Peptides with anti-inflammatory activity and their use in the preparation of anti-inflammatory compositions

Abstract

The present invention relates to a peptide having anti-inflammatory activity, a polynucleotide encoding the same, and use of the peptide in the preparation of an anti-inflammatory composition. According to the present invention, the peptide has excellent efficacy in both the suppression of inflammation and prophylactic means. Therefore, a composition comprising the peptide of the present invention can be used as an anti-inflammatory pharmaceutical composition or as a cosmetic composition, thereby treating or preventing various types of inflammatory diseases.

Description

[0001] This application is a divisional application of Chinese National Application No. 201810797270.8. The original international application number was PCT / EP2013 / 055327, and the Chinese National Application No. was 201380036618.7, filed on March 15, 2013, entitled "Anti-inflammatory Peptides and Compositions Containing the Same". A divisional application of this application (201380036618.7) with Chinese National Application No. 201810797270.8, entitled "Anti-inflammatory Peptides and Compositions Containing the Same," was filed on July 19, 2018. Technical Field

[0002] This invention relates to anti-inflammatory peptides and compositions comprising such anti-inflammatory peptides. Background Technology

[0003] Inflammation is a biological defense mechanism that protects the body from damage to biological tissues caused by external physical or chemical stimuli (such as exposure to various allergens) or invasion by microorganisms, including bacteria, fungi, and viruses.

[0004] The cyclooxygenase (COX) or lipoxygenase (LOX) pathways, which produce prostaglandins, thromboxanes, and other substances, are used for inflammatory signal transduction. Once inflammatory signals are transmitted, one of the many changes that occur in the body is vasodilation to increase blood supply to the periphery of inflammation, thereby concentrating blood cells needed for the inflammatory response, such as neutrophils. However, when an abnormal biological defense response occurs excessively, it can lead to inflammatory disease. To prevent this, drugs are currently being developed to inhibit excessive inflammatory responses by suppressing enzymes used in inflammatory signal transduction pathways (e.g., COX-1, COX-2, 5-LOX, 12-LOX, etc.).

[0005] Based on the response time, inflammation can be classified into acute inflammation (immediate response, non-specific response, lasting from days to weeks), chronic inflammation (delayed response, specific response, lasting weeks or longer), and subacute inflammation (an intermediate stage between acute and chronic inflammation, characterized by a mixture of mononuclear and polymorphic products).

[0006] In addition to peptide factors, factors such as prostaglandins, leukotrienes, lipid factors (including platelet-activating factor (PAF)), enzymes synthesizing inflammatory factors, free radicals (such as NO (nitric oxide)), various cell adhesion molecules, the immune system, and coagulation factors can all cause inflammation.

[0007] When cells are damaged by known inflammatory substances (such as external biological agents (microorganisms, viruses, parasites), physical agents (mechanical stimulation, heat, radiation, electricity), and chemical agents), they release histamine and kinins. The released histamine and kinins cause vasodilation, increased capillary permeability, and macrophage concentration at the site of inflammation, leading to increased blood flow, edema, migration of immune cells and antibodies, pain, and fever.

[0008] Currently used anti-inflammatory drugs include synthetic medications (such as ibuprofen), antihistamines, steroids, cortisone, immunosuppressants, and immune agonists; however, these drugs only provide temporary relief from inflammation. They do not address the underlying cause of inflammation and have side effects such as hypersensitivity reactions and immune system degeneration.

[0009] Therefore, research has been conducted to develop substances that inhibit the expression of the aforementioned inflammatory proteins in order to effectively alleviate inflammation. However, previously developed anti-inflammatory substances have several problems. Various types of anti-inflammatory drugs have been developed, including nonsteroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SAIDs); however, these drugs not only frequently cause side effects when taken, but they also fail to cure inflammation at its root. Therefore, there is currently a need for anti-inflammatory drugs that are both materially and economically feasible. As an example of acute or chronic inflammation (such as chronic rheumatoid arthritis), NSAIDs not only inhibit the activity of COX-2 enzymes, but they are also known to inhibit the activity of COX-1, thereby causing side effects such as gastrointestinal disturbances.

[0010] The inventors discovered that peptides derived from telomerase can possess anti-inflammatory properties, thus completing this invention.

[0011] Therefore, the 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] Another object of the present invention is to provide a peptide with anti-inflammatory activity.

[0014] Another object of the present invention is to provide an anti-inflammatory composition using this peptide as an active ingredient.

[0015] Another object of the present invention is to provide cosmetic compositions using this peptide as an active ingredient.

[0016] Another object of the present invention is to provide pharmaceutical compositions using this peptide as an active ingredient. Summary of the Invention

[0017] In one embodiment of the present invention, a peptide with anti-inflammatory activity is provided, wherein the peptide comprises at least one amino acid sequence of SEQ ID NO:2 to 179, or wherein the peptide has at least 80% sequence identity with the above sequence, or the peptide is a fragment of the above peptide.

[0018] In another embodiment, the fragment consists of three or more amino acids. For example, the fragment may consist of 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 acid residues.

[0019] In another embodiment, the peptide consists of 30 or fewer amino acids.

[0020] For example, the peptide may consist 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 acid residues.

[0021] In another embodiment, the peptide consists of any one of the amino acid sequences in SEQ ID NO:2 to 179.

[0022] In another embodiment, the peptide comprises any amino acid sequence selected from the group consisting of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:131, SEQ ID NO:140, SEQ ID NO:141, SEQID NO: 142, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177 and SEQ ID NO: 178.

[0023] In another embodiment, the peptide comprises any amino acid sequence selected from the group consisting of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:89, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:8 ... NO: 90, SEQ ID NO: 91, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:140, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:172, SEQ ID NO:173 and SEQ ID NO:174.

[0024] In another embodiment, the peptide comprises any amino acid sequence selected from the group consisting of the following sequences: SEQ ID NO:2 to SEQ ID NO:5, SEQ ID NO:7 to SEQ ID NO:36, SEQ ID NO:38 to SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:78 to SEQ ID NO:107 and SEQ ID NO:109 to SEQ ID NO:179.

[0025] In another embodiment, the peptide comprises any amino acid sequence selected from the group consisting of the following sequences: SEQ ID NO:2, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:30, SEQ ID NO:41, SEQ ID NO:112 and SEQ ID NO:113.

[0026] In another embodiment, the peptide comprises any amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:102, SEQID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQID NO:121, SEQ ID NO:123, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ IDNO:136, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:158, SEQID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178 and SEQ ID NO:179.

[0027] In another embodiment, the peptide is derived from human telomerase.

[0028] In one embodiment of the present invention, a polynucleotide encoding a peptide having anti-inflammatory activity is provided, wherein the peptide comprises at least one amino acid sequence of SEQ ID NO:2 to 179, or the peptide has at least 80% sequence identity with the above sequence, or the peptide is a fragment of the above peptide.

[0029] In another embodiment of the polynucleotide, the peptide consists of 30 or fewer amino acids. For example, the peptide may consist 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 acid residues. In another embodiment of the polynucleotide, the peptide consists of any one amino acid sequence from SEQ ID NO: 2 to 179.

[0030] In another embodiment of the polynucleotide, the peptide is derived from human telomerase.

[0031] In one embodiment of the present invention, an anti-inflammatory composition comprising a peptide as an active ingredient is provided, wherein the peptide comprises at least one amino acid sequence of SEQ ID NO:2 to 179, the amino acid sequence of the peptide having more than 80% homology with the above sequences, or the peptide being a fragment of the above peptide.

[0032] In another embodiment of the composition, the peptide consists of 30 or fewer amino acids, as described above.

[0033] In another embodiment of the composition, the peptide consists of any one of the amino acid sequences in SEQ ID NO:2 to 179.

[0034] In another embodiment of the composition, the peptide is derived from human telomerase.

[0035] In another embodiment of the composition, the composition is used to treat or prevent inflammatory diseases.

[0036] In another embodiment of the composition, the composition is a cosmetic composition for improving or preventing skin inflammation.

[0037] In another embodiment of the composition, the composition is a pharmaceutical composition for treating or preventing inflammatory diseases.

[0038] In another embodiment of the composition, the aforementioned inflammatory disease is characterized by being selected from the group consisting of: (1) generalized or localized inflammatory diseases (e.g., allergic reactions; immune complex diseases; hay fever; hypersensitivity shock; endotoxic shock; cachexia; overheating; granulomatous diseases; or sarcoidosis); (2) gastrointestinal-related diseases (e.g., appendicitis; gastric ulcer; duodenal ulcer; peritonitis; pancreatitis; ulcerative colitis, acute colitis or ischemic colitis; cholangitis; cholecystitis, steatorrhea, hepatitis, Crohn's disease). (3) Skin-related diseases (e.g., psoriasis; burns; sunburn; dermatitis; urticaria or wheals); (4) Vascular-related diseases (e.g., angiitis; vasculitis; endocarditis; arteritis; arteriosclerosis; thrombophlebitis; pericarditis; congestive heart failure; myocarditis; myocardial ischemia; periarteritis nodosa; recurrent stenosis). (5) Respiratory diseases (e.g., asthma; epiglottitis; bronchitis; emphysema; rhinitis; cystic fibrosis; interstitial pneumonia; COPD (chronic obstructive pulmonary disease); adult respiratory distress syndrome; dust sickness; alveolitis; bronchiolitis; pharyngitis; pleurisy; or sinusitis); (6) Bone, joint, muscle and connective tissue related diseases (e.g., eosinophilic granuloma; arthritis; arthralgia; osteomyelitis; dermatomyositis; fasciitis; Paget's disease). (7) Genitourinary disorders (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-Barré syndrome). (9) viral infections (e.g., influenza; respiratory syncytial virus; HIV; hepatitis B; hepatitis C; or herpesvirus), infectious diseases (e.g., dengue fever; or sepsis), fungal infections (e.g., candidiasis); or bacterial, parasitic and similar microbial infections (e.g., disseminated bacteremia; malaria; onchocerciasis; or amoebiasis); (10) autoimmune diseases (e.g., thyroiditis; lupus erythematosus; Goodpasture's syndrome; allogeneic transplant rejection; graft-versus-host disease; or diabetes); and (11) cancer or neoplastic diseases (e.g., Hodgkin's disease).

