Peptides having anti-inflammatory activity and compositions containing the same

Abstract

To provide anti-inflammatory peptides, and compositions comprising the same.SOLUTION: The present invention relates to a peptide comprising an amino acid sequence of SEQ ID NO: 1, a peptide having 80% or more sequence homology with the sequence of SEQ ID NO: 1, or a peptide being a fragment thereof, and to an anti-inflammatory composition comprising the same as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to peptides having anti-inflammatory activity and compositions containing the same. [Background technology]

[0002] Inflammation is one of the body's defense reactions to prevent damage to living tissues caused by external physical stimuli, chemical stimuli such as contact with various allergy-inducing substances, or the invasion of microorganisms such as bacteria, fungi, or viruses.

[0003] Inflammatory signals are produced via the cyclooxygenase (COX) pathway or lipoxygenase (LOX) pathway, producing prostaglandins, thromboxanes, etc. When inflammatory signals are transmitted, various changes occur in the body, including the dilation of blood vessels in the necessary areas, increasing blood supply and concentrating blood cells, such as neutrophils, that are necessary for the inflammatory response. However, if such a defense response of the body occurs abnormally or excessively, various inflammatory diseases can develop. To prevent this, drugs have been developed that can suppress excessive inflammatory responses by blocking the inflammatory signaling pathway by inhibiting enzymes in the inflammatory signaling pathway (e.g., COX-1, COX-2, 5-LOX, 12-LOX, etc.).

[0004] Depending on the duration of the reaction, inflammation can be divided into acute inflammation (an immediate, non-specific reaction lasting several days to several weeks), chronic inflammation (a delayed, specific reaction lasting several weeks or more), and subacute inflammation (an intermediate stage between acute and chronic inflammation, characterized by a mixed product of polynuclear cells and mononuclear cells).

[0005] In addition to peptide factors, factors that induce inflammation include lipid factors such as prostaglandins, leukotrienes, and platelet-activating factors, inflammatory factor-synthesizing enzymes, free radicals such as nitric oxide (NO), various types of cell adhesion molecules, the immune system, and coagulation factors.

[0006] The currently known mechanism of inflammation is that cell damage caused by external biological factors (bacteria, viruses, parasites), physical factors (mechanical stimuli, heat, radiation, electricity), or chemical factors results in the release of histamine and kinins, which in turn causes vasodilation, increased capillary permeability, and the accumulation of macrophages at the site of inflammation, resulting in increased blood flow to the infected area, edema, migration of immune cells and antibodies, pain, fever, and other symptoms.

[0007] Currently available anti-inflammatory drugs include synthetic drugs such as ibuprofen, antihistamines, steroids, cotison, immunosuppressants, and immunostimulants. However, these drugs often only provide temporary therapeutic effects, merely relieve symptoms, or have numerous side effects, such as hypersensitivity reactions and immune system deterioration, making it difficult to fundamentally treat inflammation. Therefore, research has recently been conducted into substances that can inhibit the expression of inflammation-related proteins in order to effectively alleviate inflammation. However, the anti-inflammatory substances developed through such research have been plagued by several side effects. Anti-inflammatory drugs with various mechanisms, including nonsteroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SAIDs), have been developed. However, they not only exhibit significant side effects but also do not fundamentally suppress the inflammatory response. Therefore, there remains a need for more effective, safe, and cost-effective drugs. For example, nonsteroidal anti-inflammatory drugs (NSAIDs), which are used to treat chronic inflammatory diseases such as acute arthritis or rheumatoid arthritis, are known to inhibit not only COX-2 enzyme but also COX-1 enzyme, resulting in side effects such as gastrointestinal disorders. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] KR2012-013996A [Patent Document 2] KR2012-0133661A [Patent Document 3] KR2011-0060940A [Patent Document 4] US2011-0150873A1 [Non-patent literature]

[0009] [Non-Patent Document 1] Bonaldi T et al., EMBOJ, (22)5551-60, 2003 [Non-patent document 2] Yankner BA et al., Science(New York, NY) [1990, 250(4978): 279-282] [Non-patent document 3] Dahlgren KN et al., J. Biol. Chem. 277: 32046-32053, 2002 Summary of the Invention [Problem to be solved by the invention]

[0010] The present inventors discovered that peptides derived from telomerase have anti-inflammatory activity, and have completed the present invention.

[0011] Therefore, an object of the present invention is to provide a novel peptide.

[0012] Another object of the present invention is to provide a polynucleotide encoding the novel peptide.

[0013] It is yet another object of the present invention to provide a peptide having anti-inflammatory activity.

[0014] It is still another object of the present invention to provide an anti-inflammatory composition containing a peptide having anti-inflammatory activity as an active ingredient.

[0015] It is still another object of the present invention to provide a cosmetic composition containing a peptide having anti-inflammatory activity as an active ingredient.

[0016] It is still another object of the present invention to provide a pharmaceutical composition containing, as an active ingredient, a peptide having anti-inflammatory activity. [Means for solving the problem]

[0017] According to one aspect of the present invention, there is provided a peptide having anti-inflammatory activity, the peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having at least 80% sequence identity with the sequence of SEQ ID NO: 1, or a fragment of said peptide.

[0018] According to another aspect of the invention, the fragment may be a fragment consisting of three or more amino acids, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 amino acids.

[0019] According to another aspect of the invention, the peptide may consist of 30 or fewer amino acids.

[0020] According to another aspect of the present invention, the peptide may also be a peptide consisting of the amino acid sequence of SEQ ID NO: 1. For example, the peptide may also be a fragment consisting of 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9 or 8 amino acids.

[0021] According to another aspect of the invention, the peptide is also derived from human telomerase.

[0022] According to one aspect of the present invention, there is provided a polynucleotide encoding a peptide having anti-inflammatory activity, the polynucleotide encoding a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having 80% or more sequence identity to the sequence of SEQ ID NO: 1, or a peptide which is a fragment thereof.

[0023] In another aspect of the polynucleotide of the present invention, the fragment may be a fragment consisting of three or more amino acids, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acids.

[0024] In another aspect of the polynucleotide of the present invention, the peptide may be a peptide consisting of 30 or fewer amino acids, for example, a fragment consisting of 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8 amino acids.

[0025] In another aspect of the present invention, the peptide comprises the amino acid sequence of SEQ ID NO:1.

[0026] In the polynucleotide according to another aspect of the present invention, the peptide is derived from human telomerase.

[0027] According to one aspect of the present invention, there is provided an anti-inflammatory composition comprising, as an active ingredient, a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having greater than 80% sequence identity with the sequence of SEQ ID NO: 1, or a peptide that is a fragment thereof.

[0028] In a composition according to another aspect of the invention, the fragment consists of at least 3 amino acids (see above).

[0029] In a composition according to another aspect of the invention, the peptide consists of 30 or fewer amino acids (see above).

[0030] In a composition according to another aspect of the present invention, the peptide consists of the amino acid sequence of SEQ ID NO:1.

[0031] In a composition according to another aspect of the present invention, the peptide is derived from human telomerase.

[0032] In another aspect of the present invention, the composition is for treating or preventing an inflammatory disease.

[0033] In another aspect of the present invention, the composition is a cosmetic composition for improving or preventing skin inflammation.

[0034] In another aspect of the present invention, the composition is a pharmaceutical composition for treating or preventing an inflammatory disease.

[0035] In another aspect of the present invention, the composition is a food composition for treating or preventing inflammation.

[0036] In another aspect of the present invention, the inflammatory disease is (1) a systemic or local inflammatory disease (e.g., allergy; immune complex disease; hay fever; hypersensitivity shock; toxin-resistant shock; cachexia, hyperthermia; granulomatosis; or sarcoidosis); (2) a gastrointestinal disease (e.g., appendicitis; gastric ulcer; duodenal ulcer; peritonitis; pancreatic enteritis; ulcerative, acute, or ischemic colitis; cholangitis); cholecystitis, steatorrhea, hepatitis, Crohn's disease, or Whipple's disease; (3) skin-related diseases (e.g., psoriasis, burns, sunburn, dermatitis, urticarial warts or wheals); (4) cardiovascular diseases (e.g., vasculitis, vasculitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, congestive heart failure, myocarditis, myocardial ischemia, periarteritis nodosa, recurrent stenosis, Burger's disease, or Lewy's disease) (5) respiratory diseases (e.g., asthma; epiglottitis; bronchitis; emphysema; rhinitis; cystic fibrosis; epileptic pneumonia; chronic obstructive pulmonary disease (COPD); adult respiratory syndrome; pneumoconiosis; alveolitis; bronchiolitis; pharyngitis; pleurisy; or sinusitis); (6) bone, joint, muscle, and connective tissue-related diseases (e.g., granulomatous granulomatosis; arthritis; arthralgia; osteomyelitis; dermatomyositis) (7) diseases of the genitourinary system (e.g., epididymitis, vaginitis, prostatitis, or urethritis); (8) diseases of the central or peripheral nervous system (e.g., Alzheimer's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillain-Barre syndrome, etc.) syndrome); neuritis; neuralgia; bone marrow trauma; paralysis; or uveitis; (9) viral (e.g., influenza; respiratory cytoplasmic virus; HIV; hepatitis B virus; hepatitis C virus; or herpes virus) infectious diseases (e.g., dengue fever; or septicemia), fungal infectious diseases (e.g., candidiasis), or other microbial infectious diseases such as bacterial or parasitic diseases (e.g., disseminated bacteremia; malaria; filariasis; or amebiasis); (10) autoimmune diseases (e.g., thyroiditis; lupus; Goodpasture's syndrome; allograft rejection; graft-versus-host disease; or diabetes); and (11) cancer or neoplastic diseases (e.g., Hodgkin's disease).

