Novel anti-inflammatory peptides and their uses

Abstract

To provide an anti-inflammatory peptide, an anti-inflammatory composition comprising the same, and a pharmaceutical composition for preventing or treating inflammatory diseases.SOLUTION: The present invention provides an anti-inflammatory peptide including at least one selected from the group consisting of a specific amino acid sequence and modifications thereof. An anti-inflammatory peptide according to an embodiment of the present disclosure has no cytotoxicity, and has an effect of inhibiting the activation of mitochondrial antiviral signaling protein (MAVS), immune / inflammatory activation mechanism and the expression or activity of inflammatory cytokines and inflammasomes. Therefore, the peptide can be applied to an anti-inflammatory composition or a composition for treating inflammatory diseases.SELECTED DRAWING: Figure 1a

Description

[Technical Field]

[0001] The present application relates to anti-inflammatory peptides comprising one or more selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 63 and modified amino acid sequences thereof, anti-inflammatory compositions comprising the same, and pharmaceutical compositions for preventing or treating inflammatory diseases. [Background technology]

[0002] In recent years, the proportion of elderly people has continued to increase due to advances in medical technology and longer life expectancies resulting from economic development. Furthermore, immune system abnormalities caused by environmental pollution and increased stress have led to chronic inflammatory responses, resulting in an increase in chronic inflammatory diseases such as atopy and asthma (Chang et al., 1994, Korean J. Gastroentrol., 26:907-918; Heinzemann and Daser, 2002, Int. Arch. Allergy Immunol., 127:170-180; Song et al., 1998, Korean J. Intern. Med., 55:158-168; Sung et al., 2012, J. Ethnopharmacol., 144:94-100).

[0003] In general, the inflammatory response is a defense mechanism of living tissues against external stimuli such as bacterial or viral infections (pathogen-associated molecular patterns, PAMPs) and internal stimuli such as metabolic products resulting from tissue damage (danger-associated molecular patterns, DAMPs). It occurs through the production of various intracellular inflammation-regulating factors, such as numerous cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), as well as nitric oxide (NO). Lipopolysaccharide (LPS), also known as endotoxin, is a typical example of a pathogen-associated molecular pattern. It is present on the outer cell membrane of Gram-negative bacteria and induces the activation of the intracellular transcription factor nuclear factor-κB (NF-κB) in macrophages or monocytes, thereby inducing the gene expression of inflammatory cytokines, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), resulting in the production of inflammatory mediators. Therefore, substances that regulate inflammatory responses (e.g., expression of iNOS, COX-2, or NF-κB, and secretion of cytokines and nitric oxide) induced by pathogen-associated molecular patterns or damage-associated molecular patterns have recently attracted attention as preventive and therapeutic agents for inflammatory diseases (Dela Cruz and Kang, 2018, Mitochondrion, 41:37-44; Kim et al., 2013b, J. Korean Med. Ophthalmol. Otolaryngol. Dermatol., 26:54-64).

[0004] Currently, anti-inflammatory agents used include non-steroidal agents such as flufenamic acid, ibuprofen, benzydamine, and indomethacin; and steroidal agents such as prednisolone, dexamethasone, betamethasone, and hydrocortisone. However, these agents are highly toxic and can cause serious side effects, such as liver damage, cancer, and stroke, limiting their use. Furthermore, they lack the ability to selectively target inflammation-causing substances, potentially causing severe immunosuppression. Therefore, efforts are underway to develop anti-inflammatory agents using natural products, which offer the advantages of being safe for the body and easier to take over the long term than conventional pharmaceuticals. However, natural product-derived anti-inflammatory agents have limitations, such as low effective concentrations and the need for cultivation, which increases production costs.

[0005] To address the above-mentioned problems, new anti-inflammatory agents have been developed as alternatives to existing chemical anti-inflammatory agents or anti-inflammatory agents using natural products, and in particular, much research has been conducted into the synthesis of peptides with anti-inflammatory activity.

[0006] However, although generally synthesized peptides have very good anti-inflammatory activity and do not exhibit cytotoxicity in in vitro experiments, they often have only a negligible anti-inflammatory effect in actual in vivo experiments.

[0007] There are various reasons for this, but the main one is that the physiological and anatomical conditions in vivo are very different from the experimental conditions in a test tube. First, the presence of salt, which is a physiological condition, significantly inhibits the activity of peptides with low positive charges. Second, due to their small molecular weight and size, most peptides are absorbed by the kidneys and excreted from the body. Third, peptides are easily cleaved by protein- and peptide-degrading enzymes (proteases and peptidases) present in all tissues, cells, body fluids, and blood in the body, causing them to lose their activity.

[0008] Therefore, the present inventors have made extensive efforts to develop a substance that solves the above-mentioned problems and exhibits excellent anti-inflammatory activity. As a result, they have developed a peptide that can be mass-produced economically using 5 to 15 common amino acid residues, and have confirmed that the peptide does not exhibit cytotoxicity and exhibits excellent anti-inflammatory activity, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0009] The present application provides anti-inflammatory peptides comprising one or more selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 63 and modified amino acid sequences thereof, anti-inflammatory compositions comprising the same, and pharmaceutical compositions for preventing or treating inflammatory diseases.

[0010] However, the problems to be solved by the present application are not limited to those mentioned above, and another problem not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0011] A first aspect of the present application provides anti-inflammatory peptides comprising one or more amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 63 and modified amino acid sequences thereof.

[0012] A second aspect of the present application provides a polynucleotide encoding the anti-inflammatory peptide of the present application. The overlapping content with the first aspect also applies to the polynucleotide of the second aspect.

[0013] A third aspect of the present application provides an anti-inflammatory composition comprising the anti-inflammatory peptide of the present application as an active ingredient. The overlapping content of the first and second aspects also applies to the composition of the third aspect.

[0014] A fourth aspect of the present application provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising the anti-inflammatory peptide of the present application as an active ingredient. The contents overlapping with the first to third aspects also apply to the composition of the fourth aspect. [Effects of the Invention]

[0015] The anti-inflammatory peptide according to one embodiment of the present application is non-cytotoxic and has the effect of suppressing the activation of Mitochondrial Antiviral Signaling Protein (MAVS), immune / inflammatory activation reaction mechanisms, the expression or activity of inflammatory cytokines and inflammasomes, etc., and therefore, the peptide can be applied to compositions for anti-inflammatory effects or the treatment of inflammatory diseases. [Brief explanation of the drawings]