[0039] In one embodiment of the present invention, a method for treating or preventing inflammatory diseases by applying the anti-inflammatory composition is provided.

[0040] In one embodiment of the present invention, a kit for the prevention or treatment of inflammatory diseases is provided, comprising: a peptide having anti-inflammatory activity or a composition comprising said peptide, wherein the peptide comprises at least one amino acid sequence of SEQ ID NO:2 to 179, the peptide having more than 80% homology with said sequence, or the peptide being a fragment of said peptide; and instructions for use including at least one of the following: dosage, route of administration, frequency of administration, and indications for the peptide or composition.

[0041] Industrial practicality

[0042] According to the present invention, peptides with sequences SEQ ID NO:2-179 exhibit excellent efficacy in both inhibiting inflammation and as a preventative measure. Therefore, compositions containing the peptides of the present invention can be used as anti-inflammatory pharmaceutical compositions or as cosmetic compositions, thereby treating or preventing various types of inflammatory diseases.

[0043] References

[0044] KR2012-0130996A

[0045] KR2012-0133661A

[0046] KR2011-0060940A

[0047] US2011-0150873A1

[0048] Bonaldi T et al., EMBO J, (22) 5551-60, 2003

[0049] Yankner BA et al., Science (New York, NY) [1990, 250 (4978): 279-282]

[0050] Dahlgren KN et al., J. Biol. Chem. 277:32046-32053, 2002. Attached Figure Description

[0051] Figure 1 Figure showing the results of TNF-α ELISA using monocyte cultures derived from PBMCs. Monocytes were stimulated with LPS (10 ng / ml) for 2 hours, followed by reactions with peptides FITC, FITC-TAT, PEP 1-FITC, and FITC-peptide for 2 hours, respectively (**P<0.01, compared to the negative control group (FITC and FITC-TAT)).

[0052] Figure 2 Figure showing the results of luciferase analysis after transfection of HEK293 / blank and HEK293 / TLR2 cell lines with NF-κB luciferase, followed by reaction with lipoprotein (10 ng / ml) and FITC and FITC-PEP 1 (4 μM) and incubation for 18 hours. Luciferase results were corrected using René luciferase (**P < 0.01, compared to the negative control (untreated) and compared to the lipoprotein-treated samples).

[0053] Figures 3 to 23 This is a screening result of the inhibitory effect of TNF-α on monocytes.

[0054] Figures 24-46 This is the screening result of the inhibitory effect of TNF-α on the THP-1 cell line.

[0055] Figure 47 The viability of neural stem cells treated with 0, 2.5, 5.0, 10, 20 and 40 μM amyloid-β protein was demonstrated.

[0056] Figure 48 The proliferation of neural stem cells treated with 0, 2.5, 5.0, 10, 20 and 40 μM amyloid-β protein was demonstrated.

[0057] Figure 49 The viability of neural stem cells treated with 0, 1, 10, 50, 100 and 200 μM PEP 1 was demonstrated.

[0058] Figure 50 The proliferation of neural stem cells treated with 0, 1, 10, 50, 100 and 200 μM PEP 1 was demonstrated.

[0059] Figure 51 The viability of neural stem cells treated with 1, 10, 50 and 100 μM PEP 1 was demonstrated; neural stem cells were induced to be damaged by 20 μM amyloid-β protein, and then cell viability was measured after treatment with different concentrations of PEP-1 (the control group was not treated with amyloid-β protein and telomerase peptide).

[0060] Figure 52 The toxicity of neural stem cells treated with 1, 10, 50 and 100 μM PEP 1 was demonstrated; neural stem cells were damaged by 20 μM amyloid-β protein, and cytotoxicity was then measured after treatment with different concentrations of PEP-1 (the control group was not treated with amyloid-β protein and telomerase peptides).

[0061] Figure 53The proliferation of neural stem cells treated with 1, 10, 50 and 100 μM PEP 1 was demonstrated; neural stem cells were then subjected to damage with 20 μM amyloid-β protein, and cell proliferation was measured after treatment with different concentrations of PEP-1 (the control group was not treated with amyloid-β protein and telomerase peptides).

[0062] Figure 54 The migration of neural stem cells treated with 1, 10, 50 and 100 μM PEP 1 was demonstrated; neural stem cells were induced to be damaged by 20 μM amyloid β protein, and cell migration was then measured after treatment with different concentrations of PEP-1 (the control group was not treated with amyloid β protein and PEP-1).

[0063] Figure 55 Apoptosis of neural stem cells treated with 1, 10, 50 and 100 μM PEP 1 was demonstrated; neural stem cells were damaged by 20 μM amyloid-β protein, and then apoptosis was measured after treatment with different concentrations of PEP-1 (the control group was not treated with amyloid-β protein and telomerase peptide).

[0064] Figure 56 The study demonstrated the inhibitory effect of PEP-1 on ROS (reactive oxygen species) in neural stem cells damaged by amyloid-β protein. Neural stem cells were induced to be damaged by 20 μM amyloid-β protein, and the inhibition of ROS was measured after treatment with different concentrations of PEP-1 (1, 10, 50 and 100 μM) (the control group was not treated with amyloid-β protein or PEP-1).

[0065] Figure 57 The results of protein expression level analysis using (A) 2D electrophoresis and (B) antibody array are presented. Neural stem cells were induced to be damaged by 20 μM amyloid-β protein, and then protein expression levels were measured after treatment with different concentrations of PEP-1 (1, 10 and 50 μM) (the control group was not treated with amyloid-β protein and PEP-1).

[0066] Figure 58 The results of Western blot analysis showed the expression levels of inflammation-related proteins: neural stem cells were damaged by 20 μM amyloid-β protein, followed by treatment with different concentrations of PEP-1 (1, 10 and 50 μM).

[0067] Figure 59 The inhibitory effect of PEP 1 on amyloid-β protein aggregation was demonstrated; (A) showed that oligomerization of amyloid-β protein was reduced when treated with 1 μM amyloid-β protein and PEP 1 (0.1, 1 and 10 μM); (B) showed the effect of treating induced aggregated amyloid-β protein with PEP-1.

[0068] Figure 60 The effect of the PI3K inhibitor LY294002 on the viability of cells treated with PEP 1 was presented. The increased cell viability after PEP 1 treatment was reduced after LY294002 treatment.

[0069] Figures 61 to 159 The results of Western blot analysis of the selected peptides showed the accumulation of HMGB1 in cells. Detailed Implementation

[0070] Because this invention can be adapted to various transformations and practical applications, the following is a more detailed description of the invention. However, this does not imply a limitation on practical applications; it should be understood that the intent of this invention is to encompass the technical concepts and extensions in all transformations, equivalents, and alternatives. In describing this invention, any detailed description of the prior art that would be considered to destroy the essential principles of the invention will be omitted.

[0071] Telomeres are known to be repetitive sequences of genetic material at the ends of chromosomes, preventing chromosome damage or fusion with other chromosomes. Telomeres shorten with each cell division; after a certain number of divisions, they become so short that the cell stops dividing and dies. Conversely, telomere elongation is known to extend cell lifespan. For example, cancer cells secrete an enzyme called telomerase, which prevents telomere shortening, thus leading to cancer cell proliferation. This invention is based on the discovery of peptides derived from telomerase that have anti-inflammatory effects.

[0072] In one embodiment of the present invention, a peptide with anti-inflammatory activity is provided. The peptide comprises at least one amino acid sequence from SEQ ID NO: 2 to 179, and the peptide has more than 80% homology to the aforementioned sequences, or the peptide is a fragment of the aforementioned peptide.

[0073] The peptides described in SEQ ID NO:2–179 are shown in Table 1 below. SEQ ID NO:180 lists the full-length sequence of human telomerase protein. SEQ ID NO:1 lists a telomerase-derived peptide consisting of a 16-amino acid sequence. The peptides mentioned in SEQ ID NO:2–179 are shown in Table 1 below. SEQ ID NO:180 lists the full-length sequence of human telomerase protein. SEQ ID NO:1 lists a telomerase-derived peptide consisting of a 16-amino acid sequence. The peptides in SEQ ID NO:2–77 are peptides containing SEQ ID NO:1. The peptides in SEQ ID NO:78–179 are fragments of the peptide in SEQ ID NO:1.

[0074] The “Name” in Table 1 below is used to distinguish these peptides. In another specific embodiment of the invention, more than one of the peptides described in SEQ ID NO: 2 to 179 includes a “synthetic peptide,” that is, a synthetic peptide containing a selected region of telomerase. In this specification, the term “pep” refers to: a peptide having any one of SEQ ID NO: 2 to 179, or a peptide containing an amino acid sequence having more than 80% homology with the above sequences, or a peptide fragment of the above peptides.