[0037] According to one aspect of the present invention, there is provided a method for treating or preventing inflammatory diseases by administering the anti-inflammatory composition described above.

[0038] According to one aspect of the present invention, there is provided a kit for preventing or treating an inflammatory disease, comprising: a peptide having anti-inflammatory activity, which is a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having more than 80% sequence identity with the sequence of SEQ ID NO: 1, or a peptide which is a fragment of said peptide, or a composition comprising said peptide; and instructions including at least one of the dosage, administration route, administration frequency, and indications for said peptide or composition. [Effects of the Invention]

[0039] According to the present invention, the peptide having the sequence of SEQ ID NO: 1 has excellent effects in suppressing inflammation and as a preventive measure. Therefore, the composition containing the peptide of the present invention can be used as an anti-inflammatory pharmaceutical composition or cosmetic composition, and can be used for the treatment and prevention of various different types of inflammatory diseases. [Brief explanation of the drawings]

[0040] [Figure 1] This graph shows the results of TNF-α ELISA performed on PBMC-derived monocytes that were stimulated with LPS (10 ng / ml) for 2 hours and then treated with each peptide, i.e., FITC, FITC-TAT, PEP1-FITC, and FITC-peptide, for 2 hours (**P<0.01; compared to the negative control group (FITC and FITC-TAT)). [Figure 2]HEK293 / null and HEK293 / TLR2 cell lines were transfected with NF-kB luciferase and treated with lipoprotein (10 ng / ml), FITC, and FITC-PEP1 (4 μM) for 18 hours, followed by luciferase analysis. Luciferase results were normalized with renilla (**P<0.01 compared to the negative control (untreated) and lipoprotein-treated samples). [Figure 3] 1 is a graph showing the level of cytokine inhibition in THP1 cell line with untreated; LPS, PEP1 and LPS+PEP1 treatments, respectively. [Figure 4] 1 is a graph showing the viability of neural stem cells treated with beta-amyloid protein at concentrations of 0, 2.5, 5.0, 10, and 40 μM, respectively. [Figure 5] 1 is a graph showing proliferation of neural stem cells treated with beta-amyloid protein at concentrations of 0, 2.5, 5.0, 10, and 40 μM, respectively. [Figure 6] 1 is a graph showing the viability of neural stem cells treated with 0, 1, 10, 50, 100, and 200 μM PEP1, respectively. [Figure 7] 1 is a graph showing the proliferation of neural stem cells treated with 0, 1, 10, 50, 100, and 200 μM PEP1. [Figure 8] This is a graph showing the viability of neural stem cells measured after being damaged by 20 μM beta-amyloid protein and then treated with different concentrations of PEP1 (1, 10, 50, 100 μM) (the control group is a population not treated with beta-amyloid protein or telomerase-based peptide). [Figure 9] This graph shows the toxicity of neural stem cells measured after being damaged by 20 μM beta-amyloid protein and then treated with different concentrations of PEP1 (1, 10, 50, 100 μM) (the control group is a population not treated with beta-amyloid protein or telomerase-based peptide). [Figure 10]This is a graph showing the proliferation of neural stem cells measured after being damaged by 20 μM beta-amyloid protein and then treated with different concentrations of PEP1 (1, 10, 50, 100 μM) (the control group is a population not treated with beta-amyloid protein or telomerase-based peptide). [Figure 11] This is a graph showing the migration of neural stem cells measured after being damaged by 20 μM beta-amyloid protein and then treated with different concentrations of PEP1 (1, 10, 50, 100 μM) (the control group is a population not treated with beta-amyloid protein or telomerase-based peptide). [Figure 12] This is a graph showing the apoptosis of neural stem cells measured after being damaged by 20 μM beta-amyloid protein and then treated with different concentrations of PEP1 (1, 10, 50, 100 μM) (the control group is a population not treated with beta-amyloid protein or telomerase-based peptide). [Figure 13] This graph shows the inhibitory effect of PEP1 on reactive oxygen species in damaged neural stem cells, measured after treatment with different concentrations of PEP1 (1, 10, 50, 100 μM) following damage by 20 μM beta-amyloid protein (the control group is a group not treated with beta-amyloid protein or telomerase-based peptide). [Figure 14] This figure shows the results of protein expression levels measured by (A) 2D electrophoresis and (B) antibody array analysis after treatment with different concentrations of PEP1 (1, 10, 50 μM) following damage by 20 μM beta-amyloid protein (the control group is a group not treated with beta-amyloid protein or telomerase-based peptide). [Figure 15-1] FIG. 10 shows the results of Western blot analysis showing the expression levels of inflammation-related proteins (neural stem cells were damaged by 20 μM beta-amyloid protein and then treated with different concentrations of PEP1 (1, 10, 50 μM)). [Figure 15-2]FIG. 10 shows the results of Western blot analysis showing the expression levels of inflammation-related proteins (neural stem cells were damaged by 20 μM beta-amyloid protein and then treated with different concentrations of PEP1 (1, 10, 50 μM)). [Figure 16] 1 shows the inhibitory effect of PEP1 on beta-amyloid protein aggregation. (A) shows reduced oligomerization of beta-amyloid protein when 1 μM beta-amyloid protein and PEP1 (0.1, 1, 10 μM) are treated together, and (B) shows the case where beta-amyloid protein that has already been induced to aggregate is treated with PEP1. [Figure 17] The effect of LY294002, a PI3K inhibitor, on cell viability when treated with PEP 1 was shown; cell viability increased after treatment with PEP 1 and decreased after treatment with LY294002. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention can be modified in various ways and has various embodiments, and will be described in more detail below. However, it should be understood that the present invention is not limited to a specific embodiment, but includes all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention. In describing the present invention, if a detailed description of related publicly known technology is deemed to obscure the gist of the present invention, the detailed description will be omitted.

[0042] Telomeres are genetic material found repeatedly at the ends of chromosomes and are known to prevent damage to the chromosome or its subsequent attachment to other chromosomes. Each time a cell divides, the length of the telomere gradually shortens. After a certain number of cell divisions, the telomere length becomes so short that the cell stops dividing and dies. It is known that lengthening telomeres can extend the lifespan of cells. For example, cancer cells secrete an enzyme called telomerase to prevent telomere shortening, allowing them to survive and continue to grow. The present invention was made based on the discovery of a telomerase-derived peptide with anti-inflammatory effects.

[0043] According to one aspect of the present invention, there is provided a peptide having anti-inflammatory activity, the peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having greater than 80% sequence identity with said amino acid sequence, or a fragment thereof.

[0044] In the present invention, the peptide having anti-inflammatory activity is a peptide having the amino acid sequence of SEQ ID NO: 1. The peptide of SEQ ID NO: 1 is a peptide consisting of 16 amino acids located at positions [611-626] in telomerase.

[0045] SEQ ID NO:1 EARPALLTSRLRFIPK

[0046] One aspect of the present invention provides a polynucleotide encoding a peptide having anti-inflammatory activity, the peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having greater than 80% sequence identity with the amino acid sequence, or a fragment thereof. The polynucleotide can be used to mass-produce the peptide. For example, the peptide can be mass-produced by introducing a vector comprising a polynucleotide encoding the peptide into a host cell and culturing the cell.

[0047] The peptides disclosed herein may include peptides with greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99% sequence identity, and may include peptides comprising SEQ ID NO: 1 or fragments thereof, and peptides in which more than 1 amino acid, more than 2 amino acids, more than 3 amino acids, more than 4 amino acids, more than 5 amino acids, more than 6 amino acids, or more than 7 amino acids are changed.