[0016] [Figure 1a] FIG. 1 shows the structure of the novel anti-inflammatory peptide developed in this application. [Figure 1b] FIG. 1 shows the structure of the novel anti-inflammatory peptide developed in this application. [Figure 1c] FIG. 1 shows the structure of the novel anti-inflammatory peptide developed in this application. [Figure 1d] FIG. 1 shows the structure of the novel anti-inflammatory peptide developed in this application. [Figure 1e] FIG. 1 shows the structure of the novel anti-inflammatory peptide developed in this application. [Figure 1f] FIG. 1 shows the structure of the novel anti-inflammatory peptide developed in this application. [Figure 2] FIG. 1 shows the results of an experiment confirming the MAVS aggregation-inhibiting effect of the anti-inflammatory peptide of the present invention. [Figure 3a] FIG. 1 shows the results of an experiment confirming the inhibitory effect of the anti-inflammatory peptide of the present invention on the expression of IFN-β induced by polyinosinic:polycytidylic acid (polyIC). [Figure 3b] FIG. 1 shows the results of an experiment confirming the inhibitory effect of the anti-inflammatory peptide of the present invention on the expression of IFN-β induced by polyinosinic:polycytidylic acid (polyIC). [Figure 4] FIG. 1 shows the results of an experiment confirming the cytotoxicity of the anti-inflammatory peptide of the present invention. [Figure 5a] FIG. 1 shows the results of an experiment confirming the inhibitory effect of the anti-inflammatory peptide of the present invention on IL-1β expression induced by LPS or bacterial outer membrane vesicles (OMV). [Figure 5b] FIG. 1 shows the results of an experiment confirming the inhibitory effect of the anti-inflammatory peptide of the present invention on IL-1β expression induced by LPS or bacterial outer membrane vesicles (OMV). [Figure 5c] FIG. 1 shows the results of an experiment confirming the inhibitory effect of the anti-inflammatory peptide of the present invention on IL-1β expression induced by LPS or bacterial outer membrane vesicles (OMV). [Figure 6a] FIG. 1 shows the results of an experiment confirming the inhibitory effect of the anti-inflammatory peptide of the present invention on the expression of IL-6 and TNF-α induced by LPS. [Figure 6b] FIG. 1 shows the results of an experiment confirming the inhibitory effect of the anti-inflammatory peptide of the present invention on the expression of IL-6 and TNF-α induced by LPS. [Figure 7] FIG. 1 shows the results of an experiment confirming the inhibitory effect of the anti-inflammatory peptide of the present invention on LPS-induced NF-κB phosphorylation. [Figure 8]FIG. 1 shows the results of an experiment confirming the effect of modifying the anti-inflammatory peptide of the present invention to increase resistance to proteases. [Figure 9a] FIG. 1 shows the results of an experiment confirming the inhibitory effect of peptides in which the sequences of the anti-inflammatory peptides MQP-37 and MQP-Y9 of the present application have been substituted with alanine on the production of IL-1b or IFN-b induced by LPS / ATP or polyIC. [Figure 9b] FIG. 1 shows the results of an experiment confirming the inhibitory effect of peptides in which the sequences of the anti-inflammatory peptides MQP-37 and MQP-Y9 of the present application have been substituted with alanine on the production of IL-1b or IFN-b induced by LPS / ATP or polyIC. [Figure 9c] FIG. 1 shows the results of an experiment confirming the inhibitory effect of peptides in which the sequences of the anti-inflammatory peptides MQP-37 and MQP-Y9 of the present application have been substituted with alanine on the production of IL-1b or IFN-b induced by LPS / ATP or polyIC. [Figure 10a] FIG. 1 shows the results of an experiment confirming the inhibitory effect of peptides in which the first or sixth amino acid sequence of the anti-inflammatory peptide MQP-37 of the present application has been substituted with 19 other amino acids on the production of IL-1b or IFN-b induced by LPS / ATP or polyIC. [Figure 10b] FIG. 1 shows the results of an experiment confirming the inhibitory effect of peptides in which the first or sixth amino acid sequence of the anti-inflammatory peptide MQP-37 of the present application has been substituted with 19 other amino acids on the production of IL-1b or IFN-b induced by LPS / ATP or polyIC. [Figure 11] FIG. 1 shows the results of an experiment comparing the binding strength of the anti-inflammatory peptides MQP-37 and MQP-37A6 of the present invention to MAVS protein. [Figure 12a] FIG. 1 shows the results of an experiment comparing the physiological activities (IL-6 and IL-1b production levels induced by LPS or LPS / nigericin, and NF-kB phosphorylation induced by LPS) of the anti-inflammatory peptides MQP-37 and MQP37-A6 of the present invention. [Figure 12b]FIG. 1 shows the results of an experiment comparing the physiological activities (IL-6 and IL-1b production levels induced by LPS or LPS / nigericin, and NF-kB phosphorylation induced by LPS) of the anti-inflammatory peptides MQP-37 and MQP37-A6 of the present invention. [Figure 13a] FIG. 1 shows the results of an experiment comparing the physiological activities (IFN-b production and cell death by polyIC) of the anti-inflammatory peptides MQP-37 of the present invention with those of MQP-37D6Y, MQP-36D6W, and MQP-37D6H. [Figure 13b] FIG. 1 shows the results of an experiment comparing the physiological activities (IFN-b production and cell death by polyIC) of the anti-inflammatory peptides MQP-37 of the present invention with those of MQP-37D6Y, MQP-36D6W, and MQP-37D6H. [Figure 14] FIG. 1 shows experimental results confirming the therapeutic effects of the anti-inflammatory peptides MQP-37 and MQP-37A6 of the present application on sepsis in an LPS-induced mouse sepsis animal model. BEST MODE FOR CARRYING OUT THE INVENTION

[0017] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily understand the present application. However, the present application may be embodied in various different forms and is not limited to the embodiments described herein. In order to clearly explain the present application in the drawings, parts that are not relevant to the description are omitted, and similar parts are designated by similar reference numerals throughout the specification.

[0018] Throughout this specification, when a moiety is referred to as being "linked" to another moiety, this includes not only a "direct link" but also an "indirect link" via an intermediate substance such as a linker.

[0019] Throughout this specification, when a part "comprises" a certain element, this does not mean that it excludes other elements, but that it may further include other elements, unless otherwise specified. As used throughout this specification, terms of degree such as "about," "substantially," etc., are used to mean from or close to a numerical value when inherent manufacturing and material tolerances are given, and are used to prevent unscrupulous infringers from unfairly taking advantage of the content in which precise or absolute numerical values ​​are recited to aid in the understanding of this application. As used throughout this specification, the terms of degree "(a) step" or "a step of" do not mean "a step for."

[0020] Throughout this specification, the term "combination thereof" in a maxi-phrase refers to a mixture or combination of one or more selected from the group of elements set forth in the maxi-phrase, including one or more selected from the group of elements.

[0021] Throughout this specification, the reference to "A and / or B" means "A or B, or A and B."

[0022] Hereinafter, embodiments and examples of the present application will be described in detail with reference to the accompanying drawings, but the present application may not be limited to such embodiments and examples and drawings.

[0023] A first aspect of the present application provides anti-inflammatory peptides comprising one or more amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 63 and modified amino acid sequences thereof.

[0024] The term "peptide" as used throughout the present specification refers to a linear molecule formed by the linkage of amino acid residues to each other via peptide bonds (-CO-NH-). The peptides of the present application may be produced by chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid synthesis techniques (U.S. Patent No. 5,516,891). The amino acid residues constituting the peptides of the present application may be natural or unnatural amino acid residues.

[0025] In one embodiment of the present application, the peptide may include one or more selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 63 and modified amino acid sequences thereof, and specifically, may include the amino acid sequence of SEQ ID NO: 1.

[0026] In one embodiment of the present application, the modified amino acid sequence may maintain the primary activity unchanged or may exhibit improved activity, and may include a partial mutation of the amino acid sequence by natural or artificial mutation, or one or more of the amino acids constituting the amino acid sequence may be substituted with other amino acids. Specifically, the modified amino acid sequence may include one or more of the amino acids constituting the amino acid sequences of SEQ ID NOs: 1 to 63, such as gamma-aminobutyric acid, glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), lysine (Lys, K), leucine (Leu, L), methionine (Met, M), valine (Val, V), serine (Ser, S), selenomethionine, selenocysteine ​​(Sec, U), cysteine ​​(Cys, C), citrulline, or arginine. (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), alanine (Ala, A), ornithine, isoleucine (Ile, I), taurine, threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), proline (Pro, P), pyrrolysine (Pyr, O), histidine (His, H), and unnatural amino acids may be substituted. Peptides according to an embodiment of the present application may include not only the recited SEQ ID NOs, but also peptides showing 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequences, so long as they have the same or a corresponding biological activity as the respective peptides. It is obvious that any amino acid sequence having a partial deletion, modification, substitution or addition of an amino acid sequence that is substantially identical to or has a biological activity corresponding to that of a peptide of a sequence number described as a sequence having homology to the above sequence is included in the scope of the present application.

[0027] The term "homology" as used throughout the present specification refers to the degree of identity with a given amino acid sequence or polynucleotide sequence, and may be expressed as a percentage. Herein, homologous sequences having the same or similar activity as a given amino acid sequence or polynucleotide sequence are expressed as "% homology." For example, homology may be confirmed using standard software, specifically BLAST 2.0, for calculating parameters such as score, identity, and similarity, or by comparing sequences in hybridization experiments under defined stringent conditions. Appropriate defined hybridization conditions are within the skill of the art and may be determined by methods well known to those of ordinary skill in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; F.M.A. Usubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). The term "stringent conditions" as used throughout the present specification refers to conditions that allow specific hybridization between polynucleotides. For example, such conditions are specifically described in the literature (e.g., J. Sambrook et al., supra).

[0028] The term "anti-inflammatory" used throughout the present specification means the action of suppressing or reducing inflammation, which is a defense response that occurs in the body when biological tissue is damaged and is the cause of inflammatory diseases. Therefore, the anti-inflammatory peptide of the present application can be used to prevent, treat, or improve (alleviate symptoms of) inflammatory diseases by exhibiting the activity of suppressing or reducing inflammation.