[0075] Table 1

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] In one embodiment of the invention, a polynucleotide encoding a peptide having anti-inflammatory activity is provided. This polynucleotide encodes: a peptide comprising at least one amino acid sequence from SEQ ID NO: 2 to 179, a peptide having more than 80% homology to the aforementioned sequence, or a fragment of the aforementioned peptide. This polynucleotide enables the mass production of the peptide. For example, culturing a vector comprising the polynucleotide encoding the peptide allows for the mass production of the peptide.

[0082] The peptides disclosed herein may include peptides comprising an amino acid sequence having homology of more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%. Furthermore, the peptides disclosed herein may include peptides comprising SEQ ID NO:1 or a fragment thereof, and peptides having more than one transformed amino acid, more than two transformed amino acids, more than three transformed amino acids, more than four transformed amino acids, more than five transformed amino acids, more than six transformed amino acids, or more than seven transformed amino acids.

[0083] In this specification and claims, the terms "homology" and "sequence identity" are used interchangeably to indicate the degree of sequence overlap between two amino acid (or nucleic acid, if applicable) sequences.

[0084] Unless otherwise stated, the term "sequence identity" used in this document for peptides refers to sequence identity in terms of (n...) ref -n dif )·100 / n ref The sequence identity is computed, where n difThis refers to the number of distinct residues in two sequences when comparing them to maximize the number of identical amino acids, where n ref This refers to the number of residues in the shortest of these sequences. Therefore, the DNA sequence agtcagtc and the sequence aatcaatc will have 75% sequence identity (n). dif =2 and n ref =8).

[0085] In some implementations, sequence identity is determined using conventional methods, such as: Smith and Waterman, 1981, Adv. Appl. Math. 2:482; similarity-based methods (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444); the CLUSTAL W algorithm (Thompson et al., 1994, Nucleic Acids Res 22:467380); and algorithms executed by computer (Wisconsin Genetics Software Package, GAP, BESTFIT, FASTA, and TFASTA from the Genetics Computer Group). The BLAST algorithm (Altschul et al., 1990, Mol. Biol. 215:403-10) can also be used, and its software is available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). When using any of the above algorithms, use the default parameters for "Window" length, empty space penalty, etc.

[0086] In one embodiment of the invention, the alteration of the amino acid sequence constitutes a modification of the physical and chemical properties of the peptide. For example, amino acid transformations can be performed to improve the peptide's thermal stability, alter its substrate specificity, or change its optimal pH.

[0087] In one embodiment of the present invention, a peptide comprising an amino acid sequence of at least one of SEQ ID NO:2 to 179, a peptide comprising an amino acid sequence having more than 80% homology with the above sequence, or a peptide fragment of the above peptide preferably consists of 30 or fewer amino acids.

[0088] In one embodiment of the present invention, a peptide comprising an amino acid sequence of at least one of SEQ ID NO: 2 to 179, a peptide comprising an amino acid sequence having more than 80% homology with the above sequence, or a peptide fragment of the above peptide comprising a peptide derived from telomerase (more specifically, telomerase of Homo sapiens).

[0089] The term "amino acid" as used herein includes not only the 22 standard amino acids naturally integrated into peptides, but also D-isomers and transformed amino acids. Therefore, in certain embodiments of the invention, peptides herein include peptides having D-amino acids. Alternatively, peptides may include non-standard amino acids, such as those that have undergone post-translational modifications. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including acetylation, myristylation, palmitoylation), alkylation, carboxylation, hydroxylation, glycosylation, biotinylation, ubiquitination, chemical property changes (e.g., β-elimination deimide reaction, deamidation), and structural changes (e.g., disulfide bond formation). Furthermore, changes in amino acids attributable to chemical reactions during the binding process using a crosslinking agent for forming peptide conjugates are also included.

[0090] The peptides disclosed herein may be wild-type peptides identified and isolated from natural sources. On the other hand, when compared with the peptide fragment of SEQ ID NO:1, the peptides disclosed herein may be artificially mutant peptides containing one or more substituted, deleted, and / or inserted amino acids. Amino acid variations in wild-type peptides (not only in artificially mutant peptides) include conserved amino acid substitutions that do not affect protein folding and / or activation. Examples of conserved substitutions belong to the group consisting of: 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). Amino acid substitutions that generally do not alter specific activities are known in the art to which this invention pertains. The most common changes 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, Asp / Gly, and the reverse. Other examples of conservative substitutions are shown in Table 2 below.

[0091] Table 2

[0092] Original amino acids Residue substitution examples Preferred residue substitutions Ala(A) val; leu; ile Val Arg(R) lys; gln; asn Lys Asn(N) gln; his; asp; lys; arg Gln Asp(D) glu; asn Glu Cys(C) ser;ala Ser Gln(Q) asn; glu Asn Glu(E) asp;gln Asp Gly(G) ala Ala His(H) asn; gln; lys; arg Arg Ile(I) leu;val;met;ala;phe;orleucine Leu Leu(L) Leucine; ile; val; met; ala; phe Ile Lys(K) arg; gln; asn Arg Met(M) leu; phe; ile Leu Phe(F) leu; val; ile; ala; tyr Tyr Pro(P) ala Ala Ser(S) thr Thr Thr(T) ser Ser Trp(W) tyr;phe Tyr Tyr(Y) trp; phe; thr; ser Phe Val(V) ile; leu; met; phe; ala; leucine Leu

[0093] Substantial transformation of the biological properties of peptides is achieved by selecting substitutions that differ significantly in the following aspects: (a) the ability to maintain the structure of the peptide backbone (e.g., folded or helical three-dimensional structure) in the substitution region; (b) the ability to maintain the charge or hydrophobicity of the molecule in the target region; or (c) the ability to maintain the volume of the side chain. Natural residues can be grouped into the following groups based on general side chain properties:

[0094] (1) Hydrophobicity: ortholeucine, met, ala, val, leu, ile;

[0095] (2) Neutral hydrophilicity: cys, ser, thr;

[0096] (3) Acidic: asp, glu;

[0097] (4) Alkaline: asn, gln, his, lys, arg;

[0098] (5) Residues affecting chain orientation: gly, pro; and

[0099] (6) Aromatic: trp, tyr, phe.

[0100] Non-conservative substitutions can be made by replacing members of one of the aforementioned categories with members of another category. Any cysteine ​​residue unrelated to maintaining the correct three-dimensional structure of the peptide can generally be replaced with a serine residue, thus increasing the oxidative stability of the molecule and preventing incorrect cross-linking. Conversely, stability can be improved by adding (multiple) cysteine ​​bonds to the peptide.

[0101] Other types of amino acid variations in peptides include alterations in antibody glycosylation patterns. The term "alteration" here refers to the deletion of carbohydrate residues and / or the addition of at least one glycosylated residue not present in the peptide.

[0102] Glycosylation in peptides is typically N-linked or O-linked. The term "N-linked" in this paper refers to the attachment of a carbohydrate residue to the side chain of an asparagine residue. As tripeptide sequences, asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of carbohydrate residues to the asparagine side chain. Therefore, the presence of such a tripeptide sequence in a polypeptide creates potential glycosylation sites. "O-linked glycosylation" refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid. The most common hydroxy amino acids are serine or threonine, but 5-hydroxyproline or 5-hydroxylysine can also be used.

[0103] Glycosylation sites can be conveniently added to peptides by altering the amino acid sequence to include the aforementioned tripeptide sequence (to obtain N-linked glycosylation sites). These alterations can be made by adding at least one serine or threonine residue to the first antibody sequence or by replacing these residues (to obtain O-linked glycosylation sites).

[0104] In one embodiment of the invention, the polynucleotide is a nucleic acid molecule, which may be a single-stranded or double-stranded, natural or artificial DNA or RNA molecule. The nucleic acid molecule may be one or more nucleic acids of the same type (e.g., having the same nucleotide sequence) or different types of nucleic acids. The nucleic acid molecule includes, but is not limited to, one or more DNAs, cDNA, decoy DNA, RNA, siRNA, miRNA, shRNA, stRNA, snoRNA, snRNA, PNA, antisense oligomers, plasmids, and other modified nucleic acids.

[0105] HMGB1 protein is known to be a cytokine. It first undergoes acetylation and then translocates into the cytoplasm upon external stimulation. Subsequently, it is secreted extracellularly, thus playing the role of a pro-inflammatory cytokine. Because HMGB1 protein is secreted extracellularly when this activity induces inflammation, and patients with inflammatory diseases (such as Churg Strauss syndrome, rheumatoid arthritis, and Sjogren's syndrome) exhibit elevated serum HMGB1 levels. Therefore, if the cell nucleus still contains a large amount of HMGB1 even in the presence of pro-inflammatory stimuli, it reveals the fact that HMGB1 has not been secreted extracellularly, implying that inflammation is suppressed.

[0106] In one embodiment of the present invention, when cells are treated with a peptide containing any one of the amino acid sequences of SEQ ID NO:2 to 179, with a peptide having more than 80% homology with the above sequences, or with a fragment of the above peptides, the amount of HMGB1 in the cell nucleus increases. This indicates that the above peptides have excellent anti-inflammatory or anti-inflammatory effects.

[0107] Furthermore, in a particular embodiment of the present invention, a peptide comprising any one of the amino acid sequences in SEQ ID NO:2 to 179, a peptide having an amino acid sequence having more than 80% homology with the above sequence, or a fragment of the above peptide has the following advantages: it is highly feasible because of its low intracellular toxicity.