[0048] As used herein, the terms "homology" and "sequence identity" are used interchangeably and refer to the degree of sequence overlap between two amino acids (or, for related purposes, nucleic acids).

[0049] Although the term "sequence identity" is not specifically used herein with respect to peptides or nucleic acids, sequence identity is understood to be a function of the sequence identity (n ref -n dif )*100 / n ref The formula is used to align two sequences, and when the highest number of matches is found, n dif is the total number of mismatched residues between the two sequences, ref means the total number of residues in the shorter of the two sequences. For example, the sequence identity of the DNA sequence agtcagtc with the sequence aatcaatc is 75% when calculated using the formula above (n ref =8, n dif =2).

[0050] In one aspect of the present specification, sequence identity was determined by the following conventional method: Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, using the CLUSTAL W algorithm of Thompson et al., 1994, Nucleic Acids Res 22:467380, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group). The BLAST algorithm (Altschul et al., 1990, Mol. Biol. 215:403-10), for which software may be obtained through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), may also be used. using any of the mentioned algorithms, the default parameters for "Window" length, gap penalty, etc.

[0051] In one aspect of the present invention, amino acid changes are made to alter the physicochemical properties of the peptide, for example, to improve the thermal stability of the peptide, alter its substrate specificity, or change its pH optimum.

[0052] In one aspect of the present invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having greater than 80% sequence identity to said amino acid sequence, or a peptide that is a fragment thereof may consist of 30 amino acids or less.

[0053] In one aspect of the present invention, the peptide having the sequence of SEQ ID NO: 1, a peptide having greater than 80% sequence identity to said peptide sequence, or a peptide that is a fragment thereof, comprises a peptide derived from telomerase, specifically human telomerase.

[0054] As used herein, the term "amino acid" refers not only to the 22 standard amino acids naturally incorporated into peptides, but also to D-isomers and modified amino acids. Accordingly, in one aspect of the present invention, a peptide may also contain a D-amino acid. Meanwhile, in another aspect of the present invention, a peptide may contain non-standard amino acids that have undergone post-translational modification. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including, for example, acetylation, myristoylation, and palmitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g., beta-elimination deimidation, deamidation), and structural changes (e.g., disulfide bridge formation). It also includes amino acid changes, such as changes at the amino group, carboxylic acid group, or side chain, that occur due to chemical reactions that occur during the conjugation process with a cross linker to form a peptide conjugate.

[0055] The peptides disclosed herein may be wild-type peptides identified and isolated from natural sources. Alternatively, the peptides disclosed herein may be artificial variants, which contain an amino acid sequence in which one or more amino acids have been substituted, deleted, and / or inserted compared to a peptide that is a fragment of SEQ ID NO: 1. Amino acid changes in wild-type polypeptides, as well as in artificial variants, include conservative amino acid substitutions that do not significantly affect protein folding and / or activity. Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine, and methionine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, and threonine). Generally, amino acid substitutions that do not alter specific activity are known in the art. The most commonly occurring exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, and their opposites. Other examples of conservative substitutions are set out in Table 1 below.

[0056] [Table 1]

[0057] Substantial variations in the biological properties of peptides are achieved by selecting substitutions that differ significantly in (a) their effect on maintaining the structure of the polypeptide backbone in the region of substitution, e.g., sheet or helical conformation, (b) their effect on maintaining the charge or hydrophobicity of the molecule at the target site, or (c) their effect on maintaining the bulk of the side chain. Natural residues are divided into the following groups based on common side chain properties: (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr; (3) Acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) Aromatics: trp, tyr, phe.

[0058] Non-conservative substitutions are made by exchanging a member of one of these classes for another. Any cysteine ​​residue not involved in maintaining the proper conformation of the peptide is generally substituted with serine to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bonds can be added to the peptide to improve its stability.

[0059] Another type of amino acid variant of a peptide is one that alters the glycosylation pattern of the antibody, by which is meant the deletion of one or more carbohydrate residues found in the peptide and / or the addition of one or more glycosylation sites that are not present in the peptide.

[0060] Glycosylation of peptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate residue to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate residue to the asparagine side chain. Thus, the presence of one of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0061] Glycosylation sites may be added to the peptide by altering the amino acid sequence such that it contains one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). Such alterations may also be made by adding, or substituting, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0062] In one aspect of the present invention, a polynucleotide is a nucleic acid molecule, which may be a naturally occurring or artificial DNA or RNA molecule, and may be single-stranded or double-stranded. The nucleic acid molecule may be one or more nucleic acid molecules of the same type (e.g., having the same nucleoside sequence) or of another type, including, but not limited to, DNA, cDNA, decoy DNA, RNA, siRNA, miRNA, shRNA, stRNA, snoRNA, snRNA, PNA, antisense oligomers, plasmids, and other modified nucleic acids.

[0063] HMGB1 protein is known to function as an inflammatory cytokine by being acetylated in the nucleus in response to external stimuli, translocating to the cytoplasm, and then secreting it to the extracellular space. Because HMGB1 protein is secreted to the extracellular space when inflammation is present, the serum of patients with inflammatory diseases such as Churg-Strauss syndrome, rheumatoid arthritis, and Sjögren's syndrome contains significantly higher amounts of HMGB1 protein than normal individuals. Therefore, regardless of the type of inflammation-inducing stimulus, if the amount of HMGB1 protein in the cell nucleus is high, it means that HMGB1 protein is not secreted to the extracellular space, and therefore inflammation is suppressed.

[0064] In one aspect of the present invention, when cells are treated with a peptide comprising the sequence of SEQ ID NO: 1, a peptide having greater than 80% sequence identity to the peptide sequence, or a fragment of the peptide, the amount of HMGB1 protein in the cell nucleus increases, indicating that the peptide has an excellent effect of preventing or suppressing inflammation.

[0065] Furthermore, a peptide comprising the sequence of SEQ ID NO: 1 according to one aspect of the present invention, a peptide having more than 80% sequence identity with the peptide sequence, or a fragment of the peptide has the advantage of being highly practicable due to its low intracellular toxicity.

[0066] As used herein, the term "inflammatory disease" is a broad concept that includes any disease associated with inflammation, such as a disease whose main cause is inflammation or a disease that causes inflammation.Specifically, the inflammatory diseases include (1) systemic or local inflammatory diseases (e.g., allergies; immune complex diseases; hay fever; hypersensitivity shock; toxin-resistant shock; cachexia, hyperpyrexia; granulomatosis; or sarcoidosis); (2) gastrointestinal diseases (e.g., appendicitis; gastric ulcer; duodenal ulcer; peritonitis; pancreatic enteritis; ulcerative, acute, or ischemic colitis; cholangitis; cholecystitis; steatorrhea). (3) skin-related diseases (e.g., psoriasis; burns; sunburn; dermatitis; urticarial warts or wheals); (4) cardiovascular diseases (e.g., vasculitis; vasculitis; endocarditis; arteritis; atherosclerosis; thrombophlebitis; pericarditis; congestive heart failure; myocarditis; myocardial ischemia; periarteritis nodosa; recurrent stenosis; Burger's disease; or rheumatic fever). (5) respiratory diseases (e.g., asthma, epiglottitis, bronchitis, emphysema, rhinitis, cystic fibrosis, epileptic pneumonia, chronic obstructive pulmonary disease (COPD), adult respiratory syndrome, pneumoconiosis, alveolitis, bronchiolitis, pharyngitis, pleurisy, or sinusitis); (6) bone, joint, muscle, and connective tissue-related diseases (e.g., eosinophilic granuloma, arthritis, arthralgia, osteomyelitis, dermatomyositis, fasciitis, (7) Genitourinary system diseases (e.g., epididymitis, vaginitis, prostatitis, or urethritis); (8) Central or peripheral nervous system-related diseases (e.g., Alzheimer's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillain-Barre syndrome, etc.) syndrome); neuritis; neuralgia; bone marrow trauma; paralysis; or uveitis; (9) viral (e.g., influenza; respiratory cytoplasmic virus; HIV; hepatitis B virus; hepatitis C virus, or herpes virus) infectious disease (e.g., dengue fever; or septicemia), fungal infectious disease (e.g., candidiasis), or bacterial, parasitic, and similar microbial infection (e.g., disseminated bacteremia; malaria; filariasis; or amebiasis); (10) autoimmune disease (e.g., thyroiditis; lupus; Goodpasture's syndrome; allograft rejection; graft-versus-host disease; or diabetes); or (11) cancer or neoplastic disease (e.g., Hodgkin's disease).