[0029] In one embodiment of the present application, the peptide may further include, but is not limited to, a cell-penetrating peptide.

[0030] The term "cell-penetrating peptide" as used throughout the specification of this application refers to a peptide that has the ability or property of being able to penetrate a cell membrane and enter the interior of a cell, and may exhibit cell permeability and / or skin permeability.

[0031] In one embodiment of the present application, the cell-penetrating peptide may further include a part or all of a sequence derived from a conventionally known cell-penetrating peptide or skin-penetrating peptide, for example, selected from the group consisting of dNP2, penetratin, Tat, transpotan, MAP, KALA, P1, MPG, Pep-1, Arg(7, 8, 9, 10, 11), hCT, pVEC, SPEH, YARA, WLR, VP22, MTS, FHV coat, and combinations thereof, and specifically, may be Arg(8) peptide (R8 peptide), but is not limited thereto.

[0032] In one embodiment of the present application, the cell-penetrating peptide may be contained in the anti-inflammatory peptide two or more times, specifically, the cell-penetrating peptide may be contained two, three, five, seven, or ten times, but is not limited thereto.

[0033] In one embodiment of the present application, the cell-penetrating peptide may be linked to the N-terminus or C-terminus of the anti-inflammatory peptide, specifically, to the N-terminus, but is not limited thereto as long as it can improve cell permeability or skin permeability without inhibiting the pharmacological activity of the peptide.

[0034] In one embodiment of the present application, the cell-penetrating peptide may be linked to the anti-inflammatory peptide via a linker or directly. The linker may be cleaved or degraded by various biological or chemical actions, such as enzymatic action, in cells or skin, and the cell-penetrating peptide and the anti-inflammatory peptide may be separated from each other in the target cells or skin by cleavage or degradation of the linker.

[0035] In one embodiment of the present application, the amino acids constituting the anti-inflammatory peptide may be L- or D-type, specifically D-type, but are not limited thereto as long as they do not affect the activity of the peptide.

[0036] In one embodiment of the present application, the N-terminus or C-terminus of the anti-inflammatory peptide may be bound with a protecting group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG), but the protecting group is not limited thereto as long as it does not affect the activity of the peptide.

[0037] In one embodiment of the present application, the peptide or the amino acids constituting the peptide may be acetylated or aminated in addition to the above-mentioned protecting group bond. The above-mentioned modifications significantly improve the stability of the peptide, and the stability means not only in vivo stability but also storage stability (e.g., room temperature storage stability). The above-mentioned protecting groups may act to protect the peptide of the present application from attack by protease in vivo.

[0038] In one embodiment of the present application, the peptide may inhibit the aggregation and / or function of mitochondrial antiviral-signaling (MAVS) protein.

[0039] The term "mitochondrial antiviral-signaling (MAVS) protein," as used throughout the present specification, refers to a signaling protein essential for antiviral innate immunity. It is known to be present in the outer membrane of mitochondria, peroxisomes, and the endoplasmic reticulum (ER). During viral infection, the MAVS protein aggregates and becomes activated by cytoplasmic proteins that sense the presence of the virus. The activated MAVS induces an immune response by secreting interferons and cytokines. However, if MAVS protein activation continues and excessive interferon and cytokine production increases, it may attack internal cells, leading to various pathological disorders and immune disorders. Therefore, an appropriate mechanism for regulating MAVS protein activation is needed.

[0040] In one embodiment of the present application, the anti-inflammatory peptide can suppress the inflammatory / immune activation mechanism and inflammatory / immune response induced by MAVS activation by inhibiting the aggregation or activation of MAVS, as well as treat, prevent, or ameliorate symptoms or diseases that may be caused by MAVS aggregation.

[0041] In one example of the present application, it was confirmed that the anti-inflammatory peptide of the present application effectively inhibits MAVS aggregation induced by pathogen-associated molecular patterns (PAMPs) derived from bacteria and viruses. Based on this, it can be seen that the anti-inflammatory peptide of the present application can effectively suppress inflammatory responses by inhibiting MAVS activity.

[0042] In one embodiment of the present application, the peptide may suppress the expression, production, activity, etc. of inflammatory cytokines or inflammasomes, or suppress the proliferation of inflammatory cells. Specifically, the inflammatory cytokines may be one or more selected from the group consisting of IFN-β, IL-1β, IL-6, and TNF-α, but are not limited to these, as long as they are known to cause an inflammatory response according to common technical knowledge in the art.

[0043] The term "inflammasome," as used throughout this specification, refers to a substance that induces the maturation of inflammatory cytokines, such as IL-1, which are involved in innate immune defenses against cellular infection and stress. It is a caspase-1-activating protein complex composed of 1) the sensor protein NLRP3 (NOD-like receptor family, pyrin domain-containing 3), 2) the adaptor protein ASC (adaptor protein apoptosis-associated spec-like protein containing a caspase-recruitment domain), and 3) the effector protein inactive caspase-1. The components of the inflammasome assemble upon infection by microorganisms such as bacteria or viruses or tissue injury. Once assembled and activated in the cytoplasm, the inflammasome converts inactive caspase-1 into active caspase-1. The converted, activated caspase-1 cleaves precursor IL-1β or IL-18 to produce activated IL-1β or IL-18, which are secreted extracellularly and are known to function as innate immune defenses of the host.

[0044] In one example of the present application, it was confirmed that the anti-inflammatory peptide of the present application suppresses the expression levels of various inflammatory cytokines and inflammasomes, which indicates that the anti-inflammatory peptide of the present application can effectively suppress inflammatory responses.

[0045] In one embodiment of the present application, the peptide may suppress a mechanism of inflammatory / immune activation response, and the mechanism may be caspase-1, interferon regulatory factor 3 (IRF3), or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), but is not limited thereto as long as it is a mechanism known to cause inflammatory / immune response according to common general technical knowledge in the art.

[0046] In one example of the present application, it was confirmed that the anti-inflammatory peptide of the present application inhibits the activity of the NF-κB signaling pathway by suppressing the phosphorylation of NF-κB, which indicates that the anti-inflammatory peptide of the present application can effectively suppress inflammatory responses.

[0047] In one embodiment of the present application, the peptide may suppress inflammation induced by bacteria or viruses, specifically, inflammation induced by pathogen-associated molecular patterns (PAMPs) derived from bacteria or viruses, or inflammation induced by damage-associated molecular patterns (DAMPs) secreted extracellularly due to cellular damage caused by bacterial or viral infection.

[0048] The term "pathogen-associated molecular pattern (PAMP)" as used throughout the present specification refers to a molecule derived from a pathogen that induces an immune response. Pattern recognition receptors in the immune system respond to specific molecular patterns to eliminate the source of infection through phagocytosis or induce antibody formation. Therefore, a PAMP is a molecular pattern common to all infectious sources to which PARPs react. Examples of PAMPs include endotoxin, exotoxin, lipopolysaccharide (LPS), lipoteichoic acid (LTA), muramyl dipeptide (MDP), nigericin, dsRNA (e.g., polyIC), dsDNA (e.g., polydAdT), outer membrane vesicles (OMV), and flagellin, but are not limited thereto as long as they are known to be derived from pathogens and capable of inducing an inflammatory / immune response according to the common general technical knowledge in the art.

[0049] The term "danger-associated molecular pattern (DAMP)" as used throughout the present specification refers to an intracellular metabolite or protein that is secreted extracellularly upon cellular damage and induces an immune response in surrounding cells. The DAMP may be ATP, histone protein, high mobility group box 1 (HMGB1), mitochondrial DNA (mtDNA), uric acid, etc., but is not limited thereto as long as it is a substance known to those skilled in the art to be derived from within cells and capable of inducing an inflammatory / immune response.

[0050] In one embodiment of the present application, the peptide can suppress an inflammatory response or treat or alleviate symptoms of a disease induced by an inflammatory response. Specifically, the peptide can be included in various compositions such as an anti-inflammatory pharmaceutical composition, a food composition, a cosmetic composition, a functional health food composition, and a feed composition.

[0051] A second aspect of the present application provides a polynucleotide encoding the anti-inflammatory peptide of the present application. The overlapping content with the first aspect also applies to the polynucleotide of the second aspect.