[0108] In this invention, "inflammatory disease" is a broad indication, referring to any disease with inflammation as the primary cause or inflammation caused by a disease. Specifically, inflammatory diseases include: (1) generalized or localized inflammatory diseases (e.g., allergic reactions; immune complex diseases; hay fever; hypersensitivity shock; endotoxic shock; cachexia; overheating; granulomatous diseases; or sarcoidosis); (2) gastrointestinal diseases (e.g., appendicitis; gastric ulcers; duodenal ulcers; peritonitis; pancreatitis; ulcerative colitis, acute colitis, or ischemic colitis; cholangitis; cholecystitis, steatorrhea, hepatitis, Crohn's disease; or Whipple's disease); (3) skin diseases (e.g., psoriasis; burns; sunburn; dermatitis; urticaria or wheals). (4) Vascular-related diseases (e.g., vasculitis; vasculitis; endocarditis; arteritis; arteriosclerosis; thrombophlebitis; pericarditis; congestive heart failure; myocarditis; myocardial ischemia; periarteritis nodosa; periodic stenosis; Berger's disease; or rheumatic fever); (5) Respiratory diseases (e.g., asthma; epiglottitis; bronchitis; emphysema; rhinitis; cystic fibrosis; interstitial pneumonia; COPD (chronic obstructive pulmonary disease); adult respiratory distress syndrome; dust sickness; alveolitis; bronchiolitis; pharyngitis; pleurisy; or sinusitis); (6) Bones, joints, muscles and connective tissue (7) Tissue-related diseases (e.g., eosinophilic granuloma; arthritis; arthralgia; osteomyelitis; dermatomyositis; fasciitis; Paget's disease; gout; periodontitis; rheumatoid arthritis; myasthenia gravis; ankylosing spondylitis; or synovitis); (8) Genitourinary disorders (e.g., epididymitis; vaginitis; prostatitis; or urethritis); (9) Central or peripheral nervous system-related diseases (e.g., Alzheimer's disease; meningitis; encephalitis; multiple sclerosis; cerebral infarction; cerebral embolism; Gurgbach syndrome; neuritis; neuralgia; spinal cord injury; paralysis; or uveitis); (10) Diseases Viruses (e.g., influenza; respiratory syncytial virus; HIV; hepatitis B; hepatitis C; or herpesvirus), infectious diseases (e.g., dengue fever; or sepsis), fungal infections (e.g., candidiasis); or bacterial, parasitic and similar microbial infections (e.g., disseminated bacteremia; malaria; onchocerciasis; or amoebiasis); (10) autoimmune diseases (e.g., thyroiditis; lupus erythematosus; Gudpaschury syndrome; allogeneic transplant rejection; graft-versus-host disease; or diabetes); and (11) cancer or neoplastic diseases (e.g., Hodgkin's disease), but not limited thereto.

[0109] For decades, treating the inflammatory components of these diseases has been a primary target of the global pharmaceutical industry, and a variety of useful therapeutic agents have been developed. Examples include corticosteroids (a variety of natural, semi-synthetic, and synthetic agents designed to mimic the effects of cortisol, including prednisolone, methylprednisolone, dexamethasone, betamethasone, fluticasone, etc.), cyclooxygenase inhibitors (non-selective or COX-1 selective inhibitors, such as indomethacin, sulfasalzine, and aspirin, and more recently, COX-2 selective inhibitors, such as celecoxib), leukotriene blockers (e.g., montelukast), and anti-TNF agents (e.g., modified monoclonal neutralizing antibodies, including infliximab). TM ) and adalimumab (Humira) TM ); TNF receptor fusion proteins, such as etanercept (Enbrel) TM ); and small molecule TNF-α synthesis inhibitors, such as thalidomide).

[0110] In one embodiment of the present invention, an anti-inflammatory composition comprising a peptide as an active ingredient is provided. The peptide comprises at least one amino acid sequence from SEQ ID NO:2 to 179, the peptide having more than 80% homology to the aforementioned sequence, or the peptide being a fragment of the aforementioned peptide.

[0111] In one embodiment of the invention, the anti-inflammatory composition may contain 0.1 μg / mg to 1 mg / mg, particularly 1 μg / mg to 0.5 mg / mg, and more particularly 10 μg / mg to 0.1 mg / mg of a peptide comprising at least one of the amino acid sequences of SEQ ID NO: 2 to 179, a peptide comprising an amino acid sequence having more than 80% homology to the above sequence, or a peptide fragment of the above peptide. When the composition contains peptides within the above range, all safety and stability requirements are met, and it is suitable in terms of cost-effectiveness.

[0112] In one embodiment of the invention, the composition can be applied to all animals, including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs, and monkeys.

[0113] In one embodiment of the invention, a pharmaceutical composition is provided for treating or preventing inflammatory diseases with an active ingredient comprising a peptide consisting of an amino acid sequence from SEQ ID NO:2 to 179, a peptide containing an amino acid sequence having more than 80% homology to the above sequences, or a peptide fragment of SEQ ID NO:1. In one embodiment of the invention, the pharmaceutical composition can be administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intramedullary, epidurally, or subcutaneously.

[0114] Oral administration may take the form of (but is not limited to) tablets, pills, soft capsules or hard capsules, granules, powders, solutions or emulsions. Non-oral administration may take the form of (but is not limited to) injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays.

[0115] In one embodiment of the invention, the pharmaceutical composition may contain additives, such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavorings, or sweeteners, if desired. In one embodiment of the invention, the pharmaceutical composition may be manufactured using conventional industrial methods in the art.

[0116] In one embodiment of the invention, the active ingredient of the pharmaceutical composition may vary depending on the patient's age, sex, weight, pathology and condition, route of administration, or the prescribing physician's judgment. Those skilled in the art can determine the dosage based on these factors; for example, the daily dose may be (but is not limited to) 0.1 μg / kg / day to 1 g / kg / day, particularly 1 μg / kg / day to 10 mg / kg / day, more particularly 10 μg / kg / day to 1 mg / kg / day, and more particularly 50 μg / kg / day to 100 μg / kg / day. In one embodiment of the invention, the pharmaceutical composition may be administered (but is not limited to) 1 to 3 times daily.

[0117] In one embodiment of the present invention, a topical skin composition for improving or preventing skin inflammation is provided. The topical skin composition may contain the following active ingredients: a peptide comprising an amino acid sequence from SEQ ID NO:2 to 179, a peptide comprising an amino acid sequence having more than 80% homology to the above sequence, or a peptide fragment of the above peptide.

[0118] In another embodiment of the present invention, a cosmetic composition for improving or preventing skin inflammation is provided. The cosmetic composition may contain the following active ingredients: a peptide comprising an amino acid sequence from SEQ ID NO:2 to 179, a peptide comprising an amino acid sequence having more than 80% homology to the above sequence, or a peptide fragment of the above peptide.

[0119] In one embodiment of the invention, the topical composition or cosmetic composition can be provided in all forms suitable for topical application. For example, it can be provided as a solution, an emulsion obtained by dispersing an oil phase in water, an emulsion obtained by dispersing water in an oil phase, a suspension, a solid, a gel, a powder, a paste, a foam, or an aerosol. These forms can be manufactured using conventional industrial methods in the art.

[0120] In one embodiment of the invention, the cosmetic composition may include other ingredients that can ideally enhance the main effects, to the extent that the main effects are not impaired. In another embodiment of the invention, the cosmetic composition may additionally include: moisturizers, emollients, surfactants, UV absorbers, preservatives, fungicides, antioxidants, pH adjusters, organic or inorganic pigments, fragrances, coolants, or antiperspirants. Those skilled in the art can determine the formulation ratios of the above ingredients to the extent that they do not impair the purpose and effects of the invention, and the formulation ratios based on the total weight of the cosmetic composition can be from 0.01% by weight to 5% by weight, particularly from 0.01% by weight to 3% by weight.

[0121] Those skilled in the art can determine the dosage of the above-mentioned active ingredients, and the daily dosage can be, for example, from 1 μg / kg / day to 10 mg / kg / day, more particularly from 10 μg / kg / day to 1 mg / kg / day, more particularly from 50 μg / kg / day to 100 μg / kg / day, but is not limited to these amounts, and can vary according to age, health status, complications and many other factors.

[0122] In one embodiment of the present invention, the use of a peptide comprising an amino acid sequence from SEQ ID NO:2 to 179, a peptide comprising an amino acid sequence having more than 80% homology with the above sequence, or a peptide fragment of the above peptide is provided for the prevention or treatment of inflammatory diseases.

[0123] In one embodiment of the present invention, a method is provided for preventing or treating inflammatory diseases by applying the above-mentioned peptides to a patient.

[0124] In one embodiment of the present invention, a kit for the prevention or treatment of inflammatory diseases is provided. The kit may comprise: a peptide having anti-inflammatory activity or a composition containing the peptide, wherein the peptide comprises any one amino acid sequence from SEQ ID NO:2 to 179, the peptide having more than 80% homology to the aforementioned sequence, or the peptide being a fragment of the aforementioned peptide; and instructions for use including at least one of the following: dosage, route of administration, frequency of administration, and indications for the peptide or composition.

[0125] The terminology used herein is intended to describe embodiments and not to limit the invention. Terms without preceding numbers are not intended to limit quantities, but rather to indicate that the term may refer to more than one thing. The terms "comprising," "having," "consisting of," and "including" should be interpreted openly (i.e., "including but not limited to").