[0067] Treating the inflammatory component of such diseases has been a primary goal of the global pharmaceutical industry for decades, and numerous useful treatments have been developed. These include corticosteroids (a diverse range of natural, semi-synthetic, and synthetic preparations designed to mimic the effects of cortisol, including prednisolone, methylprednisolone, dexamethasone, betamethasone, fluticasone, etc.), cyclooxygenase inhibitors (non-selective or COX-1 selective, such as indomethacin, sulfasalazine, and aspirin, and more recently COX-2 selective, such as celecoxib), leukotriene blockers (such as montelukast), and small molecule TNF-α synthesis inhibitors (such as thalidomide), as well as infliximab (Remicade). TM ) and adalimumab (Humira Tm Improved monoclonal neutralizing antibodies, including etanercept (Enbrel TM ) anti-TNF, such as TNF receptor fusion proteins.

[0068] In one aspect of the present invention, there is provided an anti-inflammatory composition comprising, as an active ingredient, a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having greater than 80% sequence identity with said amino acid sequence, or a peptide that is a fragment thereof.

[0069] In one aspect, an anti-inflammatory composition according to one aspect of the present invention may contain a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having greater than 80% sequence identity to the amino acid sequence, or a peptide that is a fragment thereof, in an amount of 0.1 μg / mg to 1 mg / mg, specifically 1 μg / mg to 0.5 mg / mg, and more specifically 10 μg / mg to 0.1 mg / mg. When contained within this range, it is not only appropriate for achieving the intended effects of the present invention, but also for satisfying both the stability and safety of the composition, and is also appropriate from the perspective of cost-effectiveness.

[0070] The compositions according to one aspect of the present invention may be applied to all animals including humans, dogs, chickens, pigs, cows, sheep, guinea pigs or monkeys.

[0071] In one aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing an inflammatory disease, comprising as an active ingredient a peptide having the sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a peptide that has more than 80% sequence identity to said peptide sequence. In one aspect of the present invention, the pharmaceutical composition may be administered via oral, rectal, transdermal, intravenous, intramuscular, intraperitoneal, intraosseous, intradural or subcutaneous means.

[0072] Dosage forms for oral administration include, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, liquids, or emulsions.Dosage forms for parenteral administration include, but are not limited to, injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches, or sprays.

[0073] The pharmaceutical composition according to one aspect of the present invention may contain, if necessary, additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavors, or sweeteners. The pharmaceutical composition according to one aspect of the present invention may be manufactured by a method commonly used in the art.

[0074] The active ingredient of a pharmaceutical composition according to one aspect of the present invention will vary depending on the age, sex, and weight of the recipient, the pathological condition and its severity, the route of administration, and the discretion of the prescriber. Determining the dosage based on such factors is within the skill of one of ordinary skill in the art. The daily dosage may be, for example, but is not limited to, 0.1 μg / kg / day to 1 g / kg / day, specifically 1 μg / kg / day to 10 mg / kg / day, more specifically 10 μg / kg / day to 1 mg / kg / day, and even more specifically 50 μg / kg / day to 100 μg / kg / day. The pharmaceutical composition according to one aspect of the present invention is administered once to three times daily, but is not limited thereto.

[0075] In one aspect of the present invention, there is provided an external skin preparation composition for improving or preventing skin inflammation, which contains as an active ingredient a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having greater than 80% sequence identity with said amino acid sequence, or a peptide that is a fragment thereof.

[0076] In another aspect of the present invention, there is provided a cosmetic composition for improving or preventing skin inflammation, which comprises as an active ingredient a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having 80% or more sequence identity with said amino acid sequence, or a peptide which is a fragment thereof.

[0077] The skin external preparation composition or cosmetic composition according to one aspect of the present invention may be provided in any dosage form suitable for topical application. For example, it may be provided in the form of a solution, an emulsion obtained by dispersing an oily phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oily phase, a suspension, a solid, a gel, a powder, a paste, a foam, or an aerosol. Such dosage forms may be prepared by methods commonly known in the art.

[0078] The cosmetic composition according to one aspect of the present invention may contain other ingredients that can provide a synergistic effect to the main effect, as long as the ingredients do not impair the main effect. The cosmetic composition according to one aspect of the present invention may further contain a moisturizer, an emollient, a surfactant, an ultraviolet absorber, a preservative, a bactericide, an antioxidant, a pH adjuster, an organic or inorganic pigment, a fragrance, a cooling agent, or an antiperspirant. The amount of the ingredients can be easily determined by those skilled in the art within a range that does not impair the purpose and effects of the present invention, and the amount may be 0.01 to 5 wt %, specifically 0.01 to 3 wt %, based on the total weight of the cosmetic composition.

[0079] In one aspect of the present invention, the composition provides a food composition for preventing or suppressing inflammation, which comprises as an active ingredient a peptide having the sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a peptide that has more than 80% sequence identity with the peptide sequence.

[0080] The dosage form of the food composition according to one aspect of the present invention is not particularly limited, and may be, for example, a tablet, granule, powder, liquid, solid preparation, etc. In addition to the active ingredient, each dosage form may be formulated by a person skilled in the art by appropriately selecting and incorporating ingredients commonly used in the art according to the dosage form or intended use, and when used simultaneously with other ingredients, a synergistic effect occurs.

[0081] Determining the dosage of the active ingredient is within the level of a person skilled in the art, and the daily dosage may be, for example, specifically 1 μg / kg / day to 10 mg / kg / day, more specifically 10 μg / kg / day to 1 mg / kg / day, and even more specifically 50 μg / kg / day to 100 μg / kg / day, but is not limited thereto, and may vary depending on various factors such as the age, health condition, and complications of the subject to be administered.

[0082] In one aspect of the present invention, there is provided a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having more than 80% sequence identity with said amino acid sequence, or a peptide which is a fragment thereof, for use in the prevention or treatment of inflammatory diseases.

[0083] According to one aspect of the present invention, there is provided a method for preventing or treating an inflammatory disease using a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having greater than 80% sequence identity with said amino acid sequence, or a peptide that is a fragment thereof.

[0084] In one aspect of the present invention, there is provided a kit for preventing or treating an inflammatory disease. The kit may include a peptide having anti-inflammatory activity or a composition containing the peptide, and instructions including one or more of the dosage, administration route, administration frequency, and indication of the peptide or composition. The peptide comprises the amino acid sequence of SEQ ID NO: 1, has more than 80% sequence identity with the amino acid sequence, or is a fragment of the peptide.

[0085] The terminology used herein is intended for the purpose of describing particular embodiments only and is not intended to limit the invention. The use of terms preceded by a noun without a number does not denote a limitation of quantity, but rather indicates that there are one or more of the referenced item. The terms "including," "having," and "comprising" are to be construed as open terms (i.e., meaning "including, but not limited to").

[0086] Reciting ranges of values ​​is merely an easy way of referring to each separate value falling within the range individually, and unless expressly stated otherwise, each separate value is incorporated herein as if it were individually set forth in the specification. All range endpoints are included within the range and are independently combinable.

[0087] All methods referred to herein may be performed in any suitable manner unless otherwise specified or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as"), unless otherwise included in the claims, is merely to better describe the invention and does not limit the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the invention belongs.

[0088] The preferred embodiment of the present invention includes the best mode known to the inventors for carrying out the invention. Variations of the preferred embodiment will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that those skilled in the art will take advantage of such variations and will implement the invention in ways other than as described herein. Accordingly, this invention includes equivalents and all modifications of the subject matter recited in the appended claims as permitted by the patent laws. Furthermore, any combination of the above-described elements, within all possible variations, is encompassed by this invention unless expressly stated to the contrary herein or clearly contradicted by context. While the invention has been particularly shown and described with reference to illustrative embodiments, it will be readily apparent to those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

[0089] Tumor necrosis factor (TNF), particularly TNF-α, is known to be released from inflammatory cells and induce a variety of cytotoxic, immune, and inflammatory responses. TNF-α is involved in the onset and prolongation of many inflammatory and autoimmune diseases, and if released into the blood and acts systemically, it can cause severe sepsis and septic shock. Because TNF-α is a factor widely involved in the immune system, active development of drugs that inhibit TNF-α is underway. TNF-α is biosynthesized in an inactive form and converted to an active form by protease cleavage. The enzyme responsible for its activation is called tumor necrosis factor-converting enzyme (TACE). Therefore, substances that inhibit TACE can treat, ameliorate, or prevent diseases, pathologies, abnormal conditions, and adverse symptoms caused by TNF-α.