[0052] The term "polynucleotide" as used throughout this specification means DNA, a polymeric substance made up of linked nucleotides that encodes genetic information.

[0053] In one embodiment of the present application, the polynucleotide may include a base sequence encoding one or more of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 63.

[0054] In one embodiment of the present application, the nucleotide sequence encoding the anti-inflammatory peptide includes, without limitation, not only the nucleotide sequence encoding the amino acid sequence set forth in each SEQ ID NO., but also any nucleotide sequence showing 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more homology with the above sequence, and encoding a protein exhibiting substantially the same or corresponding efficacy as the above peptide. It is also clear that the scope of the present invention includes amino acid sequences with partial deletions, modifications, substitutions, or additions, as long as the amino acid sequence is substantially the same as or corresponding to the peptide of the SEQ ID NO. as a sequence having homology to the above sequence. Furthermore, polynucleotides encoding the above peptides may be modified in various ways in the coding region, taking into account the codons preferred in the organism in which the peptide is to be expressed, due to codon degeneracy, without changing the amino acid sequence of the peptide expressed from the coding region. Therefore, the polynucleotide may include, without limitation, any polynucleotide sequence encoding the respective peptide. Furthermore, probes that can be prepared from known sequences, for example, sequences that hybridize under stringent conditions with a complementary sequence to all or part of the above polynucleotide sequence and encode a protein having the activity of the above peptide, may also be included without limitation.

[0055] The term "stringent conditions" as used herein refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see, for example, Sambrook et al., supra, pp. 9.50-9.51, pp. 11.7-11.8). For example, these conditions may be used under conditions that allow hybridization between highly homologous genes, specifically genes with a homology of 40% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, and particularly 99% or more, but not between genes with lower homology. Alternatively, these conditions may be used under conditions that allow washing once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to the washing conditions for standard Southern hybridization: 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS. Hybridization requires that two polynucleotides have complementary sequences, even though mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present application may include not only substantially similar polynucleotide sequences, but also isolated polynucleotide fragments that are complementary to the entire sequence.

[0056] Specifically, homologous polynucleotides may be detected using the hybridization conditions described above, including a hybridization step at a Tm of 55°C. The Tm may be, but is not limited to, 60°C, 63°C, or 65°C, and may be adjusted appropriately by those skilled in the art depending on the purpose. The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0057] In another embodiment of the second aspect of the present application, there is provided an expression vector comprising the above polynucleotide.

[0058] The term "expression vector" as used throughout the specification of this application is a recombinant vector that can be introduced into a suitable host cell to express a target protein, and refers to a genetic construct that contains the necessary regulatory elements operably linked to allow the gene insert to be expressed.

[0059] The term "operably linked" as used throughout the specification of this application means that an expression control sequence of a nucleic acid and a nucleic acid sequence encoding a protein of interest are functionally linked to each other so as to perform their common functions. Operable linkage with a recombinant vector may be produced using recombinant gene technology well known in the art, and site-specific DNA cleavage and ligation can be easily performed using enzymes generally known in the art.

[0060] In addition to expression regulatory elements such as a promoter, initiation codon, termination codon, polyadenylation signal, and enhancer, an expression vector suitable for the present application may also contain a signal sequence for membrane targeting or secretion. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein, and must be in-frame with the coding sequence so that the gene construct is effective in an individual when administered. Common promoters may be constitutive or inducible, and include, but are not limited to, lac, tac, T3, and T7 promoters for prokaryotic cells, and simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV) promoters, such as the HIV long terminal repeat (LTR) promoter, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Rous sarcoma virus (RSV) promoters, as well as promoters derived from β-actin, human hemoglobin, human muscle creatine, and human metallothionein, for example.

[0061] The expression vector may also contain a selectable marker for selecting host cells containing the vector. The selectable marker is used to select cells transformed with the vector, and a marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein, may be used. In an environment treated with a selective agent, only cells expressing the selectable marker survive, allowing the selection of transformed cells. Furthermore, if the vector is a replicable expression vector, it may contain a replication origin, which is a specific nucleic acid sequence from which replication is initiated.

[0062] Various types of vectors may be used as recombinant expression vectors for inserting foreign genes, such as plasmids, viruses, cosmids, etc. The type of recombinant vector is not particularly limited as long as it functions to express a desired gene in various prokaryotic and eukaryotic host cells and produce a desired protein, but specifically, a vector that has a highly active promoter and strong expression ability and is capable of mass-producing a foreign protein in a form similar to that in its natural state may be used.

[0063] Various host and vector combinations may be used to express the anti-inflammatory peptides of the present application. Expression vectors suitable for eukaryotic hosts may include, but are not limited to, expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors that can be used in bacterial hosts may include, but are not limited to, bacterial plasmids derived from Escherichia coli, including pET, pRSET, pBluescript, pGEX2T, pUC, colE1, pCR1, pBR322, pMB9, and their derivatives; plasmids with broader host ranges, such as RP4; phage DNA, such as phage lambda derivatives, such as λgt10, λgt11, or NM989; and other DNA phages, such as M13 and filamentous single-stranded DNA phages. For yeast cells, the 2°C plasmid or its derivatives may be used, and for insect cells, pVL941 may be used.

[0064] In another embodiment of the second aspect of the present application, there is provided a non-human transformant comprising the above expression vector.

[0065] The term "transformant" used throughout the present specification may refer to a host cell into which the expression vector can be introduced. Specifically, the transformant of the present application may be, but is not limited to, a non-human transformant.

[0066] Suitable host cells for introducing the vectors may be prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis, or Staphylococcus sp., or fungi such as Aspergillus sp., yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp., or Neurospora crassa, or other lower eukaryotic cells, or cells of higher eukaryotes such as plant or insect cells. Mammalian cells may also be used, specifically, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT78 cells, or HEK293 cells, but are not limited thereto.

[0067] The transformation method of the present application includes any method for introducing nucleic acid into an organism, cell, tissue, or organ, and may be performed using standard techniques known in the art that are suitable for the host cell, including, but not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO) precipitation, calcium chloride (CaCl) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-, dextran sulfate-, lipofectamine-, and desiccation / repression-mediated transformation.

[0068] In another embodiment of the second aspect of the present application, there is provided a method for producing an anti-inflammatory peptide, which comprises the step of culturing the above-mentioned transformant.

[0069] The method for producing the anti-inflammatory peptide includes a step of culturing the transformant of the present application, and may specifically include a step of producing an expression vector by inserting a polynucleotide sequence encoding the anti-inflammatory peptide into a vector, a step of producing a transformant by introducing the expression vector into a host cell, a step of culturing the transformant, and a step of separating and purifying the anti-inflammatory peptide from the cultured transformant.

[0070] More specifically, the transformant can be cultured in a nutrient medium to produce a large amount of the peptide. The medium and culture conditions may be appropriately selected from those commonly used depending on the host cell. During the culture, conditions such as temperature, medium pH, and culture time may be appropriately adjusted to suit cell growth and large-scale protein production.

[0071] Recombinantly produced peptides or proteins as described above may be recovered from the culture medium or cell lysates. If membrane-bound, they may be released from the membrane using a suitable detergent solution (e.g., Triton-X100) or by enzymatic cleavage. Cells used to express anti-oskar antibodies or fragments thereof can be disrupted by various physical or chemical means, such as freeze-thawing, sonication, mechanical disruption, or cell lysing agents, and then isolated and purified by conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182, Academic Press, Inc., San Diego, CA (1990)). Methods that can be used include, but are not limited to, electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunoadsorption chromatography, size exclusion chromatography, etc.), isoelectric focusing, and various variations and hybrid methods thereof.

[0072] A third aspect of the present application provides an anti-inflammatory composition comprising the anti-inflammatory peptide of the present application as an active ingredient. The overlapping content of the first and second aspects also applies to the composition of the third aspect.

[0073] In one embodiment of the present application, the anti-inflammatory composition may be used in pharmaceutical, quasi-drug, cosmetic, food and feed compositions.

[0074] In one embodiment of the present application, the composition may further include a pharmaceutically acceptable salt, and specific examples of the pharmaceutically acceptable salt include hydrochloride, sulfate, phosphate, acetate, citrate, stannate, succinate, lactate, maleate, fumarate, oxalate, methanesulfonate, and paratoluenesulfonate.

