Novel anti-inflammatory peptides and uses thereof

Abstract

The present application relates to an anti-inflammatory peptide comprising one or more selected from the group consisting of amino acid sequences of SEQ ID NO. 1 to SEQ ID NO. 63 and modified amino acid sequences thereof, an anti-inflammatory composition comprising the same, and a pharmaceutical composition for preventing or treating an inflammatory disease. The anti-inflammatory peptide according to an embodiment of the present application has no cytotoxicity and has an effect of inhibiting the expression or activity of MAVS activation, immune / inflammatory activation reaction mechanisms, inflammatory cytokines, and inflammasomes, and the like, and the above peptide can be applied to an anti-inflammatory composition or a composition for treating an inflammatory disease.

Description

Technical Field

[0001] This application relates to an anti-inflammatory peptide comprising one or more amino acid sequences selected from SEQ ID NO.1 to SEQ ID NO.63 and modified amino acid sequences thereof, an anti-inflammatory composition comprising the same, and a pharmaceutical composition for the prevention or treatment of inflammatory diseases. Background Technology

[0002] Recently, with economic development, advancements in medical technology, and increased life expectancy, the proportion of elderly people has continued to increase. Furthermore, due to environmental pollution and increased stress leading to immune system abnormalities, inflammatory responses have become chronic, with chronic inflammatory diseases such as atopic dermatitis and asthma showing an increasing trend (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.; Sunget et al., 2012, J. Ethnopharmacol. 144: 94-100).

[0003] Generally, the inflammatory response is a defense mechanism of biological tissues against external stimuli such as bacterial or viral infections (pathogen-associated molecular pattern, PAMP) or internal stimuli such as metabolites caused by tissue damage (danger-associated molecular pattern, DAMP). It occurs due to the production of various intracellular inflammatory regulators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and other cytokines, as well as nitric oxide (NO). Lipopolysaccharide (LPS), known as an endotoxin, is a representative example of a pathogen-associated molecular pattern. It is present on the outer membrane of Gram-negative bacteria and induces the activation of the intracellular transcription factor nuclear factor-κB (NF-κB) in macrophages or monocytes. This, in turn, induces the gene expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which are inflammatory cytokines, and generates inflammatory mediators. Therefore, substances that regulate inflammatory responses based on pathogen-associated molecular patterns or damage-associated molecular patterns (e.g., expression of iNOS, COX-2, or NF-κB, and secretion of cytokines and nitric oxide) have recently attracted much 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 substances include nonsteroidal anti-inflammatory drugs (NSAIDs) such as flufenamic acid, ibuprofen, benzyldamine, and indomethacin; and steroidal anti-inflammatory drugs such as prednisolone, dexamethasone, betamethasone, and hydrocortisone. However, these substances are highly toxic and can cause serious side effects such as liver damage, cancer, and stroke, thus limiting their use. Furthermore, their inability to selectively target inflammatory substances can lead to severe immunosuppression. Therefore, the development of anti-inflammatory agents using natural substances is underway. These natural substances have the advantages of being safe for organisms and easier to take long-term compared to traditional medicines. However, anti-inflammatory substances extracted from natural substances tend to have lower effective concentrations and require cultivation in agricultural land, resulting in high production costs.

[0005] To address these issues, a novel concept of anti-inflammatory agents is being developed as an alternative to existing chemical anti-inflammatory agents or those using natural substances. In particular, extensive research is being conducted on the synthesis of peptides with anti-inflammatory activity.

[0006] However, although synthetic peptides often exhibit excellent anti-inflammatory activity and do not show cytotoxicity in in vitro experiments, their anti-inflammatory effects are often negligible in in vivo experiments.

[0007] While there are various reasons for this, the main one is the significant difference between the physiological and anatomical conditions within an organism and those in vitro. First, the presence of salt, a physiological condition, significantly inhibits the activity of peptides with low positive charges. Second, due to their small molecular weight and size, peptides are almost entirely absorbed by the kidneys and excreted. Third, they are easily cleaved and inactivated by proteins and peptide-degrading enzymes (proteases and peptidases) present in all tissues, cells, body fluids, and blood within the body.