[0126] Mentioning a range of values ​​is used in place of stating individual numbers within that range; therefore, unless explicitly stated otherwise, each number can be interpreted as an individual number within that set. All endpoints of the range are included within it and can be combined independently.

[0127] Unless otherwise specified in the context or clearly contradicted, all methods described herein may be performed in a suitable order. Unless included in the claims, the use of any and all embodiments or exemplary language (such as the use of "for example") is for the purpose of more clearly describing the invention and not for limiting its scope. No language other than that used in the claims herein should be construed as essential to the invention. Unless otherwise defined, the technical terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0128] The preferred embodiments of the present invention are the best modes known to the inventors for carrying out the invention. Variations of the preferred embodiments will be clear to those skilled in the art after reading the preceding description. The inventors intend that those skilled in the art will fully utilize these variations, and that the invention can be practiced in ways other than those listed herein. Therefore, to the extent permitted by patent law, the invention includes equivalents and variations of the key points of the invention as described in the appended claims. Furthermore, unless expressly stated or contradicted in the context, all possible variations in any combination of the foregoing components are included in the invention. Although the invention has been illustrated and shown through exemplary embodiments, those skilled in the art will fully understand that various changes in form and detail are possible without departing from the spirit and scope of the invention as defined by the claims.

[0129] Inflammatory cells are known to release tumor necrosis factor (TNF), particularly TNF-α, which causes various cytotoxic, immune, and inflammatory responses. TNF-α is known to be involved in the development and prolongation of many inflammatory diseases and autoimmune diseases, and when released into the bloodstream and acting systemically, it can further lead to severe sepsis and septic shock. Because TNF-α is a factor widely associated with the living immune system, TNF-α inhibitors are being actively developed. TNF-α is biosynthesized in its inactive form and is cleaved into its active form by proteases; the enzyme responsible for this activation is called tumor necrosis factor converting enzyme (TACE). Therefore, substances that inhibit TACE can treat, improve, or prevent diseases, pathological states, abnormalities, distress, and adverse symptoms attributed to TNF-α (KR2011-0060940A).

[0130] High-mobility group box 1 (HMGB1) is present in high concentrations in the thymus, lymph nodes, testes, and fetal liver, and is typically found in the cell nucleus, except in hepatocytes and brain cells. The HMGB1 protein has three domains: an A-box, a B-box, and a C-terminus.

[0131] According to Tracey et al., 1999, the HMGB1 protein plays the role of an inflammation-inducing cytokine, and the inflammation-inducing mechanism of HMGB1 is as follows: HMGB1 is acetylated by external stimuli, and then moves from the nucleus into the cytoplasm. It is then known to be secreted extracellularly, or secreted into necrotic extracellular spaces (Bonaldi T et al., EMBO J, (22) 5551-60, 2003).

[0132] The present invention will be further described with reference to the accompanying drawings, the following embodiments, and experiments. The drawings, embodiments, and experiments are only for illustrating specific implementations of the present invention and should in no way be construed as limiting the scope of the present invention.

[0133] Example 1

[0134] Synthesis of PEP-1 and measurement of the anti-inflammatory activity of PEP-1 (SEQ ID NO:1)

[0135] Experiment 1: Synthesis of PEP-1 (SEQ ID NO:1)

[0136] A peptide (PEP-1) consisting of 16 amino acids with the following chemical structure was synthesized. This peptide is derived from human telomerase and its sequence is SEQ ID NO:1:

[0137] <Chemical Structure 1>

[0138]

[0139] SEQ ID NO:1 (PEP-1) was synthesized according to existing solid-phase peptide synthesis methods. Specifically, the peptide was synthesized via Fmoc solid-phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, ROK) to couple each amino acid from the C-terminus. The following peptides were used (the first amino acid at the C-terminus was attached to the resin):

[0140] NH2-Lys(Boc)-2-chloro-triphenylmethyl resin

[0141] NH2-Ala-2-chloro-triphenylmethyl resin

[0142] NH2-Arg(Pbf)-2-chloro-triphenylmethyl resin

[0143] All amino acid materials used in the synthesis of peptides are protected at the N-terminus by Fmoc, and the amino acid residues are protected by acid-soluble Trt, Boc, t-Bu (tert-butyl ester), and Pbf (2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl). For example:

[0144] 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(tB u)-OH, Fmoc-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-mercaptoacetic acid.

[0145] HBTU [2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate] / HOBt [N-hydroxybenzotriazine] / NMM [4-methylmorpholine] was used as the coupling agent. Fmoc was removed using piperidine in 20% DMF. A cleavage mixture [TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / EDT (ethylene dithiol) / H2O = 92.5 / 2.5 / 2.5 / 2.5] was used to remove protection from residues or to separate the synthesized peptide from the resin.

[0146] Peptides were synthesized using a solid-phase scaffold and by repeating the following process: amino acid protection was initiated, allowing the corresponding amino acids to react individually, followed by solvent washing and removal of the protection. After cleaving the synthesized peptides from the resin, they were purified by HPLC, and the synthesis was verified by MS, followed by lyophilization.

[0147] The specific synthesis process of PEP 1 is described below.

[0148] 1) Coupling

[0149] The amino acid (8 equivalents) protected by NH2-Lys(Boc)-2-chloro-triphenylmethyl resin and the coupling agent HBTU (8 equivalents) / HOBt (8 equivalents) / NMM (16 equivalents) were melted and added to DMF, and then allowed to react at room temperature for 2 hours. The mixture was then washed with DMF, MeOH and DMF in sequence.

[0150] 2) Fmoc protection

[0151] 20% piperidine was added to DMF and the reaction was carried out twice at room temperature for 5 minutes each time. The mixture was then washed with DMF, MeOH and DMF in sequence.

[0152] 3) The basic framework of the peptide is created by repeating reactions 1 and 2.

[0153] 4) Cutting: Add the cutting mixture to the fully synthesized peptide to separate the peptide from the resin.

[0154] 5) Add ether for cooling to the obtained mixture, and then precipitate the collected peptides by centrifugation.

[0155] 6) After purification by preparative HPLC, the molecular weight was checked by LC / MS, and the product was lyophilized to produce a powder.

[0156] Experiment 2: Measurement of the anti-inflammatory activity of PEP-1

[0157] Cell line culture

[0158] Raw 264.7 macrophages (KCBL, 40071) from the Korea Cell Bank were maintained at 37°C and 5% CO2 in Dalberg modified Eagle medium (DMEM; PAA, Austria) containing 10% fetal bovine serum (FBS; Gibco Laboratories) and 100 units / mL streptomycin-penicillin (Gibco Laboratories). Cells were grown at a concentration of 1 × 10⁻⁶ cells / mL. 6 Raw 264.7 cells were seeded into 96-well plates at a density of 10 cells / mL and incubated overnight.

[0159] The following day, the culture medium was replaced with fresh medium, and 5 μg / mL of peptide (obtained as described in Experimental Example 1) was added to the cells. After incubating the cells with the peptide for 30 minutes, 50 μL of LPS was added (to a final concentration of 1 μg / mL), and the cells were incubated for another 24 hours. Samples inducing an inflammatory response were treated with 1 μg / mL lipopolysaccharide (LPS; Sigma, USA), and control samples were treated with phosphate-buffered saline (PBS; pH 7.2). Supernatants from each condition were collected in eppendorf tubes and further analysis was performed.

[0160] Experiment 2-1: Analysis of NO levels

[0161] Raw 264.7 cells (1×10⁻⁶) were measured using the Griess reagent system (Promega, USA). 6 The nitric oxide (NO) level was measured at cells / ml. 50 μl of culture medium was added to a 96-well plate, followed by equal volumes of Griess Reagent I (NED) solution and Griess Reagent II (p-aminobenzenesulfonamide solution). Cells were incubated with these reagents for 10 minutes, after which the optical density at 540 nm was measured over 30 minutes using a microplate reader (Molecular Devices, USA). The NO concentration was calculated using a sodium nitrite standard curve (0–100 μM).

[0162] As shown in Table 3, LPS stimulation of cells increased NO expression, but when treated with LPS and PEP-1 together, the NO expression level decreased. NO is produced during inflammation, and the results showed that PEP-1 reduced NO levels to 65% of the control, which strongly supports the anti-inflammatory effect of PEP-1.

[0163] Table 3: Measurement of the anti-inflammatory effect of PEP 1 derived from human telomerase

[0164]

[0165] Experiment 2-2: Analysis of the inhibitory effect on cytokines

[0166] To investigate the effect of PEP-1 on inhibiting the production of pro-inflammatory cytokines, RAW 264.7 cells excited by LPS at a concentration of 1 μg / mL were pretreated with 5 μg / mL PEP-1 and then incubated for 24 hours. The supernatant containing cell culture medium was collected, and cytokine levels were analyzed using an ELISA kit (eBioscience, San Diego).

[0167] Spread 100 μL of capture antibody (diluted to the concentration recommended by the manufacturer's operating procedure) onto a 96-well plate overnight at 4°C. Next, after washing the plate five times, add 200 μL of assay diluent to each well and incubate at room temperature for 1 hour for blocking. After washing each well five times with wash buffer, dilute the cell culture sample or each cytokine standard protein sample and add 100 μL of each sample to each well. Incubate the plate containing the samples overnight at 4°C. Next, after washing the plate five times with wash buffer, add 100 μL of avidin-conjugated secondary antibody and incubate at room temperature for 1 hour.

