0211] 3) Experimental results

[0212] The results confirmed that when the peptide was administered, the growth of tumors formed by implantation of the CT-26 cell line was inhibited. This indicates that the peptide of the present invention can inhibit tumor growth through immunomodulation based on the inflammation-resolving function. The analysis results of the changes in the severity scores of the peptides are shown in FIG. Figure 7 shown.

[0213] The present invention has been described in conjunction with the above embodiments and experimental examples, but this is merely exemplary, and those skilled in the art should understand that various modifications and equivalent embodiments and experimental examples can be made therefrom. Therefore, the true scope of the present invention should be determined according to the technical essence of the appended claims.

Claims

1. A peptide consisting of an amino acid sequence represented by X1-X2-X3-X4-X5-X6-m, wherein X1 is absent, or X1 is arginine (R), lysine (K), histidine (H) or proline (P); X2 is methionine (M) or tryptophan (W); X3 is a basic amino acid, such as arginine (R) or lysine (K); X4 is tyrosine (Y); X5 is a basic amino acid, such as arginine (R) or lysine (K), or an aromatic amino acid, such as tryptophan (W) or tyrosine (Y); X6 is proline (P), tyrosine (Y) or valine (V); and m is D-methionine.

2. The peptide according to claim 1, wherein X1 is absent, or X1 is arginine (R), lysine (K), histidine (H) or proline (P); X2 is methionine (M) or tryptophan (W); X3 is a basic amino acid, such as arginine (R) or lysine (K); X4 is tyrosine (Y); X5 is arginine (R) or lysine (K); X6 is valine (V), tyrosine (Y) or proline (P); and m is D-methionine.

3. The peptide according to claim 1, wherein X1 is absent, or X1 is a basic amino acid, such as arginine (R) or lysine (K); X2 is methionine (M) or tryptophan (W); X3 is a basic amino acid, such as arginine (R) or lysine (K); X4 is tyrosine (Y); X5 is an aromatic amino acid, or tryptophan (W); X6 is valine (V), tyrosine (Y) or proline (P); and m is D-methionine.

4. The peptide according to claim 1, wherein X1 is absent, or X1 is a basic amino acid, such as arginine (R) or lysine (K); X2 is methionine (M) or tryptophan (W); X3 is a basic amino acid, such as arginine (R); X4 is tyrosine (Y); X5 is tyrosine (Y); X6 is valine (V), tyrosine (Y) or proline (P); and m is D-methionine. 5 . The peptide according to claim 1 , wherein any one selected from the group consisting of an acetyl group, a butyryl group, a hexanoyl group, and an octanoyl group is added to the N-terminus of the peptide. The peptide according to claim 1 , wherein the C-terminus of the peptide is amidated.

7. The peptide according to claim 1, which consists of any one of the amino acid sequences of SEQ ID NOs: 1-127.

8. A peptide consisting of an amino acid sequence represented by X1-X2-X3-X4-X5-X6-m, wherein X1 is a basic amino acid, such as arginine (R) or lysine (K); X2 is an aromatic amino acid, or tryptophan (W); X3 is a basic amino acid, such as arginine (R) or lysine (K); X4 is tyrosine (Y); X5 is arginine (R), lysine (K), leucine (L), asparagine (N), phenylalanine (F), or threonine (T); X6 is isoleucine (I), threonine (T), tyrosine (Y) or valine (V); and m is D-methionine.

9. A pharmaceutical composition for preventing or treating an immune disease or cancer, comprising the peptide according to any one of claims 1 to 8 as an active ingredient.

10. The pharmaceutical composition of claim 9, wherein the immune disease is atopic dermatitis, psoriasis, infantile eczema, sepsis, conjunctivitis, keratitis, dry eye, asthma, lung injury, lung inflammation, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, peritonitis, systemic sclerosis, neuroinflammation, Parkinson's disease or Alzheimer's disease.

11. An anti-inflammatory cosmetic composition comprising the peptide according to any one of claims 1 to 8 as an active ingredient.

Images