[0008] Therefore, in order to solve the above problems and as a result of efforts to develop a substance that exhibits excellent anti-inflammatory activity, the inventors developed an economical peptide that can be mass-produced using 5 to 15 common amino acid residues, and confirmed that the peptide does not exhibit cytotoxicity and exhibits excellent anti-inflammatory activity, thus completing the present invention. Summary of the Invention

[0009] The problem that the invention aims to solve

[0010] The purpose of this application is to provide an anti-inflammatory peptide comprising one or more amino acid sequences selected from SEQ ID NO.1 to SEQ ID NO.63 and modified amino acid sequences thereof, an anti-inflammatory composition comprising the same, and a pharmaceutical composition for the prevention or treatment of inflammatory diseases.

[0011] However, the problems to be solved by this application are not limited to those described above, and those skilled in the art can clearly understand other problems not mentioned in the following description.

[0012] Solution for solving the problem

[0013] According to a first aspect of this application, an anti-inflammatory peptide is provided, comprising one or more amino acid sequences selected from SEQ ID NO.1 to SEQ ID NO.63 and modified amino acid sequences thereof.

[0014] According to a second aspect of this application, a polynucleotide is provided that encodes the anti-inflammatory peptide of this application.

[0015] According to a third aspect of this application, an anti-inflammatory composition is provided, comprising the anti-inflammatory peptide of this application as an active ingredient.

[0016] According to a fourth aspect of this application, a pharmaceutical composition for the prevention or treatment of inflammatory diseases is provided, comprising the anti-inflammatory peptide of this application as an active ingredient.

[0017] Invention Effects

[0018] The anti-inflammatory peptide according to one embodiment of this application is non-cytotoxic and has the effects of inhibiting the activation of mitochondrial antiviral signaling protein (MAVS), immune / inflammatory activation response mechanisms, inflammatory cytokines and the expression or activity of inflammasomes. The above peptide can be used in anti-inflammatory compositions or compositions for treating inflammatory diseases. Attached Figure Description

[0019] Figures 1a to 1f This is a diagram showing the structure of the novel anti-inflammatory peptide developed in this application.

[0020] Figure 2 This is a graph showing the experimental results confirming the MAVS aggregation inhibition effect of the anti-inflammatory peptide of this application.

[0021] Figure 3a and Figure 3b This is a graph showing the experimental results confirming the inhibitory effect of the anti-inflammatory peptide of this application on the expression of IFN-β induced by polyinosine:polycytidylic acid (polyIC).

[0022] Figure 4 This is a graph showing the experimental results confirming the cytotoxicity of the anti-inflammatory peptide of this application.

[0023] Figures 5a to 5c This is a graph showing the experimental results confirming the inhibitory effect of the anti-inflammatory peptide of this application on the expression of IL-1β induced by LPS or bacterial outer membrane vesicles (OMV).

[0024] Figure 6a and Figure 6b This is a graph showing the experimental results confirming the inhibitory effect of the anti-inflammatory peptide of this application on the expression of LPS-induced IL-6 and TNF-α.

[0025] Figure 7 This is a graph showing the experimental results confirming the inhibitory effect of LPS-induced NF-κB phosphorylation on the anti-inflammatory peptide of this application.

[0026] Figure 8 This is a graph showing the experimental results confirming the enhanced resistance to proteolytic enzymes modified with the anti-inflammatory peptides of this application.

[0027] Figures 9a to 9c This is a graph showing the experimental results confirming the inhibitory effect of LPS / ATP or polyIC on the production of IL-1β or IFN-β by peptides whose sequences of the anti-inflammatory peptides MQP-37 and MQP-Y9, which are replaced by alanine, in this application.

[0028] Figure 10a and Figure 10b The figure shows the experimental results confirming the inhibitory effect of LPS / ATP or polyIC on the production of IL-1β or IFN-β by peptides whose first or sixth sequence of the anti-inflammatory peptide MQP-37, which is replaced by 19 other amino acids.

[0029] Figure 11 This is a graph showing the experimental results comparing and confirming the binding ability of the anti-inflammatory peptides MQP-37 and MQP-37A6 of this application to MAVS protein.

[0030] Figure 12a and Figure 12b The figure shows the experimental results that comparatively confirm the physiological activities of the anti-inflammatory peptides MQP-37 and MQP37-A6 of this application (the production of IL-6 and IL-1β induced by LPS or LPS / nigericin, and the phosphorylation of NF-κB induced by LPS).

[0031] Figure 13a and Figure 13bThis is a figure showing the experimental results that comparatively confirm the physiological activities (polyIC-induced IFN-β production and apoptosis) of the anti-inflammatory peptides MQP-37 and MQP-37D6Y, MQP-36D6W and QP-37D6H of this application.