[0168] After incubation with the secondary antibody, the plate was washed five times and incubated with 100 μL of avidin-HRP (BDBioscience) at room temperature for 30 minutes. After washing the plate seven times, 100 μL of TMB solution (Pierce) was added and the plate was incubated at room temperature for 15 minutes. The reaction was terminated by adding 50 μL of 2N H2SO4 to each well. The optical density at 450 nm was measured using a microplate reader. Statistical analysis was performed using ANOVA in SPSS, and Duncan's multivariate test was used to verify the significance of the analyses.

[0169] Experiment 2-3: Measurement of IL-6 secretion

[0170] As shown in Table 4 below, LPS treatment alone increased the secretion of the cytokine IL-6 (interleukin-6). However, co-treatment with LPS and PEP-1 showed a decrease in the secretion level of the pro-inflammatory cytokine IL-6. More importantly, after treatment with PEP-1, the secretion level of pro-inflammatory cytokines decreased by more than 70%, indicating the robust anti-inflammatory effect of PEP-1.

[0171] Table 4: PEP-1 inhibits the production of the cytokine IL-6

[0172]

[0173] Experiments 2-4: Inhibition of HMGB1, TNF-α, and COX-2 expression

[0174] Western blotting analysis was used to determine protein expression levels. Cells grown in PEP-1 medium were washed with PBS, treated with 0.05% trypsin-EDTA, and collected by centrifugation. The collected cells were dissolved in an appropriate volume of lysis buffer. Intracellular debris was centrifuged to form a precipitate, and equal amounts of proteins from each sample were separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH, USA), and antibody specificity for each protein was then tested. The membranes were incubated with ECL (enhanced chemiluminescence) solution (Amersham Life Science Corp., Arlington Heights, IL, USA), exposed to X-rays, and protein expression levels were analyzed based on the exposure levels shown on the X-ray membranes.

[0175] Western blot analysis was performed to determine the inhibitory effect of PEP-1 on cytokine protein expression. As shown in Table 5, LPS stimulation of cells increased the expression of cytokines (HMGB1, TNF-α, and COX). However, when cells were treated with both LPS and PEP-1, the expression levels of these pro-inflammatory cytokines decreased. The results showed that treatment with PEP-1 reduced the levels of pro-inflammatory cytokines by more than 70%, providing strong evidence to support the anti-inflammatory effect of PEP-1.

[0176] Table 5: Measurement of the inhibitory effect of PEP-1 on the expression levels of pro-inflammatory cytokines

[0177]

[0178] Experiment 3: Study on the inhibitory effect of Pep1 on TNF-α levels in HepG2 cells

[0179] Experiment 3-1: Cell Culture

[0180] PBMCs (peripheral blood mononuclear cells) were isolated from blood samples (50 ml) collected from healthy subjects using a Ficoll-Paque™ PLUS (GE Healthcare Life Sciences, Piscataway, NJ, USA). The PBMCs were then enriched in complete RPMI 1640 medium containing 20% ​​human serum and transferred to 100 mm polystyrene cell culture plates coated with human serum for 30 minutes. After incubation at 37°C and 5% CO2 for 2 hours, monocytes were detached from the bottom of the cell culture plate using cold PBS (Gibco / Life Technologies, Carlsbad, CA, USA) and cultured overnight in RPMI 1640 medium (supplemented with penicillin-streptomycin 100 mg / ml; 20% human serum) for 1 × 10⁶ cells per well of a 96-well plate. 5 Each cell.

[0181] In the luciferase assay, HEK293 / blank (human embryonic kidney) cells and HEK293 / TRL cells stably expressing TLR2 (Toll-like receptor 2) were used. One day prior to the luciferase experiment, 2.5 × 10⁻⁶ cells were prepared. 5 Cells were seeded into each well of a 12-well plate and cultured overnight in DMEM (Dalberke modified Eagle medium) supplemented with 10 μg / ml blast fungicide and 10% fetal bovine serum (Invitrogen / Life Technologies, Carlsbad, CA, USA).

[0182] Experiment 3-2: Cytokine Assay

[0183] To observe the effect of PEP-1 on TNF-α levels in terms of protein expression, an ELISA (enzyme-linked immunosorbent assay) was performed. The cells were cultured overnight in 96-well plates at a density of 1×10⁶ cells / well. 5Mononuclear cells derived from PBMCs were used. They were then treated with LPS (lipopolysaccharide; 10 ng / ml, Sigma) for 2 hours, followed by washing three times with PBS. Next, they were treated with OPTI-MEM medium (Invitrogen / Life Technologies, Carlsbad, CA, USA) for 1 hour to induce starvation, and then treated with 4 μM FITC (fluorescein isothiocyanate), FITC-TAT, PEP-1-FITC, and FITC-PEP-1 for 2 hours, after which TNF-α levels were measured. After culture, cell soup was collected, and TNF-α levels were measured using an ELISA kit (R&D, Minneapolis, MN, USA) as follows:

[0184] TNF was measured using a sandwich ELISA method. 100 μl of TNF-α primary antibody was added to each well of a pre-coated 96-well plate, and the plate was incubated overnight at 4°C. The next day, the plate was washed three times with 0.5% Tween 20 washing solution for 5 minutes each time, followed by the addition of 100 μl of each sample and standard solution, and incubated at room temperature for 2 hours. After washing the plate as described above, 100 μl of HRP-conjugated secondary antibody was added to each well, and the plate was incubated at room temperature for 2 hours. The 96-well plate was washed again, and avidin / biotin was added for absorbance measurement. The amount of TNF-α in each sample was quantitatively determined using a standard graph calculated from the absorbance of the standard solutions.

[0185] PBMC-derived monocytes were stimulated with endotoxin LPS (10 ng / ml) for 2 hours, starved with OPTI-MEM for 1 hour, and then treated with 4 μM FITC, FITC-TAT, PEP-1-FITC, and FITC-PEP-1 for 2 hours. After incubation, TNF-α levels in the cell culture medium were measured using ELISA. The results showed that in the FITC and FITC-TAT cases, TNF-α levels increased due to LPS (6.2 ng / ml and 6.7 ng / ml, respectively), but in the PEP-1-FITC and FITC-PEP-1 cases, TNF-α levels significantly decreased (0.17 ng / ml and 0.25 ng / ml, respectively), and this difference was statistically significant (P<0.01). Figure 1 ).

[0186] Experiment 3-3: Luciferase Analysis

[0187] To investigate the role of PEP 1 in the inflammatory response, the inventors evaluated the NF-κB expression pattern using luciferase analysis. First, HEK293 / blank and HEK293 / TLR2 (Seoul National University College of Dentistry) were incubated in 12-well plates for 24 hours to obtain 2.5 × 10⁻⁶ PEP ...5 Cells / well. After washing three times with PBS, the medium was replaced with OPTI-MEM (Invitrogen / Life Technologies, Carlsbad, CA, USA) and incubated for 4 hours. Then, a mixture of 3 μl Lipofectamine (Invitrogen / Life Technologies), 1 μg NF-κB luciferase, and 10 ng Renisar luciferase (Promega, Madison, WI, USA) was added to each well and incubated again for 4 hours. Lipoprotein pam3cys (10 ng / ml, Sigma-Aldrich, St. Louis, MO, USA) was placed in all wells except the negative control and treated with FITC (4 μM) and FITC-PEP1 (4 μM) for 18 hours, followed by washing three times with PBS. NF-κB activation was confirmed using a TD-20 / 20 illuminometer (Turner designs, Sunnyvale, CA, USA) after cells were lysed (dissolved) by adding 50 μl of passive lysis buffer provided by the Promega dual luciferase reporter assay system to each well. Transfection efficacy was confirmed by co-transfection with pCMV-Rhizoglossum luciferase (Promega), and the results were analyzed by calibrating luciferase values.

[0188] After transfecting HEK293 / blank and HEK293 / TLR2 cell lines with NF-κB luciferase, cells were treated with pam3cys (a synthetic lipoprotein) and FITC (4 μM) (negative control), and then again with pam3cys and FITC-PEP-1 (4 μM) and cultured for 18 hours. Cells were lysed using passive lysis buffer provided by a dual luciferase reporter assay system (Promega), and NF-κB expression patterns were measured using luciferase intensity. The results showed no difference between lipoprotein-treated, FITC-PEP-1-treated, and untreated HEK293 / blank cells. However, when HEK293 / TLR2 cell lines were treated with lipoprotein (an agonist of TLR2), NF-κB expression was increased compared to untreated cells (P < 0.01), confirming the occurrence of an inflammatory response. Furthermore, compared with untreated cells, NF-κB expression was increased when treated with FITC-PEP1; and decreased compared with the negative control treated with lipoprotein and FITC (P<0.01). Figure 2 Ultimately, it was confirmed that treatment with PEP 1 reduced the inflammatory response that could be induced by TLR 2.

[0189] Example 2

[0190] TNF-α inhibitory effect of PEPRIA series (SEQ ID NO: 2~179) peptides

[0191] Based on the results of Example 1 (wherein SEQ ID NO:1 (PEP1) exhibits TNF-α inhibitory effects), experiments were conducted using peptides SEQ ID NO:2–179 to confirm their TNF-α inhibitory effects. The peptides SEQ ID NO:2–179 were synthesized using the same method as described in Example 1 (the method used to synthesize PEP1), but with different added amino acids.