[0075] In one embodiment of the present application, the pharmaceutical composition contains a pharmaceutically acceptable carrier in addition to the active ingredient, and may be prepared into an oral dosage form or a parenteral dosage form depending on the route of administration by a conventional method known in the art. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity greater than that applicable to the subject to which it is applied (prescribed).

[0076] In one embodiment of the present application, the pharmaceutical composition may be formulated and used in the form of an oral dosage form such as powder, granules, tablets, capsules, suspension, emulsion, syrup, aerosol, external preparation, suppository, or sterile injection solution by a conventional method, but is not limited thereto.

[0077] In one embodiment of the present application, when the pharmaceutical composition is formulated, it may be prepared using a commonly used diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant, but is not limited thereto.

[0078] In one embodiment of the present application, when the pharmaceutical composition is prepared into an oral dosage form, it may be prepared with a suitable carrier into dosage forms such as powder, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, suspensions, wafers, etc., by methods known in the art. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; celluloses such as methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, and vegetable oils. When formulated, diluents and / or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added, if necessary.

[0079] In one embodiment of the present application, when the pharmaceutical composition is prepared into a parenteral dosage form, it may be formulated with a suitable carrier into the form of an injection, transdermal administration, nasal inhalation, or suppository by methods known in the art. When formulated into an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof. Preferably, infusion solutions, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, isotonic solutions such as 5% dextrose, etc. When formulated into a transdermal administration, it may be formulated into the form of an ointment, cream, lotion, gel, topical solution, paste, liniment, aerosol, etc. In the case of nasal inhalants, suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. may be used to formulate the agent into an aerosol spray form. When the agent is formulated into a suppository, the base may be witepsol, Tween 61, polyethylene glycols, cocoa butter, laurin butter, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc.

[0080] In one embodiment of the present application, the pharmaceutical composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level may be determined based on factors including the severity of the disease, drug activity, the patient's age, weight, health, sex, and drug sensitivity, the administration time, route of administration, and excretion rate of the composition of the present invention used, the duration of treatment, drugs used in combination with or concomitantly with the composition of the present application, and other factors well known in the medical field. The pharmaceutical composition of the present application may be administered alone or in combination with an ingredient known to have a therapeutic effect on known intestinal diseases. Taking all of the above factors into consideration, it is important to administer an amount that will achieve maximum effect at the minimum dose without side effects.

[0081] In one embodiment of the present application, the dosage of the pharmaceutical composition may be determined by one skilled in the art, taking into consideration the purpose of use, the toxicity of the disease, the age, weight, sex, and medical history of the patient, and the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present application may be administered at about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg, per adult. The administration frequency of the composition of the present application is not particularly limited, but may be once a day or may be administered in divided doses several times. The above dosage amount or frequency of administration does not limit the scope of the present application in any aspect.

[0082] A fourth aspect of the present application provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising the anti-inflammatory peptide of the present application as an active ingredient. The contents overlapping with the first to third aspects also apply to the composition of the fourth aspect.

[0083] The term "treatment" as used throughout this specification means any action that reverses or alters the symptoms of an inflammatory disease by administering the compositions of this application.

[0084] The term "prevention" as used throughout this specification refers to any action that inhibits or delays an inflammatory disease or its possible onset by administering the compositions of this application.

[0085] The term "inflammatory disease" as used throughout this specification may be defined as a pathological condition caused by an inflammatory response, which is a local or systemic defense response of the body against external physical or chemical stimuli or infections or autoimmunity caused by external infectious agents such as germs, bacteria, fungi, viruses, and various allergens. Such an inflammatory response involves a series of complex physiological responses, such as activation of various inflammatory mediators and immune cell-associated enzymes (e.g., iNOS, COX-2, etc.), secretion of inflammatory mediators (e.g., secretion of NO, TNF-α, IL-6, etc.), fluid infiltration, cell migration, and tissue destruction, and is externally manifested by symptoms such as erythema, pain, edema, fever, and the decline or loss of specific bodily functions. Such inflammatory diseases may be acute, chronic, ulcerative, allergic, or necrotizing, and as long as a disease falls within the above definition of an inflammatory disease, it does not matter whether it is acute, chronic, ulcerative, allergic, or necrotizing.

[0086] In one embodiment of the present application, the inflammatory disease is sepsis, septic shock, systemic inflammatory response syndrome, acute respiratory distress syndrome, asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, or the like. The inflammatory disease may be one or more selected from the group consisting of inflammatory bowel syndrome (IFS), inflammatory bowel disease (IGF), inflammatory bowel syndrome (IGS), migraine, headache, lower back pain, fibromyalgia, fascial disease, viral infection (e.g., Hepatitis C infection), bacterial infection, fungal infection, burns, wounds from surgical or dental operations, prostaglandin E excess syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis, atopic dermatitis, eczema, systemic lupus erythematosus (SLE), and multiple sclerosis, and is not limited to the above, as long as it is a disease induced by an inflammatory response, specifically, a disease induced by an inflammatory response due to a bacterial or viral infection.

[0087] A fifth aspect of the present application provides a method for preventing or treating inflammation or an inflammatory disease, comprising administering to an individual an anti-inflammatory composition or a pharmaceutical composition for preventing or treating an inflammatory disease of the present application. The content that overlaps with the first to fourth aspects also applies to the method of the fifth aspect.

[0088] The term "individual" as used throughout the specification of this application may include, without limitation, mammals, including mice, livestock, humans, farmed fish, etc., that develop or are at risk of developing an inflammatory response or inflammatory disease.

[0089] In one embodiment of the present application, the individual may be a non-human.

[0090] In one embodiment of the present application, the method may involve administering a pharmaceutically effective amount of the composition to prevent or treat inflammation or an inflammatory disease. This amount may vary depending on various factors, such as the progression of the inflammatory response or inflammatory disease, the patient's age and weight, the characteristics and severity of symptoms, the type of current treatment, the course of treatment, and the form and route of administration, and can be easily determined by a person skilled in the art. The composition of the present application may be administered together with the above-mentioned pharmacological or physiological components or sequentially, or may be administered in combination with an additional conventional therapeutic agent, or may be administered sequentially or simultaneously with the conventional therapeutic agent. Such administration may be single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that will achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0091] The term "administration" as used throughout the present specification means introducing a substance into an individual by an appropriate method. The administration route of the compositions of the present application may be any common route that can reach the target tissue. Administration may include, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, and rectal administration. However, because proteins are digested during oral administration, oral compositions are preferably formulated to coat the active agent or protect it from digestion in the stomach. Furthermore, the compositions may be administered by any device that can deliver the active agent to target cells.

[0092] The present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative and are not intended to limit the scope of the present invention.

[0093] [Example] Example 1: Synthesis of a novel peptide with anti-inflammatory effects To develop an anti-inflammatory peptide that can alleviate inflammatory responses by inhibiting the aggregation of mitochondrial anti-viral signaling (MAVS) protein, which is known as a major hub protein in the innate immune response, we derived an anti-inflammatory peptide sequence that inhibits MAVS signaling in the following manner.

[0094] Specifically, in vivo protein synthesis occurs when one of the 5'-3' strands of DNA in a double helix structure is transcribed into mRNA (mRNA), which then synthesizes the protein. However, when a complementary DNA sequence in the opposite direction (3'-5') is transcribed in the same direction as the original protein, it has been shown to have electrostatic properties opposite to those of the original protein, resulting in specific binding between the two (hydropathic complementarity). Furthermore, aggregation occurs when the MAVS protein is activated in response to external stimuli. Based on the hydropathic complementarity theory described above, we synthesized new peptides complementary to the amino acid sequences of a partial sequence of the MAVS protein domain that appears to play a key role in this step (SEQ ID NOS: 1-4, 48-52).

[0095] Example 2: Confirmation of MAVS aggregation inhibitory efficacy of novel peptides In order to confirm whether the novel peptide prepared in Example 1 above has the effect of inhibiting the aggregation phenomenon of MAVS protein, the following experiment was carried out.