[0032] Figure 14 This is a diagram showing the experimental results confirming the therapeutic effects of the anti-inflammatory peptides MQP-37 and MQP-37A6 of this application on sepsis in an LPS-induced mouse sepsis animal model. Detailed Implementation

[0033] Hereinafter, embodiments of this application will be described in detail with reference to the accompanying drawings to enable those skilled in the art to readily implement them. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Furthermore, for the purpose of clearly describing this application in the drawings, parts unrelated to the description have been omitted, and similar reference numerals have been used for similar parts throughout the specification.

[0034] Throughout the instruction manual, when it is mentioned that one part is "connected" to another part, it includes not only the case of "direct connection" but also the case of "indirect connection" in which the two are separated by other linking groups or other substances.

[0035] Throughout this specification, when a part is referred to as "comprising" a component, unless otherwise stated, it means that other components may also be included, not that other components are excluded. The terms "about," "substantially," etc., used throughout this specification, are used in a sense equal to or close to the permissible tolerances of manufacture and materials inherent in the meaning as stated in the disclosure, and are intended to prevent unethical infringers from improperly using disclosures that mention precise or absolute values ​​to aid in understanding this application. The terms "~(of) step" or "~ step" used throughout this specification to indicate degree do not mean "a ~ step."

[0036] Throughout the specification, the term “their (multiple) combinations” included in the Markush form of expression refers to a mixture or combination of one or more of the constituent elements of the Markush form of expression, which means including one or more of the constituent elements of the aforementioned group.

[0037] In the full text of the instruction manual, the description of "A and / or B" means "A or B, or A and B".

[0038] The embodiments and examples of this application will now be described in detail with reference to the accompanying drawings. However, this application is not limited to these embodiments, examples, and drawings.

[0039] According to a first aspect of this application, an anti-inflammatory peptide is provided, comprising one or more amino acid sequences selected from SEQ ID NO.1 to SEQ ID NO.63 and modified amino acid sequences thereof.

[0040] The term "peptide" as used throughout this specification refers to a linear molecule formed by the binding of amino acid residues together through peptide bonds (-CO-NH-). The peptides of this application can be prepared according to 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-phase synthesis techniques (U.S. Patent No. 5516891), and the amino acid residues constituting the peptides of this application can be natural or non-natural amino acid residues.

[0041] According to one embodiment of this application, the peptide may comprise one or more of the amino acid sequences selected from SEQ ID NO.1 to SEQ ID NO.63 and their modified amino acid sequences, specifically the amino acid sequence of SEQ ID NO.1.

[0042] According to one embodiment of this application, the modified amino acid sequence can be an amino acid sequence that maintains its main activity or exhibits enhanced activity, may include a portion of the amino acid sequence that has been modified due to natural or artificial variation, or a sequence in which one or more of the amino acids constituting the amino acid sequence are replaced by another amino acid. Specifically, the modified amino acid sequence can be constituting SEQ ID NO.1 to SEQ ID NO. The amino acid sequence of NO.63 contains one or more amino acids selected from the group consisting of γ-aminobutyric acid, glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), lysine (Lys, K), leucine (Leu, L), methionine (Met, M), (Val, V), serine (Ser, S), selenomethionine, selenocysteine ​​(Sec, U), cysteine ​​(Cys, C), citrulline, 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), pyrrolidone (Pyr, O), histidine (His, H), and non-natural amino acids. If a peptide according to an example of this application has the same or corresponding biological activity as the respective peptide, it may include not only the described sequence number, 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 above-described amino acid sequence. It is obvious that sequences homologous to the above-described sequences are included in the scope of this application as long as they are amino acid sequences that substantially have the same or corresponding biological activity as the peptide with the described sequence number, including those with partially deleted, modified, substituted, or added amino acid sequences.

[0043] The term "homology" as used throughout this specification refers to the degree of similarity to a given amino acid or polynucleotide sequence, expressed as a percentage. In this specification, "%homology" indicates a homologous sequence having the same or similar activity as a given amino acid or polynucleotide sequence. For example, sequences can be compared and confirmed by standard software, specifically BLAST 2.0, to calculate parameters such as score, identity, and similarity, or by hybridization experiments under defined stringent conditions. Appropriate hybridization conditions, as defined within the art, can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, ColdSpring Harbor Laboratory press, ColdSpring Harbor, New York, 1989; F.M. Usubelet et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). The term "stringent conditions" as used throughout this 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., ibid.).