[0192] Experiment 1: Cell Culture

[0193] PBMCs (peripheral blood mononuclear cells) were isolated from blood samples (50 ml) collected from healthy subjects using Biocoll Separating Solution (Biochrom AG, Berlin, Germany). The collected PBMCs were enriched in RPMI 1640 medium containing 20% ​​human serum for 30 min, then transferred to 100 mm polystyrene cell culture plates coated with human serum and incubated at 37°C, 5% CO2 for 2 h. Mononuclear cells were detached from the bottom of the plate using cold PBS and incubated overnight in 96-well plates with RPMI 1640 medium (supplemented with penicillin-streptomycin 100 mg / ml; 20% human serum) to achieve a cell density of 1 × 10⁻⁶ cells / well. 5 The number of cells / well.

[0194] Experiment 2: Analysis of TNF-α Inhibition Effect

[0195] ELISA was performed to determine how the PEP RIA peptides affect TNF-α levels. Mononuclear cells derived from PBMCs were incubated in 96-well plates to a density of 1 × 10⁻⁶. 5 The number of cells / well was determined, and the cells were then treated with LPS (lipopolysaccharide; 10 ng / ml, Sigma) for 2 hours. OPTI-MEM medium was added to the mononuclear cells, which had been washed three times with PBS, to induce cell starvation for 1 hour. 4 μM peptide was then incubated for 2 hours. Three negative control groups were present. The first group was untreated. The second group was treated with estrogen (estradiol was used as an estrogen in this experiment). The third group was treated with LPS (10 ng / ml) or LPS (10 ng / ml) + estrogen (20 nM). PEP1, confirmed to have TNF-α inhibitory activity, was used as a positive control to measure TNF-α inhibitory activity. After incubation, TNF-α was measured according to the ELISA kit manual (R&D, Minneapolis, MN, USA). Details of the quantification method can be found in Experiment 2.2 of Example 1.

[0196] Using the above method, peptides with TNF-α inhibitory effects were screened. PBMC-derived monocytes were stimulated with endotoxin LPS (10 ng / ml) for 2 hours, followed by OPTI-MEM-induced cell starvation for 1 hour, and then treated with 4 μM of 179 peptides and incubated for 2 hours. The amount of TNF-α in the cell culture medium was measured using ELISA, and peptides with TNF-α inhibitory effects were screened by comparing with negative and positive controls. Figures 3 to 23 ).

[0197] The following peptide sequences showed TNF-α inhibitory effects compared to the control group treated only with LPS: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:124, SEQ ID NO:131, SEQ ID NO:140, SEQ ID NO:141, SEQ IDNO: 142, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177 and SEQ ID NO: 178.

[0198] Furthermore, the following peptide sequences showed TNF-α inhibitory effects compared to the group treated with LPS + estrogen: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 140, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 172, SEQ ID NO:173, SEQ ID NO:174.

[0199] Experiment 3: Analysis of peptides affecting TNF-α levels in the THP1 cell line

[0200] Experiments were conducted using the human acute monocytic leukemia cell line THP-1 (American Center for Type Culture Collection (ATCC) (Manassas, VA, USA)).

[0201] THP-1 cells were incubated in RPMI 1640 medium in 96-well plates for 24 hours to reach a cell density of 1×10⁻⁶ cells / well. 5 The number of cells / well was determined, and then 100 μM PMA (12-myristate-13-acetic acid phorbol ester) was added to differentiate them into macrophages. After THP-1 cells were differentiated into macrophages by PMA for 1 day, they were treated with LPS for 2 hours and washed away. They were starved for 1 hour and then treated with PEP1.

[0202] THP-1 cells differentiated under PMA were treated with LPS (lipopolysaccharide; 10 ng / ml, Sigma) for 2 hours, followed by washing twice with PBS. OPTI-MEM medium was added to the cells to induce cell starvation for 1 hour, then 1 μM of 179 peptides was incubated for 1 hour. After incubation, TNF-α levels were measured using an ELISA kit, and peptides that decreased TNF-α levels were screened. Figures 24 to 46 ).

[0203] The results showed that, compared with the control group treated with LPS alone, peptide sequences SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:107, SEQ ID NO:109, and SEQ ID NO:179 appeared to reduce TNF-α levels. Furthermore, compared with the group treated with LPS plus estrogen, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:30, SEQ ID NO:41, SEQ ID NO:112, and SEQ ID NO:113 were selected as peptides that reduced TNF-α expression levels.

[0204] Example 3

[0205] Analysis of the inflammatory response induced by amyloid-β protein

[0206] HMGB1 first undergoes acetylation and then translocates into the cytoplasm upon external stimulation. It is subsequently secreted extracellularly, thus playing the role of a pro-inflammatory cytokine. Because HMGB1 protein is secreted extracellularly when this activity induces inflammation, and patients with inflammatory diseases (such as Churg Strauss syndrome, rheumatoid arthritis, and Sjögren's syndrome) exhibit elevated serum HMGB1 levels. Therefore, if the cell nucleus still contains a large amount of HMGB1 even in the presence of pro-inflammatory stimuli, it indicates that HMGB1 has not been secreted extracellularly, suggesting that inflammation is suppressed.

[0207] Experiment 1: Analysis of neural stem cell survival and proliferation induced by the anti-inflammatory effect of PEP-1

[0208] First, PEP-1 was prepared according to the manufacturing method described in Example 1.

[0209] Experiment 1-1: Culture of Neural Stem Cells and Assessment of Amyloid-β Toxicity

[0210] The cortex was removed from the head of a 13-day-old gestational rat embryo and then cultured with basic fibroblast growth factor (bFGF) for one week to obtain neural stem cells. To analyze the effect of amyloid-β protein on neural stem cells, neural stem cells were treated with pre-oligomeric amyloid-β protein at concentrations ranging from 0 to 40 μM for 48 hours, followed by cytotoxicity assessment using CCK-8, BrdU, and TUNEL assays (refer to BA Yankner et al., 1990 and KN Dahlgren et al., 2002). The inventors confirmed that cell viability decreased to 60% upon treatment with 20 μM amyloid-β protein (see [link to previous section]). Figure 47 and Figure 48 Subsequently, the same concentration of amyloid β protein was used in subsequent experiments.

[0211] Experiments 1-2: Cytotoxicity assessment when treated with PEP-1

[0212] To assess the effects of PEP-1 on cultured neural stem cells, neural stem cells were first cultured using well-known methods (BA Yankner et al., 1990 and KN Dahlgren et al., 2002). Cells were then treated with different concentrations of PEP-1 (0, 1, 10, 50, 100, 200 μM) for 48 hours, followed by assessment of cell viability and proliferation using MTT, BrdU, and TUNEL assays. PEP-1 concentrations from 0 to 200 μM appeared stable in the neuronal system because these concentrations did not inhibit the survival and proliferation of neural stem cells (see [link to study]). Figure 49 and Figure 50 ).

[0213] Experiments 1-3: Cytotoxicity assessment of co-treatment with amyloid-β protein and telomerase peptide

[0214] To determine whether PEP-1 has an inhibitory effect on neurotoxicity induced by amyloid-β protein, cells were co-treated with 20 μM amyloid-β protein and multiple concentrations of PEP-1 for 48 hours. Cell viability and apoptosis were measured using MMT, CCK-8, LDH, and TUNEL assays, and neural stem cell proliferation was measured using BrdU assay.

[0215] The results of MMT and CCK-8 assays confirmed that 10 μM PEP-1 began to protect neural stem cells from neurotoxicity induced by amyloid-β, and that 100 μM provided the most effective protection (see [link to relevant documentation]). Figure 51LDH assays were performed as an alternative method for assessing the extent of cell death, and it was confirmed that the increase in cell death induced by amyloid-β was reduced by PEP-1, with efficacy observed starting from a concentration of 1 μM (see [link to study]). Figure 52 ).

[0216] The inventors also used BrdU assays to confirm that cell proliferation reduced by amyloid β protein was restored upon treatment with PEP-1 (see [link to BrdU assay]). Figure 53 ).

[0217] Due to the nature of neural stem cells, cell motility is a crucial factor. Based on the experimental results on cell motility, the inventors confirmed that, compared to the control, cell proliferation reduced by amyloid-β protein was restored when treated with PEP-1, and cell motility increased even more at a concentration of 10 μM. This suggests that pretreatment before stem cell transplantation could yield more effective results in future clinical trials (see [link to clinical trial]). Figure 54 ).

[0218] To confirm the extent of neural stem cell damage, a TUNEL assay was performed. A significant increase in neural stem cell death was observed in the 20 μM amyloid-β protein treatment group; however, neural stem cell death decreased when treated with PEP 1 ranging from 1 μM to 100 μM (see [link to study]). Figure 55 ).

[0219] The mechanism by which PEP-1 protects against apoptosis induced by amyloid-β protein was investigated. First, it was examined whether PEP-1 could minimize oxidative damage caused by amyloid-β protein. Changes in reactive oxygen species (ROS) production after treatment with amyloid-β protein and PEP-1 were observed using DCF-DA staining (Molecular Probes, Eugene, OR). In the group where ROS increased due to 20 μM amyloid-β protein, treatment with PEP-1 (1 μM, 10 μM, 50 μM) reduced the increase in ROS (see [link to study]). Figure 56 ).