[0096] Specifically, to induce MAVS protein aggregation, pathogen-associated molecular patterns (PAMPs) were used as stimuli. Examples of PAMPs used were endotoxin (lipopolysaccharide, LPS) and nigericin. First, 5 mM of MQP-15, MQP-23, MQP-31, or MQP-37 was added to serum-free cell culture medium containing 1 mg / ml of endotoxin. Then, 1 × 10 mouse peritoneal macrophage cell line (IC21) was cultured. 5 The cells were treated for 4 hours. Then, 5 mM MQP-15, MQP-23, MQP-31, or MQP-37 was added to serum-free cell culture medium containing 5 mM nigericin, and the mixture was further treated with mouse peritoneal macrophage cell lines for 1 hour. Mitochondria were then isolated from the macrophage cell lines using hypotonic buffer and centrifugation. To confirm MAVS aggregation by Western blot, the isolated mitochondria were prepared using semi-denaturing detergent and subjected to electrophoresis. The electrophoretically separated mitochondrial proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and further treated with a primary antibody recognizing MAVS protein and a secondary antibody recognizing the primary antibody. MAVS protein aggregation was then confirmed by chemiluminescence induction of the secondary antibody.

[0097] As a result, it was confirmed that the MAVS protein aggregation phenomenon observed in the control group treated with endotoxin and nigericin was reduced in the experimental groups treated with MQP-15, MQP-23, MQP-31, or MQP-37 (Figure 2), indicating that MQP-15, MQP-23, MQP-31, or MQP-37 produced in this application effectively inhibit the MAVS protein aggregation phenomenon.

[0098] Example 3: Confirmation of the efficacy of novel peptides in suppressing antiviral responses In order to confirm whether the novel peptide prepared in Example 1 above has the effect of suppressing antiviral responses caused by viral infection, the following experiment was carried out.

[0099] Specifically, to induce a viral infection response, we used polyIC (polyinosinic:polycytidylic acid, polyIC), a synthetic ribonucleic acid known to be involved in viral infection and known to promote the production of interferon (IFN), an RNA virus gene analog. First, we cultured 1 x 10 mouse lung epithelial cell line (MLE-12) in serum-free cell culture medium supplemented with 5 mg / ml of polyIC and MQP-15, MQP-23, MQP-31, MQP-37, MQP-Y9, or MQP-T234. 5 The cells were treated for 16 hours, after which the supernatant was collected and the expression level of interferon-beta (IFN-b) was determined using enzyme-linked immunosorbent assay (ELISA).

[0100] The results showed that IFN-b expression levels were reduced in cells treated with MQP-15, MQP-23, MQP-31, MQP-37, MQP-Y9, or MQP-T234 compared to the control group treated with Poly IC alone. In particular, MQP-37 significantly reduced IFN-b expression levels compared to the other peptides (Figure 3a, A). Furthermore, the same experiment was performed using various concentrations of MQP-37, which showed a superior effect, and a concentration-dependent reduction in IFN-b expression was confirmed (Figure 3a, B). The same experiment was performed using various concentrations of the MQP-Y9 peptide, and a concentration-dependent reduction in IFN-b expression was confirmed (Figure 3b, C). The same experiment using the MQP-T234 peptide also reduced IFN-b expression (Figure 3b, D). Based on the above results, it can be seen that MQP-15, MQP-23, MQP-31, MQP-37, MQP-Y9, and MQP-T234 produced in the present application effectively suppress antiviral responses caused by viral infection.

[0101] Example 4: Confirmation of cytotoxicity of novel peptides In order to confirm whether the anti-inflammatory activity of the novel peptide prepared in Example 1 above is due to the cytotoxicity of the peptide itself, the following experiment was carried out.

[0102] Specifically, to confirm the cytotoxicity of the above peptides, we used an XTT experiment to measure intracellular mitochondrial activity. First, a serum-free cell culture medium containing 5 mM MQP-15, MQP-23, MQP-31, or MQP-37 was treated in a mouse peritoneal macrophage cell line for 16 hours. The cells were then treated with XTT for 1 hour, and the amount of water-soluble formazan produced by reduction with mitochondrial dehydrogenase was measured.

[0103] As a result, it was confirmed that the novel peptides of the present invention did not exhibit cytotoxicity within the concentration range used in the cell experiments (Figure 4A). In particular, MQP-37 and MQP-Y9, which were found to be peptides that very effectively suppress the inflammatory response caused by polyIC, did not exhibit cytotoxicity even at a concentration of 10 mM (Figure 4B and C). Therefore, it is clear that the inflammatory response inhibitory effect described above is not due to the cytotoxicity of the peptides themselves, and that the use of these peptides as pharmaceuticals is not harmful to individuals.

[0104] Example 5: Confirmation of the efficacy of novel peptides in inhibiting inflammasome responses In order to confirm the inhibitory effect of the novel peptide prepared in Example 1 on the inflammasome response caused by bacterial infection, the following experiment was carried out.

[0105] Specifically, to induce the inflammasome response due to bacterial infection, lipopolysaccharide (LPS) and outer membrane vesicles (OMV) derived from Gram-negative bacteria were used as endotoxins. First, 5 mM of MQP-15, MQP-23, MQP-31, or MQP-37 was added to serum-free cell culture medium containing 0.1 mg / ml of LPS or OMV, respectively, and 1 × 10 mouse peritoneal macrophage cell line was cultured. 5 The cells were treated for 6 hours. Alternatively, MQP-37 or MQP-Y9 was added to serum-free cell culture medium containing 1 mg / ml LPS and 5 mM nigericin, and a mouse peritoneal macrophage cell line was treated for 5 hours (LPS treatment: 4 hours; nigericin treatment: 1 hour). The supernatant was then collected and interleukin-1 beta (IL-1b) expression levels were determined using ELISA. The cells were then lysed, subjected to SDS-PAGE, and Western blot analysis for inflammasome factors was performed.

[0106] The results confirmed that IL-1b expression levels were reduced by treatment with the novel peptides of the present invention compared to the control group treated with LPS or OMV alone. In particular, MQP-37 significantly reduced IL-1b expression levels compared to the other peptides (Figures 5a-A and 5b-C). Furthermore, the same experiment was performed using various concentrations of MQP-37, which showed excellent effects. The results confirmed that LPS treatment reduced IL-1b expression levels in a concentration-dependent manner (Figure 5a-B). IL-1b production by LPS and nigericin treatment was suppressed in an MQP-Y9 concentration-dependent manner (Figure 5b-D). Furthermore, MQP-37 treatment inhibited caspase-1 activation (Figure 5c-E). These results demonstrate that the novel peptides developed in the present invention effectively suppress the inflammasome response induced by bacterial infection.

[0107] Example 6: Confirmation of the efficacy of novel peptides in suppressing inflammatory responses In order to confirm the inflammatory response suppressing effect of the novel peptide prepared in Example 1 above, the following experiment was carried out.

[0108] Specifically, LPS was used to induce an inflammatory response due to bacterial infection. First, MQP-15, MQP-23, MQP-31, MQP-37, or MQP-Y9 was added to a serum-free cell culture medium containing 0.1 mg / ml of LPS, and 1 × 10 macrophage cell lines derived from mouse peritoneal cavity were cultured. 5 The cells were treated for 6 hours, after which the supernatant was collected and the expression levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were determined using ELISA.

[0109] As a result, it was confirmed that MQP-37, one of the novel peptides developed herein, significantly reduced the expression levels of IL-6 and TNF-α compared to the control group treated with LPS alone (Figure 6a-A and Figure 6b-C). Furthermore, when the same experiment as above was performed using various concentrations of MQP-37, which showed excellent effects, it was confirmed that the IL-6 expression level due to LPS treatment was reduced in a concentration-dependent manner (Figure 6a-B). When the same experiment as above was performed using various concentrations of MQP-Y9, it was confirmed that the IL-6 expression level due to LPS treatment was reduced in a concentration-dependent manner (Figure 6b-D). Based on these results, it can be seen that the novel peptide developed herein effectively suppresses inflammatory responses.

[0110] Example 7: Confirmation of the inhibitory effect of novel peptides on immune activation reaction mechanisms In order to confirm the inhibitory effect of the novel peptide prepared in Example 1 on immune activation reaction mechanisms, the following experiment was carried out.