[0090] High-mobility group box 1 (HMGB1) protein is present at high concentrations in the thymus, lymph nodes, testes, fetal liver, etc., and is primarily present in the nucleus of most cells, except for hepatocytes and brain cells. The HMGB1 protein has three domains: an A-box, a B-box, and a C-terminal.

[0091] The role of HMGB1 protein as an inflammation-inducing cytokine was reported by Tracey et al. in 1999. The inflammation-inducing mechanism of HMGB1 is that, upon external stimuli, HMGB1 present in the nucleus is acetylated and then translocated to the cytoplasm, where it is then secreted extracellularly or secreted from cells undergoing necrosis (Bonaldi T et al., EMBO J, (22)5551-60, 2003).

[0092] The configuration and effects of the present invention will be described in more detail below with reference to examples and experimental examples. However, the following examples and experimental examples are provided for illustrative purposes only to aid in understanding the present invention, and the scope and spirit of the present invention are not limited thereto. [Example]

[0093] Example 1: Synthesis of PEP 1 (SEQ ID NO: 1) and measurement of anti-inflammatory activity Experimental Example 1: Synthesis of PEP 1 (SEQ ID NO: 1) A peptide consisting of 16 amino acids having the following chemical structure 1 and the following sequence SEQ ID: 1 (PEP 1) selected from human telomerase was synthesized.

[0094] [ka]

[0095] The peptide PEP 1 of SEQ ID NO: 1 was prepared by a conventional solid phase peptide synthesis method. Specifically, the peptide was synthesized by coupling amino acids one by one from the C-terminus via Fmoc solid phase peptide synthesis (SPPS) using an ASP48S (Peptron, Inc., Daejeon, Republic of Korea). The peptide was used with the first amino acid at the C-terminus attached to the resin as follows. For example:

[0096] NH2-Lys(Boc)-2-chloro-Trityl Resin NH2-Ala-2-chloro-Trityl Resin NH2-Arg(Pbf)-2-chloro-Trityl Resin

[0097] All amino acid starting materials used in peptide synthesis were protected at the N-terminus with Fmoc, and the residues were protected with Trt, Boc, t-butylester (t-Bu), 2,2,4,6,7-pentamethyl dihydrobenzofuran-5-sulfonyl (Pbf), etc., all of which can be removed with acid. For example:

[0098] Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, F moc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ahx-OH, Trt-Mercaptoaceticacid

[0099] The coupling reagents used were HBTU [2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetamethylaminium hexafluorophosphate], HOBt [N-Hydroxxybenzotriazole], and NMM [4-Methylmorpholine]. Fmoc removal was performed using 20% ​​piperidine in DMF. The synthesized peptide was separated from the resin and the protecting groups of the residues were removed using a cleavage cocktail [TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / EDT (ethanedithiol) / HO = 92.5 / 2.5 / 2.5 / 2.5].

[0100] Each peptide was synthesized by repeatedly reacting the starting amino acids with protecting groups bound to the solid support, washing with solvent, and deprotecting the amino acids. The synthesized peptides were cleaved from the resin, purified by HPLC, and their synthesis was confirmed by MS. They were then lyophilized.

[0101] The specific synthesis process of PEP 1 is as follows. 1) Coupling An amino acid (8 equivalents) protected by NH2-Lys(Boc)-2-chloro-Trityl Resin and coupling reagents HBTU (8 equivalents), HOBt (8 equivalents), and NMM (16 equivalents) were dissolved in DMF and added, and the mixture was reacted at room temperature for 2 hours. The mixture was then washed with DMF, MeOH, and DMF, in that order. 2) Fmoc deprotection 20% piperidine in DMF was added, and the mixture was reacted twice for 5 minutes at room temperature, and then washed with DMF, MeOH, and DMF in that order. 3) Reactions 1 and 2 were repeated to produce the peptide backbone NH2-E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(Boc)-2-chloro-Trityl Resin. 4) Cleavage: After synthesis, a cleavage cocktail was added to the peptide resin, and the peptide was separated from the resin. 5) Cooling diethyl ether is added to the resulting mixture, which is then centrifuged to precipitate the resulting peptide. 6) After purification by Prep-HPLC, the molecular weight was confirmed by LC / MS, and the product was frozen and made into powder.

[0102] Experimental Example 2: Measurement of the anti-inflammatory activity of PEP 1 Cell line culture Raw 264.7 macrophage cells (KCBL, 40071) provided by the Korean Cell Line Bank were cultured at 1 × 10 cells / well in Dulbecco's modified Eagle's medium (DMEM; PAA, Austria) supplemented with 10% fetal bovine serum (FBS; Gibco Laboratories) and 100 units / mL of streptomycin and penicillin (Gibco Laboratories). 6 After adjusting the concentration to cells / ml, the cells were seeded into a 96-well plate and incubated at 37°C, 5% CO 2 The mixture was pre-cultured under the conditions

[0103] The next day, the medium was removed and replaced with fresh medium. 5 μg / mL of the peptide sample obtained in Experiment 1 was added to the cells. After 30 minutes of incubation in a CO2 incubator, 50 μL of medium containing LPS (final concentration 1 μg / mL) was added and the cells were incubated in an incubator (5% CO2, 37°C) for 24 hours. The inflammatory response model was treated with 1 μg / mL lipopolysaccharide (LPS; Sigma, USA) per well, while the control group was treated with phosphate-buffered saline (PBS; pH 7.2). The cell culture medium obtained from the LPS-treated model and control group was stored in Eppendorf tubes for further analysis.

[0104] Experimental Example 2-1. Nitric oxide (NO) analysis Nitric oxide was measured using Raw 264.7 cells (1 × 10 6 The measurement method was performed using the Griess reagent system (Promega, USA) with 100 μL of culture medium (cells / ml). Equal amounts of Griess Reaction Solution I (naphthylethylenediamide (NED) solution) and Griess Reaction Solution II (sulfanilamide solution) were mixed and added to a 96-well plate. After a 10-minute incubation, the optical density was measured at 540 nm within 30 minutes using a microplate reader (Molecular Devices, USA). The concentration of nitric oxide (NO) was calculated using a sodium nitrite standard curve (0–100 μM). As shown in Table 3 below, LPS increased NO production, but simultaneous treatment with LPS and the peptide PEP 1 reduced NO production. In particular, treatment with PEP 1 reduced the amount of NO produced during inflammation induction by 65%, indicating that inflammation induction was suppressed.

[0105] [Table 2]

[0106] Experimental Example 2-2. Analysis of cytokine production suppression effect To investigate the inhibitory effect of PEP 1 on inflammatory cytokine production, RAW 264.7 cells were first treated with human telomerase-derived PEP at a concentration of 15 μg / mL, followed by LPS at a concentration of 1 μg / mL and cultured for 24 hours. Supernatant samples containing the cell culture medium were collected and analyzed for cytokine levels using an ELISA kit (eBioscience, San Diego, CA).

[0107] A 96-well plate was coated with 100 μl of capture antibodies (dissolved in coating buffer at the concentration recommended by the manufacturer's protocol) and incubated overnight at 4°C. After washing the plate five times, 200 μl of assay diluents was added to each well and incubated for 1 hour at room temperature during blocking. After washing each well five times with wash buffer, cell culture medium samples or cytokine standard protein samples were diluted and added in 100 μl volumes to each well. The sample-containing plate was incubated overnight at 4°C. After washing the plate five times with wash buffer, 100 μl of secondary antibody and avidin conjugate was added and incubated for 1 hour at room temperature.

[0108] After incubation with the secondary antibody, the plate was washed five times and incubated with 100 μl of avidin-HRP (BD Bioscience) for 30 minutes at room temperature. After washing the plate seven times, 100 μl of TMB solution (Pierce) was added and incubated for 15 minutes at room temperature. The reaction was terminated by adding 50 μl of 2N sulfuric acid (H2SO4) to each well. Optical density was measured at 450 nm using a microplate reader. Statistical analysis was performed using the ANOVA procedure in the SPSS program, with significance between analyses verified using Duncan's multiple range test.

[0109] Experimental Example 2-3: IL-6 secretion measurement As shown in Table 4 below, LPS increased the secretion of the cytokine IL-6 (interleukin-6), but simultaneous treatment with LPS and PEP 1 reduced the secretion of the inflammation-related cytokine IL-6. In particular, treatment with PEP 1 reduced the secretion of inflammation-related cytokines by more than 70%, demonstrating excellent anti-inflammatory activity.