[0220] Experiments 1-4: Comparison of protein expression levels between the PEP-1-treated group and the non-PEP-1-treated group Sexual analysis

[0221] Protein expression levels in the PEP-1-treated and untreated groups were analyzed using 2D electrophoresis and antibody microarray techniques. 200 μg of proteome was prepared from neural stem cells cultured in Experiment 1-1 of Example 3. Furthermore, the untreated group was used as a control group under the same conditions.

[0222] 2D electrophoresis was performed using a 12% acrylamide gel. Electrophoresis was first performed on an 8.5 × 7 cm gel at P1 4–10 N. After electrophoresis, the gel was stained with colloidal Coomassie blue, and each spot was analyzed using PDQuest software to compare expression levels.

[0223] The expression level differences exceeding 1.5-fold were identified using MALDI-TOF MS (matrix-assisted laser desorption / ionization time-of-flight mass spectrometry). Proteins associated with inflammation-related signaling, such as i-NOS and HMGB-1, were identified (see Table 6). Changes in protein expression levels were either increased or decreased by 1.5-fold due to amyloid-β protein, but it was also confirmed that the expression levels were modulated to near the negative control level upon the addition of PEP-1 (see Table 6). Figure 57 ).

[0224] Antibody microarrays were created using the Panorama™ Ab Microarray Cell Signaling Kit (CSAA1). The array slides were scanned using a GenePix Personal 4100A scanner (Molecular Devices), and the data were analyzed using GenePix Pro 5.0 (Molecular Devices).

[0225] Table 6 below shows the analysis of expression levels of inflammation-related proteins using 2D electrophoresis. The control group represents the protein expression levels of cells that were neither treated with amyloid-β nor with PEP-1. Table 6 shows the fold increase or decrease in protein expression levels based on the control group expression levels.

[0226] Based on the analysis results, the inventors confirmed that, as shown in Table 6 below, the overexpression or underexpression of inflammation-related proteins was controlled by PEP-1; the protein expression level was close to that of the negative control group.

[0227] Table 6

[0228]

[0229] The phosphatidylinositol 3-kinase (PI3K) / AKT signaling pathway plays a crucial role in the growth and survival of neural stem cells. The PI3K pathway is activated by growth factors and regulatory factors and participates in the normal regulation of neural stem cell growth and survival. The AKT signaling pathway inactivates various pro-apoptotic factors, including the well-known apoptosis signaling molecule GSK3β.

[0230] To further investigate the anti-inflammatory effects of PEP-1, the inventors performed Western blotting on HMGB1, as HMGB1 showed significant changes in protein analysis. The results showed that PEP-1 treatment increased the protein expression levels of anti-apoptotic proteins such as Ki67, pAKT, PI3K, HSTF-1, and Bcl-2, and decreased the protein expression levels of apoptotic signaling proteins such as Bax, GSK3β, cytochrome c, and caspase-3 (see [link to study]). Figure 58 ).

[0231] HMGB1, a DNA-binding non-histone structural protein, plays multiple roles within cells, such as stabilizing nucleosome structure and regulating gene expression. As one of the pro-inflammatory substances secreted in the later stages of the inflammatory response, it is secreted by macrophages and monocytes when stimulated by inflammation. However, when neurons are significantly damaged, leading to cell necrosis, HMGB1 is secreted extracellularly, resulting in a severe inflammatory response. In the cytoplasm of nerve cells, HMGB1 is reduced by amyloid-β treatment, but increased by PEP-1 treatment. This reflects the fact that PEP-1 inhibits the extracellular secretion of HMGB1 due to neuronal cell death; thus indicating that PEP-1 has a strong anti-inflammatory effect (see [link to relevant documentation]). Figure 58 ).

[0232] Furthermore, the inventors investigated the response of PEP-1 to amyloid β-aggregation. When PEP-1 was used to induce amyloid β-aggregation, protein aggregation was inhibited (see [link to study]). Figure 59 (A)), while treatment with PEP-1 on induced aggregated amyloid β protein causes protein depolymerization (see [A]). Figure 59 (B)).

[0233] Regarding the mechanism of action of PEP-1, the inventors had previously confirmed the increase in PI3K-mediated cell survival signaling and the decrease in PI3K-mediated apoptosis signaling. To investigate whether these effects were direct or indirect, the inventors treated patients with the PI3K inhibitor LY294002 (Promega). The results showed that treatment with LY294002 reduced the increased cell viability following PEP-1 treatment. Therefore, the inventors were able to conclude that PI3K is directly related to the neuroprotective effect of PEP-1 (see [link to article]). Figure 60 ).

[0234] PEP-1 inhibits apoptosis of neural stem cells induced by amyloid-β protein. Furthermore, it confirmed an increase in neural stem cell motility, thus indicating various possibilities for clinical application. The inhibitory effect on neurotoxicity caused by β-amyloid protein was confirmed by the following phenomena: the anti-inflammatory effect of PEP-1's mechanism of action, the increase of neural stem cell survival factors and the decrease of apoptosis factors, particularly the activation of the PI3K signaling pathway and antioxidant effects.

[0235] Example 4

[0236] The effects of PEP RIA series peptides (SEQ ID NO: 2~179) on inflammation caused by amyloid-β protein.

[0237] Experiment 1: Cell Culture

[0238] Undifferentiated PC12 cells (ATCC, Rockville, MD, USA) were kept in logarithmic growth phase on RPMI 1640 medium (GIBCO, Grand Island, NY, USA) pre-coated with poly-L-lysine (Sigma, Saint Louis, MO, USA) in 100 mm plates (Corning, PA, USA) containing 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum, 100 units / ml penicillin, and 100 g / ml streptomycin. The cultures were incubated at 37°C in a humid atmosphere containing 5% CO2. After reaching 50% confluence, the cultures were collected in a Ca-free medium. 2+ / Mg 2+ Cells were placed in a Hank's balanced salt solution containing 1 mM EDTA. 6 PC12 cells were seeded at a density of 100 cells / 100 mm² plate and incubated for 24 hours. To induce neuronal differentiation, PC12 cells were serum-starved for 12 hours (RPMI 1640 medium containing 100 units / ml penicillin and 100 g / ml streptomycin, but without horse serum or fetal bovine serum); subsequently, the cells were kept in serum-free medium. After two days, the medium was replaced with fresh serum-free medium. On the third day, NGF (50 ng / ml, Sigma, Saint Louis, MO, USA) was added to the medium, and the culture was kept for another three days. After differentiation, nPC12 cells were incubated for 48 hours with 20 μM amyloid β protein and various concentrations of peptides [0 (control), 1, 10, and 50 μM].

[0239] Experiment 2. Western blot analysis

[0240] HMGB1 levels were analyzed using Western blotting. In short, the protein was washed 5 × 10⁶ times with cold PBS.6 Cells were incubated twice on ice for 10 minutes in lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 100 μg / ml phenylmethylsulfonyl fluoride (PMSF), 50 μl / ml aprotinin, 1% Igepal 630, 100 mM NaF, 0.5% sodium deoxycholate, 0.5 mM EDTA, 0.1 mM EGTA]. The lysate was then centrifuged at 2000 × g for 10 minutes to precipitate unbroken cells and nuclei, and further centrifuged at 10,000 × g to clarify the lysate. The antibodies used were anti-HMGB1 (1:1000, Cell Signaling, Beverly, MA, USA) and anti-β-tubulin (1:1000, Cell Signaling, Beverly, MA, USA). The membrane was washed with Tris-buffered saline containing 0.05% Tween-20 (TBST), then treated with an anti-rabbit antibody conjugated with HRP (Amersham Pharmacia Biotech, Piscataway, NJ, USA), followed by ECL detection (Amersham Pharmacia Biotech). The blot was quantified using an image analyzer (GE Healthcare, ImageQuant LAS4000).

[0241] As a result of Western blot analysis, peptides that showed HMGB1 accumulation in cells were selected. Figures 60 to 159 These are the Western blot results of the selected peptides. The tubulin in these images is used to confirm protein expression. The sequences of the selected peptides are as follows:

[0242] SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ IDNO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ IDNO:16、SEQ ID NO:17、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:26、SEQ IDNO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:35、SEQ ID NO:36、SEQ IDNO:37、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:46、SEQ IDNO:47、SEQ ID NO:48、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:61、SEQ ID NO:64、SEQ IDNO:65、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:73、SEQ ID NO:74、SEQ IDNO:75、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ IDNO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:102、SEQID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118、SEQ ID NO:119、SEQ ID NO:120、SEQID NO:121、SEQ ID NO:123、SEQ ID NO:127、SEQ ID NO:128、SEQ ID NO:130、SEQ ID NO:132、SEQ ID NO:133、SEQ ID NO:134、SEQ ID NO:135、SEQ ID NO:136、SEQ ID NO:142、SEQID NO:143、SEQID NO: 144, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, and SEQ ID NO: 179.

Claims

1. A peptide with anti-inflammatory activity, wherein, The peptide consists of any one of the amino acid sequences in SEQ ID NO: 112 and SEQ ID NO:

113.

2. The peptide of claim 1, wherein the peptide is derived from human telomerase.

3. A polynucleotide encoding the peptide of claim 1 or 2.

4. Application of peptides in the preparation of anti-inflammatory compositions, wherein, The peptide is the peptide described in claim 1 or 2.

5. The application as described in claim 4, wherein, The composition is used to improve or treat inflammation.

6. The application as described in claim 5, wherein, The composition is formulated into a pharmaceutical composition.

7. The application as described in claim 6, wherein, The pharmaceutical composition is used to improve or treat inflammatory damage that leads to the abnormal release of high-mobility group 1 proteins.

Images