[0111] Specifically, LPS was used to induce immune activation by bacterial infection, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) phosphorylation was examined to confirm the mechanism of immune activation. First, 5 mM of MQP-15, MQP-23, MQP-31, MQP-37, MQP-Y9, MQP-91, MQP-T234, or MQP-341 was added to serum-free cell culture medium containing 10 ng / ml endotoxin, and 1 × 10 cells of a mouse peritoneal macrophage cell line were cultured. 6 The cells were treated for 6 hours. After washing with PBS, the cells were lysed in RIPA buffer and the supernatant was separated. Western blotting was performed as described in Example 2 above to measure changes in the phosphorylation level of nuclear factor kappa B protein in the separated supernatant.

[0112] As a result, it was confirmed that the novel peptides MQP-37 and MQP-Y9 significantly reduced the phosphorylation level of NF-kB compared to the control group treated with LPS only (Figure 7). Based on these results, it can be seen that the novel peptides developed in this study effectively suppress the mechanism of immune activation responses.

[0113] Example 8: Confirmation of improved resistance of D-type novel peptides to protease degradation The novel peptide MQP-37, whose anti-inflammatory activity was confirmed through the experiments in Examples 2 to 7, was synthesized with D-amino acids to enhance its resistance to proteases, and the following experiment was carried out to confirm its resistance to proteases.

[0114] Specifically, LPS was used to induce an inflammatory response due to bacterial infection. First, 0.1 mg / ml of LPS was mixed with serum-free cell culture medium or 10% serum (fetal bovine serum, FBS) cell culture medium. Next, 5 mM of L-type MQP-37 peptide and 5 mM of D-type MQP-37 peptide were added to each LPS-containing cell culture medium, and 1 × 10 cells of a mouse peritoneal macrophage cell line were cultured. 5 The cells were treated for 6 hours, after which the supernatant was collected and the expression levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were determined using ELISA.

[0115] As a result, it was confirmed that both the novel L-type and D-type peptides of the present invention exhibited the same anti-inflammatory response in serum-free cell culture medium conditions, in the absence of proteases, compared to a control group treated with LPS only. However, in cell culture medium conditions containing 10% serum, in the presence of proteases, the novel L-type peptide of the present invention did not exhibit anti-inflammatory activity, while the novel D-type peptide still retained its anti-inflammatory activity (Figure 8). Based on these results, it can be seen that the novel D-type peptide prepared in this invention effectively suppresses inflammatory responses regardless of the presence or absence of proteases.

[0116] Example 9: Confirmation of improved anti-inflammatory effect of alanine-substituted novel peptides In order to identify the key amino acids of the novel peptides MQP-37 and MQP-Y9, whose anti-inflammatory activity was confirmed through the experiments in Examples 2 to 8 above, peptides were synthesized in which each sequence was sequentially substituted with alanine (MQP-37 alanine-substituted peptides [MQP-37 derivatives] - SEQ ID NOs: 5 to 11; MQP-Y9 alanine-substituted peptides [MQP-Y9 derivatives] - SEQ ID NOs: 53 to 62), and the following experiments were conducted to confirm their anti-inflammatory effects.

[0117] Specifically, LPS and ATP were used to induce the inflammasome response due to bacterial infection, and polyIC was used to induce the inflammatory response due to viral infection. First, 1 mg / ml of LPS and 5 mM or 1 mM of the alanine-substituted novel peptide were mixed in serum-free cell culture medium, and then 1 × 10 5 The cells were treated with a mouse peritoneal macrophage cell line. Then, 5 mM ATP and 5 mM or 1 mM of the alanine-substituted novel peptide were mixed in serum-free cell culture medium and treated for 30 minutes. The supernatant was then collected, and the expression level of interleukin-1 beta (IL-1b) was confirmed using ELISA. Furthermore, 5 mg / ml of polyIC and 25 mM or 2 mM of the alanine-substituted novel peptide were mixed in serum-free cell culture medium and treated with 1 x 10 mouse lung epithelial cell line (MLE-12). 5 The cells were treated for 16 hours, after which the supernatant was collected and the expression level of interferon-beta (IFN-b) was confirmed using ELISA.

[0118] As a result, MQP-37 (A1 and A6), in which the amino acids at positions 1 and 6 were substituted with alanine, was confirmed to have a significant effect in suppressing the inflammasome response induced by bacterial infection and the inflammatory response induced by viral infection compared to the original MQP-37 peptide (Figure 9a, A and B). Furthermore, MQP-Y9 (A2, A7, and A9), in which the amino acids at positions 2, 7, and 9 were substituted with alanine, was confirmed to exhibit a superior inhibitory effect against the inflammasome response induced by bacterial infection compared to the original peptide (Figure 9b, C). MQP-Y9 (A10), in which the amino acid at position 10 was substituted with alanine, was confirmed to more effectively suppress the inflammatory response induced by viral infection compared to the original peptide (Figure 9c, D). Based on these results, it can be seen that the novel alanine-substituted peptides developed in this study effectively suppress the inflammasome response induced by bacterial infection and the inflammatory response induced by viral infection (SEQ ID NOS: 5-11; 53-62).

[0119] Example 10: Confirmation of anti-inflammatory effect of a novel peptide in which the first and sixth amino acids of the sequence are replaced with other amino acids The novel peptide MQP-37A6 (SEQ ID NO: 9), which was confirmed to have improved anti-inflammatory activity through the experiment in Example 9, was synthesized by substituting 19 amino acids other than serine at position 1 (MQP-37A1 derivatives - SEQ ID NOs: 12-29, 63). MQP-37A6 was also synthesized by substituting 18 amino acids (excluding aspartic acid) at position 6 (MQP-37A6 derivatives - SEQ ID NOs: 30-47). The following experiment was performed to confirm the anti-inflammatory activity of the above peptides.

[0120] Specifically, LPS and ATP were used to induce the inflammasome response due to bacterial infection, and polyIC was used to induce the inflammatory response due to viral infection. First, 1 mg / ml of LPS and 2 mM of the novel amino acid-substituted peptide were mixed in serum-free cell culture medium, and then 1 × 10 5The cells were treated with a mouse peritoneal macrophage cell line. Then, 5 mM ATP and 2 mM of the amino acid-substituted novel peptide were mixed in serum-free cell culture medium and incubated for 30 minutes. The supernatant was then collected and the expression level of interleukin-1 beta (IL-1b) was confirmed using ELISA. Furthermore, 5 mg / ml of polyIC and 2 mM of the amino acid-substituted novel peptide were mixed in serum-free cell culture medium and incubated with 1 x 10 mouse lung epithelial cell line (MLE-12). 5 The cells were treated for 16 hours, after which the supernatant was collected and the expression level of interferon-beta (IFN-b) was confirmed using ELISA.

[0121] As a result, MQP-37A1, in which the first amino acid of MQP-37A6 was substituted with alanine, was the most effective inhibitor of the inflammasome response induced by bacterial infection. Substitutions with phenylalanine (F), leucine (L), arginine (R), and tyrosine (Y) also effectively inhibited the inflammasome response induced by bacterial infection (Figure 10a, A). Furthermore, substitutions of the sixth amino acid of MQP-37 with other amino acids, such as alanine (A), phenylalanine (F), histidine (H), leucine (L), and methionine (M), as shown in Figure 10a, B, effectively inhibited the inflammasome response induced by bacterial infection. Among these, the novel peptide substituted with alanine was the most effective inhibitor. It was confirmed that when the sixth amino acid of MQP-37 was substituted with tryptophan (W) or tyrosine (Y), the inflammatory response caused by viral infection was more effectively suppressed than the original peptide, even at low peptide concentrations (Figure 10b, C). Based on these results, it can be seen that the novel amino acid-substituted peptides developed in this study effectively suppress the inflammasome response caused by bacterial infection and the inflammatory response caused by viral infection.

[0122] Example 11: Comparative confirmation of binding strength between novel peptide MQP-37 and alanine-substituted novel peptide MQP-37A6 The following experiment was carried out to compare the binding ability of the novel alanine-substituted peptide MQP-37A6, whose anti-inflammatory activity was confirmed through the experiments in Examples 9 and 10, with that of the original peptide MQP-37A6, to the MAVS protein.

[0123] Specifically, to confirm the binding strength with MAVS protein, recombinant MAVS protein and fluorescently labeled MQP-37 and MQP-37A6 peptides were used. First, 250 ng of recombinant MAVS protein was coated onto a 96-well ELISA plate and blocked with 1% BSA / PBS solution. Various concentrations of fluorescently labeled peptides were then added and incubated for 2 hours, after which the amount of peptide bound to MAVS protein was determined by fluorescence measurement.