[0110] [Table 3]

[0111] Experimental Example 2-4: Measurement of the ability to suppress the expression of HMGB1, TNF-α, and COX-2 Protein expression was analyzed by Western blot analysis. Cells grown in medium containing human telomerase-derived peptides were washed with PBS, treated with 0.05% trypsin-EDTA, and then collected by centrifugation. The collected cells were lysed in an appropriate volume of lysis buffer, and the intracellular debris was separated. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA) and reacted with specific antibodies and secondary antibodies. After application of ECL (enhanced chemiluminescence) solution (Amersham Life Science Corp., Arlington Heights, IL, USA), the membrane was exposed to X-ray film and analyzed for protein expression.

[0112] To confirm the relationship between TNF-α, which is a mechanism for inhibiting NO production, and COX-2 protein, expression levels were measured via Western blot. As shown in Table 5 below, LPS increased the expression of cytokines HMGB1, TNF-α, and COX, but simultaneous treatment with LPS and PEP 1 reduced the expression levels of inflammation-related proteins. In particular, treatment with PEP 1 of the present invention reduced the expression levels of inflammation-related cytokine proteins by more than 70%, demonstrating excellent anti-inflammatory activity.

[0113] [Table 4]

[0114] Experimental Example 3: Verification of TNFα inhibitory activity of PEP 1 in liver cancer cell lines Experimental Example 3-1: Cell line culture Blood (50 ml) was collected from healthy volunteers and then Ficoll-Paque TM The peripheral blood mononuclear cell (PBMC) layer was collected using a PLUS (GE Healthcare Life Sciences, Piscataway, NJ, USA). The collected PBMCs were enriched in RPMI 1640 medium (Invitrogen / Life Technologies, Carlsbad, CA, USA) supplemented with 20% human serum, transferred to a 100-mm polystyrene cell culture plate coated with human serum for 30 minutes, and cultured for 2 hours at 37°C in a 5% CO2 incubator. Afterwards, monocytes attached to the bottom of the cell culture plate were detached with cold phosphate buffered saline (PBS) (Gibco / Life Technologies, Carlsbad, CA, USA) and plated at 1 × 10 per well in a 96-well plate. 5 To obtain cells, the cells were cultured in RPMI 1640 medium (supplemented with penicillin-streptomycin; 100 mg / ml, human serum; 20%) the day before the experiment.

[0115] In addition, HEK293 (human embryonic kidney 293) cell line (HEK293 / TLR 2) stably expressing TLR 2 (toll-like receptor 2) and the general HEK293 (HEK293 / null) cell line were provided by the Graduate School of Dentistry, Seoul National University, and used for luciferase analysis. The HEK293 / null and HEK293 / TLR 2 cell lines were plated in 12-well plates at 2.5 × 10 cells per well on the day before the luciferase analysis experiment. 5 The cells were enriched with Dulbecco's modified Eagle's medium (DMEM) (supplemented with blasticidin; 10 μg / ml, fetal bovine serum; 10%) (Invitrogen / Life Technologies, Carlsbad, CA, USA) and cultured the day before the experiment.

[0116] Experimental Example 3-2: Cytokine assay To investigate the effect of PEP 1 on the expression of TNF-α protein, an enzyme-linked immunosorbent assay (ELISA) experiment was performed. PBMC-derived monocytes were cultured in a 96-well plate at 1 × 10 cells per well. 5The cells were cultured the day before the experiment to obtain a uniform TNF-α concentration. They were then treated with lipopolysaccharide (LPS; 10 ng / ml, Sigma) for 2 hours and washed three times with PBS. OPTI-MEM medium (Invitrogen / Life Technologies, Carlsbad, CA, USA) was added and starved for 1 hour. Then, they were treated with 4 μM FITC (fluorescein isothiocyanate), FITC-TAT, PEP 1-FITC, and FITC-PEP 1, and cultured for 2 hours before measuring TNF-α levels. After incubation, the cell culture medium was collected and TNF-α was quantified using an ELISA kit (R&D, Minneapolis, MN, USA). The specific quantification method is as follows.

[0117] TNF measurement is performed using a sandwich ELISA. 100 μl of TNFα primary antibody is added to a pre-coated 96-well plate and incubated at 4°C the previous day. The next day, the plate is washed three times with 0.5% Tween 20 wash solution for 5 minutes each, and then 100 μl of the sample to be measured and the standard solution are added and incubated for 2 hours at room temperature. After washing the plate as described above, 100 μl of HRP-conjugated secondary antibody is added and incubated for 2 hours at room temperature. The plate is then washed again, and avidin / biotin is added to develop color, and the absorbance is measured. A standard curve is generated using the absorbance of the standard solution, and the TNF-α in each sample is quantified using this.

[0118] PBMC-derived monocytes were stimulated with the endotoxin LPS (10 ng / ml) for 2 hours, starved in OPTI-MEM for 1 hour, and then treated with FITC, FITC-TAT, PEP 1-FITC, and FITC-PEP 1 at 4 μM for 2 hours. After the incubation, TNF-α levels in the cell culture medium were measured by ELISA. FITC and FITC-TAT significantly increased TNF-α levels (6.2 and 6.7 ng / ml) due to LPS, whereas PEP 1-FITC and FITC-PEP 1 significantly decreased TNF-α levels (0.17 and 0.25 ng / ml), a difference that was statistically significant (P<0.01) (Figure 1).

[0119] Experimental Example 3-3: Luciferase Assay To investigate the role of PEP 1 in inflammatory responses, NF-kB expression patterns were assessed via luciferase assay. First, 2.5x10 cells per well were cultured in a 12-well plate. 5HEK293 / null and HEK293 / TLR 2 (Graduate School of Dentistry, Seoul National University) cells were cultured for 24 hours. After washing the plates three times with PBS, the medium was replaced with OPTI-MEM (Invitrogen / Life Technologies, Carlsbad, CA, USA). After 4 hours of incubation, a mixture of 3 μl lipofectamine (Invitrogen / Life Technologies), 1 μl NF-kB luciferase, and 10 ng renilla luciferase (Promega, Madison, WI, USA) was added to each well and the cells were further incubated for 4 hours. All wells, except the negative control, received Lipoprotein pam3cys (10 ng / ml, Sigma-Aldrich, St. Louis, MO, USA) and were treated with FITC (4 μM) and FITC-PEP 1 (4 μM) for 18 hours before being washed three times with PBS. Cells were lysed using 50 μl of passive lysis buffer (Promega) in each well, and NF-κB activation was monitored using a TD-20 / 20 luminometer (Turner Designs, Sunnyvale, CA, USA). Transfection efficacy was confirmed by cotransfection with Pcmv-renilla luciferase (Promega), and luciferase activity was calibrated for analysis.

[0120] NF-kB luciferase was transfected into HEK293 / null and HEK293 / TLR 2 cell lines, and then treated with synthetic lipoprotein, FITC (4 μM), or a negative control. Pam3cys cells were further treated with FITC-PEP 1 (4 μM) for 18 hours. Cell lysis was performed using passive lysis buffer, and NF-kB expression patterns were measured using a dual-luciferase reporter assay system. No significant differences were observed between lipoprotein- and FITC-PEP 1-treated or untreated HEK293 / null cells. However, treatment of HEK293 / TLR 2 cells with lipoprotein, a TLR 2 activator, resulted in increased NF-kB expression (P<0.01) compared to untreated cells, confirming an anti-inflammatory response. Furthermore, NF-kB expression was increased when treated with FITC-PEP 1 compared to when not treated, and was decreased compared to the negative control group treated with lipoprotein and FITC (P<0.01) (Figure 2). Consequently, it was confirmed that the anti-inflammatory response induced by TLR 2 was reduced when PEP 1 was also treated.

[0121] Experimental Example 3-4: Reanalysis of peptides affecting cytokine levels in THP1 cell line The efficacy of PEP 1 was confirmed using THP-1 cells (American Type Culture Collection, Manassas, VA, USA), a human acute monocytic leukemia cell line. The cells were cultured at a density of 0.5-0.7x10 5 THP-1 cells were cultured in RPMI 1640 containing 10% FBS, 0.05 mM 2-mercaptoethanol, 100 U / ml penicillin, and 100 μg / ml streptomycin and maintained at 37°C in 5% CO. THP-1 cells were differentiated into macrophages by treatment with 100 ng / ml phorbol myristate acetate (PMA) for 24 hours at 37°C.

[0122] All test drugs and culture media were purchased from Gibco BRL. PMA, LPS, and 2-mercaptoethanol were purchased from Sigma (St. Louis, MO, USA). Peptide RIA was synthesized by Peptron (Daejeon, South Korea). Reverse transcription PCR kit was purchased from Promega (Madison, WI, USA). RT 2 SYBR® Green qPCR master mix test agent and QIAzold were purchased from QIAGEN (Valencia, CA, USA).