[0124] As a result, when MQP-37 was treated at different concentrations and the binding affinity with MAVS protein was measured, the binding affinity (K d The binding strength of MQP-37A6 was measured to be approximately 16 mM, whereas that of MQP-37A6 was measured to be approximately 0.75 mM, confirming that it has approximately 20 times stronger binding strength (Figure 11). Based on these results, it is clear that the novel peptide MQP-37A6 produced in this study has improved binding strength with the MAVS protein.

[0125] Example 12: Comparative confirmation of physiological activities of novel peptide MQP-37 and alanine-substituted novel peptide MQP-37A6 The following experiment was carried out to compare the physiological activity of the novel alanine-substituted peptide MQP-37A6, whose anti-inflammatory activity was confirmed through the experiments in Examples 9 and 10, with that of the original peptide MQP-37.

[0126] Specifically, LPS was used to induce an inflammatory response due to bacterial infection. First, MQP-37 or MQP-37A6 was added to a serum-free cell culture medium containing 0.1 mg / ml of LPS, and 1 × 10 macrophage cell lines derived from mouse peritoneal cavity were cultured. 5The cells were treated for 6 hours. The supernatant was then collected and the expression level of interleukin-6 (IL-6) was confirmed using ELISA. Furthermore, to confirm the mechanism of immune activation, Western blot was performed to confirm the phosphorylation of nuclear factor kappa B (NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells). Next, LPS and ATP were used to induce the inflammasome response due to bacterial infection. First, 1 mg / ml LPS and L- or D-type MQP-37 and MQP-37A6 were mixed in serum-free cell culture medium, and 1 x 10 cells were cultured. 5 The mouse peritoneal macrophage cell line (MCL1 cells) was treated with ATP (5 mM) and MQP-37 or MQP-37A6 in serum-free culture medium for 30 minutes. The supernatant was collected and the expression level of interleukin-1 beta (IL-1b) was determined using ELISA.

[0127] As a result, it was confirmed that MQP-37A6, in which the sixth amino acid was substituted with alanine, in both the L-type and D-type, more effectively suppressed the inflammatory response (Fig. 12a, A) and inflammasome response (Fig. 12a, B and C), as well as the immune activation response (NF-kB phosphorylation, Fig. 12b, D to F), caused by bacterial infection, compared to the original MQP-37. Based on the above results, it can be seen that the novel alanine-substituted peptide (MQP-37A6) developed in this application effectively suppresses inflammatory responses.

[0128] Example 13: Comparative confirmation of viral inflammatory responses in novel peptide MQP-37 and novel amino acid-substituted peptides MQP-37D6Y, MQP-37D6W, and MQP-37D6H The following experiment was carried out to compare the activity of the novel amino acid substituted peptides MQP-37D6Y and MQP-37D6W, whose viral inflammatory response inhibitory activity was confirmed through the experiment in Example 10 above, with that of the original peptide MQP-37.

[0129] Specifically, polyIC was used to induce viral infection. First, serum-free cell culture medium containing 5 mg / ml of polyIC and MQP-37, MQP-37A6, MQP-37D6W, MQP-37D6Y, MQP-37D6H, MQP-37D6K, MQP-37D6R, or MQP-37D6V was added to 1 × 10 mouse lung epithelial cell line (MLE-12) cells. 5 The cells were treated for 16 hours, after which the supernatant was collected and the expression level of interferon-beta (IFN-b) was determined using enzyme-linked immunosorbent assay (ELISA).

[0130] As a result, it was confirmed that MQP-37D6W and MQP-37D6Y exhibited superior inhibitory effects against viral infection-induced inflammatory responses in a concentration-dependent manner compared to MQP-37 and MQP-37A6 (Figure 13a, A and B). Furthermore, it was confirmed that MQP-37D6Y effectively inhibited the cell killing effect of polyIC (Figure 13b, E), and that no cytotoxicity was observed at the maximum concentration of MQP-37D6Y used in the experiment (Figure 13b, F). Furthermore, it was confirmed that MQP-37D6H also inhibited viral infection-induced inflammatory responses in a concentration-dependent manner (Figure 13a, C and D). Based on these results, it can be seen that the novel peptides containing amino acid substitutions developed in this application, including MQP-37D6Y, effectively inhibit inflammatory responses caused by viral infection.

[0131] Example 14: Confirmation of the effect of novel peptides in a mouse sepsis animal model The following experiment was conducted to confirm the effects of D-type or L-type MQP-37 and MQP-37A6, whose inhibitory activity against inflammatory responses caused by bacterial infection was confirmed through the experiments in Examples 2 to 12 above, in a mouse sepsis animal model.

[0132] Specifically, 7-week-old C57BL / 6 wild-type mice were intraperitoneally administered 2 mg / kg and 5 mg / kg LPS at 6-hour intervals. One hour later, the mice were intraperitoneally administered 0.4 mg / kg, 2 mg / kg, and 10 mg / kg of the novel peptide. The mice were then monitored for survival over four days.

[0133] As a result, it was confirmed that the survival rate was 40-80% when 0.4 mg / kg was administered, whereas 100% of mice survived when 2 mg / kg and 10 mg / kg were administered (Figure 14, A to C). It was also confirmed that the amount of IL-6, an inflammatory cytokine in the blood, was reduced in the group treated with the novel peptide compared to the control group (Figure 14, D). Based on these results, it is clear that D-type or L-type MQP-37 and MQP-37A6 are effective in treating sepsis, an inflammatory disease.

[0134] The above description of the present application is for illustrative purposes only, and those skilled in the art will understand that the present application may be easily modified into other specific forms without changing the technical spirit or essential features of the present application. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not restrictive. For example, each component described as a single component may be implemented in a distributed form, and similarly, each component described as a distributed component may be implemented in a combined form.

[0135] The scope of the present application is defined by the claims set forth below rather than the above detailed description, and all modifications and variations derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present application.

Claims

1. X １ LVLAX ６ An anti-inflammatory peptide consisting of an amino acid sequence represented by ARW, The amino acid X １ is S, and the amino acid X ６ is D, A, F, G, H, I, L, M, Q, S, T, or W; or The amino acid X ６ is A, and the amino acid X １ is A, C, F, H, I, L, M, R, or Y.

2. The peptide is mutated at the N-terminus or C-terminus, The mutation is bound to a protecting group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG); The anti-inflammatory peptide according to claim 1 , wherein the mutation is acetylation or amination.

3. The anti-inflammatory peptide of claim 1 , further comprising a cell-penetrating peptide.

4. The anti-inflammatory peptide according to claim 1, wherein the peptide inhibits aggregation of MAVS (mitochondrial antiviral-signaling) protein.

5. The anti-inflammatory peptide according to claim 1 , wherein the peptide suppresses the expression of inflammatory cytokines or inflammasomes.

6. The anti-inflammatory peptide according to claim 1 , wherein the peptide suppresses inflammation induced by bacteria or viruses.

7. A polynucleotide encoding the anti-inflammatory peptide according to any one of claims 1 to 6.

8. An anti-inflammatory composition comprising the anti-inflammatory peptide according to any one of claims 1 to 6 as an active ingredient.

9. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising the anti-inflammatory peptide according to any one of claims 1 to 6 as an active ingredient.

10. The inflammatory diseases include sepsis, septic shock, systemic inflammatory response syndrome, acute respiratory distress syndrome, asthma, allergic rhinitis, non-allergic rhinitis, chronic rhinitis, acute rhinitis, chronic gastritis, acute gastritis, chronic enteritis, acute enteritis, ulcerative gastritis, acute nephritis, chronic nephritis, acute hepatitis, chronic hepatitis, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.

10. The composition of claim 9, wherein the inflammatory bowel syndrome (IGS), inflammatory bowel disease (IPF), inflammatory pain syndrome, headache, lower back pain, fibromyalgia, fascial disorders, viral infection, bacterial infection, fungal infection, burns, wounds from surgical or dental procedures, prostaglandin E excess syndrome, atherosclerosis, gout, arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis, eczema, systemic lupus erythematosus (SLE), and multiple sclerosis.

Images