[0123] After differentiation into macrophages, THP-1 cells were washed twice with RPMI 1640 (5 min per wash), then treated with 10 ng / ml LPS and / or 4 μM peptide RIA for 4 hours in RPMI 1640 without FBS.

[0124] Total RNA samples were isolated from peptide-treated THP-1 cells using Trizol (QIAzol) reagent, and cDNA was synthesized by reverse transcription-PCR using a Promega reverse transcription-PCR kit according to the manufacturer's protocol.

[0125] Real-time (RT) qPCR was performed using a CFX96 (Bio-Rad) instrument with the SYBR Green system. The primers used in the experiment are listed in Table 5. PCR cycling conditions were set at 95°C for 10 minutes with HotStart DNA Taq polymerase activity, followed by 45 cycles consisting of 10 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C. All samples were measured in triplicate, and differences in gene expression were calculated using the 2-cycle threshold method. All data were normalized to β-actin (a housekeeping gene) and used as means + / - standard error (SE) from three independent experiments.

[0126] [Table 5]

[0127] As can be seen from Figure 3, cytokines involved in inflammatory responses were significantly reduced by LPS and PEP 1 treatment.

[0128] Experimental Example 4: Analysis of inflammatory responses induced by beta-amyloid HMGB1 protein is known to act as an inflammatory cytokine by being acetylated in the nucleus and translocated to the cytoplasm in response to external stimuli, where it is then secreted to the extracellular space. Because HMGB1 protein is secreted to the extracellular space in the presence of inflammation, the serum of patients with inflammatory diseases such as Churg-Strauss syndrome, rheumatoid arthritis, and Sjögren's syndrome contains significantly higher amounts of HMGB1 protein than normal individuals. Therefore, regardless of the type of inflammation-inducing stimulus, if the amount of HMGB1 protein in the cell nucleus is high, this indicates that HMGB1 protein is not secreted to the extracellular space, suggesting that inflammation is suppressed.

[0129] Experimental Example 4-1: Analysis of the survival and proliferation effects of neural stem cells due to the anti-inflammatory effect of PEP 1 First, PEP 1 was prepared by the peptide production method described in Example 1.

[0130] Experimental Example 4-2: Cultivation of neural stem cells and evaluation of beta-amyloid toxicity Cerebral cortices were isolated from the heads of fetal rats on day 13 of gestation and treated with basic fibroblast growth factor (bFGF) for one week under the following culture conditions to obtain neural stem cells. To analyze the effects of beta-amyloid protein on the cultured neural stem cells, neural stem cells were treated with pre-oligomerized beta-amyloid protein at concentrations of 0 μM and 40 μM for 48 hours, and cytotoxicity was evaluated using CCK-8, BrdU, and TUNEL assays. Treatment with 20 μM beta-amyloid protein confirmed a decrease in cell viability of approximately 60%, and the same concentrations (0 μM to 40 μM) were used in subsequent experiments (see Figures 47 and 48).

[0131] Experimental Example 4-3: Evaluation of cytotoxicity by PEP 1 treatment To analyze the effect of PEP1 on the cultured neural stem cells, the cells were treated with various concentrations of PEP1 (0, 1, 10, 50, 100, and 200 μM) according to known methods, and cell viability and proliferation were evaluated using MTT assay, BrdU assay, and TUNEL assay. PEP1 at concentrations of 0 to 200 μM did not inhibit cell viability or proliferation, confirming its stability in neural cell lines (see Figures 49 and 50).

[0132] Experimental Example 4-4: Cytotoxicity evaluation after simultaneous treatment with beta-amyloid protein and telomerase peptide (GV1001) To determine whether PEP 1 has the effect of suppressing the neurotoxicity of beta-amyloid protein, cells were treated with 20 μM beta-amyloid protein and various concentrations of PEP 1 for 48 hours, and then cell viability and death were assessed using MTT assay, CCK-8 assay, LDH assay, and TUNEL assay, and neural stem cell proliferation was analyzed using BrdU assay.

[0133] MTT assay and CCK-8 assay results showed that 10 μM PEP 1 began to protect neural stem cells from neurotoxicity caused by beta-amyloid protein, with the best protective effect observed at 100 μM (see Figure 8). Alternatively, an LDH assay was performed to assess the degree of cell death, confirming that PEP 1 effectively reduced cell death caused by beta-amyloid protein, with the effect beginning at a concentration of 1 μM (see Figure 9).

[0134] BrdU assay confirmed that cell proliferation, which was reduced by beta-amyloid protein, was restored by treatment with PEP 1 (see FIG. 10).

[0135] Cell migration is a very important characteristic of neural stem cells. Experimental results on cell migration showed that cell migration, which was reduced by beta-amyloid protein, was restored by PEP 1 treatment and was even increased at a concentration of 10 μM compared to the control group. This suggests that if PEP 1 is used as a pretreatment before stem cell transplantation in clinical trials, even more effective results will be obtained (see Figure 11).

[0136] To directly assess the extent of neural stem cell damage, a TUNEL assay was performed. We observed a significant increase in neural stem cell death in the 20 μM beta-amyloid protein treatment group, and confirmed that neural stem cell death was reduced by treatment with 1 to 100 μM PEP 1 (see Figure 12).

[0137] We investigated the mechanism of action of PEP 1's protective effect on apoptosis induced by beta-amyloid protein. First, we investigated whether PEP 1 reduces oxidative damage caused by beta-amyloid protein. Using DCF-DA stained samples (Molecular Probes, Eugene, OR), we observed changes in the generation of reactive oxygen species after treatment with beta-amyloid protein and PEP 1. We confirmed that 20 μM beta-amyloid protein increased reactive oxygen species, and that the increased reactive oxygen species were reduced in the groups treated with PEP 1 (1 μM, 10 μM, and 50 μM) (see Figure 13).

[0138] Experimental Example 4-5: Comparative analysis of protein expression levels between a PEP 1-treated group and an untreated control group The protein expression levels in the PEP 1-treated and untreated groups were analyzed and quantified using 2-D gel electrophoresis and antibody microarray. 200 μg of proteome was extracted from the neural stem cells cultured in Experimental Example 1-1 of Example 3, and a group not treated with PEP 1 was used as a control group and analyzed under the same conditions.

[0139] For 2D electrophoresis, primary electrophoresis was performed using an 8.5x7 12% acrylamide gel at PI 4-10 N. After electrophoresis, the gels were stained with Colloidal Coomassie Blue staining reagent, and the expression levels were compared by analyzing each spot using PDQuest software.

[0140] Proteins with a difference in expression level of more than 1.5-fold were identified using MALDI-TOF MS (Matrix Desorption / Ionization Time of Flight Mass Spectroscopy). Among these, proteins known to be correlated with inflammation-related signal transduction, such as i-NOS and HMGB-1, were identified (see Table 6). While the expression level of beta-amyloid protein increased or decreased by 1.5-fold, the addition of PEP-1 confirmed that the expression level was regulated to a level close to that of the negative control group (see Figure 14).

[0141] The antibody microarray technique uses a cell signaling kit (CSAA1, Panorama TM The array slides were scanned using a GenePix Personal 4100A scanner, and the data were analyzed using GenePix Pro 5.0 (Molecular Devices).

[0142] Table 6 below shows the expression analysis results of inflammation-related proteins obtained through 2D electrophoresis. The control group shows the expression level of proteins extracted from normal cells that were not treated with beta-amyloid protein or PEP 1, and the fold increase or decrease in expression level based on the expression level of the control group is shown.

[0143] The analysis results shown in Table 6 below confirmed that PEP 1 controls the over-expression and under-expression of inflammation-related proteins, and that the protein expression level in the control group was close to that of the negative control group.

[0144] [Table 6]

[0145] The PI3K (phosphatidylinositol-3-kinase) / AKT signaling pathway plays a crucial role in the growth and survival of neural stem cells. The PI3K pathway is activated by various growth factors and regulatory factors and is involved in the normal regulation of neural stem cell growth and survival. The AKT signaling pathway inactivates many pro-apoptotic factors and suppresses GSK3β, a known representative apoptosis signal.

[0146] To further confirm the anti-inflammatory effect of PEP 1, we performed Western blot analysis of HMGB1, which showed clear changes in protein analysis. The results showed that PEP 1 treatment increased the expression levels of cell survival signals Ki67, pAKT, PI3K, HSTF-1, and Bcl-2 proteins, and decreased the expression levels of cell death signals Bax, GSK-3β, Cytochrom-c, and caspase-3 proteins (see Figure 58).

[0147] HMGB1, a non-histone protein that binds to DNA, plays a variety of roles within cells, including stabilizing nucleosome structures and regulating gene expression. It is one of the inflammatory inducers released in the late stages of an inflammatory response. When an inflammatory response occurs, it is initially released by macrophages and monocytoides. However, when neurons are severely damaged and undergo necrosis, it is released extracellularly, inducing a very intense inflammatory response. HMGB1 decreased in the cytoplasm of neurons after processing beta-amyloid protein. The telomerase peptide (GV1001) inhibits the extracellular release of HMGB1 due to neuronal death, enhancing its important role within the cell, suggesting that GV1001 may have a potent anti-inflammatory effect (see Figure 15).

[0148] We investigated the effect of PEP 1 on beta-amyloid aggregation. We found that when beta-amyloid protein was treated with PEP 1 during the aggregation induction process, protein aggregation was inhibited (see Figure 16(A)), and that when beta-amyloid protein that had already been aggregated was treated with PEP 1, the protein was degraded (see Figure 16(B)).

[0149] The mechanism of action of PEP 1 was confirmed to be an increase in cell survival signals and a decrease in cell death signals in the PI3K pathway. To determine whether this phenomenon was direct or indirect, cells were pretreated with the PI3K inhibitor LY294002 (Promega). As a result, the cell viability rate, which increased after treatment with the telomerase peptide (GV1001), appeared to decrease with pretreatment with LY294002, confirming that the neuroprotective effect of GV1001 involves a direct effect on the PI3K pathway (see Figure 17).

[0150] The inhibitory effect of PEP 1 on neural stem cells caused by beta-amyloid protein was further confirmed in neural stem cells. It was also confirmed that it contributes to improving the migration ability of neural stem cells, suggesting various possibilities for clinical application. The mechanism of action of PEP 1 has been confirmed to include anti-inflammatory effects, an increase in neural stem cell survival factors and a decrease in death factors, particularly activation of the PI3K signaling system and antioxidant effects, demonstrating its ability to inhibit neurotoxicity caused by beta-amyloid protein through a wide range of mechanisms.

[0151] Experimental example 5: qPCR array Experimental Method THP-1 cell culture Experiments were carried out using THP-1 cells (ATCC: American Type Culture Collection (ATCC), Manassas, VA, USA), a human acute monocytic leukemia cell line. THP-1 cells were plated in a 96-well plate at 1 × 10 per well.5 To differentiate into THP-1 cells, THP-1 cells were enriched in RPMI 1640 medium and cultured for 24 hours. THP-1 cells, which grow normally in suspension, were differentiated into an adherent macrophage-like phenotype for 24 hours in differentiation medium (complete growth medium conditions containing 100 ng / mL phorbol 12-myristate 13-actate (PMA; Sigma-Aldrich)). During differentiation, THP-1 cells (3 x 10 6 cells / plate, ~95% confluency (measure of proliferation) were seeded onto 10 / cm tissue culture plates and cultured in differentiation medium.

[0152] Treatment of THP-1 cells with the anti-inflammatory peptide PEP-1 After differentiation, macrophage-like THP-1 cells were washed twice with complete growth medium, and then treated with 10 ng / mL lipopolysaccharide (Sigma-Aldrich) and / or 4 μM PEP-1 for 4 h at 37°C.

[0153] RNA isolation and cDNA synthesis in THP-1 cells Total RNA was extracted and purified using the RNeasy minikit (purchased from Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. cDNA was synthesized via reverse transcription using the Reverse Transcription System (purchased from Madison, WI, USA) according to the manufacturer's protocol.

[0154] PCR array The cDNA samples obtained from THP-1 cells were then used as templates for real-time quantitative PCR (qPCR) analysis. For qPCR analysis, the RT2 Profilier PCR Array Kit was purchased from SABiosciences / Qiagen (Valencia, CA, USA). Four different PCR array kits for analyzing isolated signal transduction were used in the experiment, with the following details: Human Signal Transduction Mechanism Finder, Human Inflammatory Cytokines and Receptors, Human Transcription Factors, and Human NF-κB Signaling Mechanism. PCR was performed using a Bio-Rad CFX 96 real-time PCR instrument (Mercules, CA, USA) with the SYBR Green detection system (Qiagen).

[0155] Thermal cycling conditions were as follows: 95°C for 10 seconds, 55°C for 30 seconds, 95°C for 10 minutes, 95°C for 10 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by 50 amplification cycles. Data represent the average of three independent experiments, and percent reductions were determined by comparing gene expression in LPS-treated samples with LPS + PEP-1-treated samples. Of the 336 genes analyzed, only the following showed a statistically significant (p<0.05, Student's t-test) percent reduction, as shown in Table 7 below.

[0156] [Table 7]

[0157] The genes whose transcription was suppressed by PEP-1, shown in Table 7, are expressed as percent inhibition, which was calculated as the percent change in transcription level between LPS-treated and LPS + PEP-1-treated samples (THP-1 cells). Of the 336 genes analyzed, only 13 genes shown in Table 7 showed a statistically significant decrease after PEP-1 treatment. These genes were grouped into different functional categories, including chemokine and cytokine, TNFα receptor signaling, lipid metabolism, cell apoptosis, and NF-κB signaling. Importantly, genes in the chemokine and cytokine categories are known target genes of NF-κB and contain NF-κB-cognate DNA binding sites in their promoter regions. In addition, the PCR array data also support the idea that the anti-inflammatory effects of PEP-1 are achieved by manipulating NF-κB, a key regulator of inflammation, and therefore support the use of PEP-1 as an anti-inflammatory therapeutic agent in a wide range of inflammatory diseases.

Claims

1. A pharmaceutical composition for treating or preventing an inflammatory disease, comprising as an active ingredient a peptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein the peptide exhibits activity of suppressing the expression of at least one of HMGB1, TNF-α, and COX-2, and the inflammatory disease is associated with the expression of at least one of HMGB1, TNF-α, and COX-2; and (1) Systemic or local inflammatory diseases; (2) gastrointestinal diseases; (3) cardiovascular disease; (4) Respiratory diseases; (5) bone, joint, muscle, and connective tissue-related diseases; A pharmaceutical composition selected from the group consisting of:

2. The composition according to claim 1, wherein the inflammatory diseases (1) to (5) are selected from the group consisting of: (1) The systemic or local inflammatory disease is selected from the group consisting of allergies; immune complex diseases; hay fever; hypersensitivity shock; toxic shock; hyperthermia; granulomatosis; and sarcoidosis; (2) The gastrointestinal disease is selected from the group consisting of appendicitis; gastric ulcer; duodenal ulcer; peritonitis; pancreatic enteritis; ulcerative, acute, or ischemic colitis; cholangitis; cholecystitis, steatorrhea, hepatitis, Crohn's disease; and Whipple's disease; (3) the cardiovascular disease is selected from the group consisting of vasculitis; vasculitis; endocarditis; arteritis; atherosclerosis; thrombophlebitis; pericarditis; congestive heart failure; myocarditis; myocardial ischemia; periarteritis nodosa; restenosis; Burger's disease; or rheumatic fever; (4) The respiratory disease is selected from the group consisting of asthma; epiglottitis; bronchitis; emphysema; rhinitis; cystic fibrosis; epileptic pneumonia; chronic obstructive pulmonary disease (COPD); adult respiratory syndrome; pneumoconiosis; alveolitis; bronchiolitis; pharyngitis; pleurisy; and sinusitis; (5) The composition, wherein the bone, joint, muscle, and connective tissue-related disease is selected from the group consisting of eosinophilic granuloma; osteomyelitis; dermatomyositis; fasciitis; Paget's disease; gout; periodontal disease; myasthenia gravis; ankylosing spondylitis; and lumbago.

3. 2. The composition of claim 1, wherein the composition comprises 0.1 μg / mg to 1 mg / mg of the peptide.

4. 10. The composition of claim 1, wherein the peptide is administered in a single administration dose at a concentration of 0.1 μg / kg to 1.0 g / kg.

5. 10. The composition of claim 1, wherein the peptide is administered in a single administration dose at a concentration of 1 μg / kg to 10 mg / kg.

6. The composition of any one of claims 1 to 5, which is administered 1 to 3 times daily.

7. 2. The composition of claim 1, wherein the peptide is administered at a daily dosage of 0.1 μg / kg to 1.0 g / kg.

8. The composition of any one of claims 1 to 7, administered via oral, rectal, transdermal, intravenous, intramuscular, intraperitoneal, intraosseous, intradural or subcutaneous means.

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