[0044] The term "anti-inflammatory" as used throughout this specification refers to the ability to inhibit or reduce inflammation, while "inflammation" is a defensive response that occurs in the body when biological tissues are damaged, and is a cause of inflammatory diseases. Therefore, the anti-inflammatory peptides of this application can be used for the prevention, treatment, or improvement (symptom relief) of inflammatory diseases by exhibiting activity that inhibits or reduces inflammation.

[0045] According to one example of this application, the aforementioned peptides may also include, but are not limited to, cell-penetrating peptides.

[0046] The term "cell-penetrating peptide" as used throughout this specification refers to a peptide that has the ability or property to penetrate cell membranes and permeate into the cell interior, and may be a peptide exhibiting cell-penetrating and / or skin-penetrating properties.

[0047] According to one embodiment of this application, the aforementioned cell-penetrating peptide may be a peptide that also includes a portion or all of a sequence derived from a previously known cell-penetrating peptide or skin-penetrating peptide, for example, it may be a peptide 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, specifically an Arg(8) peptide (R8 peptide), but is not limited thereto.

[0048] According to one embodiment of this application, the cell-penetrating peptide may be contained in the anti-inflammatory peptide more than twice. Specifically, the cell-penetrating peptide may be contained two, three, five, seven, or ten times, but is not limited thereto.

[0049] According to one embodiment of this application, the cell-penetrating peptide may be a cell-penetrating peptide linked to the N-terminus or C-terminus of the anti-inflammatory peptide, specifically a cell-penetrating peptide linked to the N-terminus, but it is not limited thereto as long as it does not inhibit the pharmacological activity of the peptide and can improve cell penetration or skin penetration.

[0050] According to one embodiment of this application, the cell-penetrating peptide can be bound to the anti-inflammatory peptide via a linker group, or it can be directly linked to the anti-inflammatory peptide. Furthermore, the linker group can be cleaved or decomposed within cells or skin through various biological and chemical processes such as enzymatic action. Based on the cleavage or decomposition of the linker group, the cell-penetrating peptide and the anti-inflammatory peptide can be separated from each other in the desired cells or skin.

[0051] According to one example of this application, the amino acids constituting the above-mentioned anti-inflammatory peptides can be L-type or D-type, specifically D-type, but are not limited thereto as long as they do not affect the activity of the above-mentioned peptides.

[0052] According to one embodiment of this application, the N-terminus or C-terminus of the above-mentioned anti-inflammatory peptide may be bound with a protecting group selected from the group consisting of acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl and polyethylene glycol (PEG), and the above-mentioned protecting group is not limited thereto as long as it does not affect the activity of the above-mentioned peptide.

[0053] According to one embodiment of this application, the peptide or the amino acids constituting it may not only be bound to the above-mentioned protecting group, but may also be acetylated or amidated. The above modifications significantly improve the stability of the peptide. The above stability not only means stability in vivo, but also means storage stability (e.g., room temperature storage stability). The above-mentioned protecting group can protect the peptide of this application from the attack of proteolytic enzymes in vivo.

[0054] According to one example of this application, the above-mentioned peptide can inhibit the aggregation of MAVS protein and / or the function of the above-mentioned protein.

[0055] The term "MAVS protein" as used throughout this specification refers to a signal transduction protein essential for antiviral innate immunity, known to be present in the outer mitochondrial membrane, peroxisome, and endoplasmic reticulum (ER). Upon viral infection, the aforementioned MAVS protein is activated by the formation of aggregates (assemblages) of cytoplasmic proteomes in response to viral presence. Activated MAVS induces an immune response by triggering the secretion of interferons and cytokines. However, if MAVS protein activation is prolonged, it can lead to an excessive increase in the production of interferons and cytokines, which may attack cells in the body, potentially inducing various pathological abnormalities and immune diseases. Therefore, appropriate regulation of MAVS protein activation is necessary.

[0056] According to one example of this application, the above-mentioned anti-inflammatory peptides, by inhibiting MAVS aggregation or activation, can not only suppress the inflammatory / immune activation response mechanism and inflammatory / immune response induced by MAVS activation, but also treat, prevent or improve symptoms or diseases that may occur due to MAVS aggregation.

[0057] According to one embodiment of this application, it has been confirmed that the anti-inflammatory peptide of this application effectively inhibits MAVS aggregation induced by pathogen-associated molecular patterns (PAMPs) derived from bacteria and viruses. Thus, it can be seen that the anti-inflammatory peptide of this application can effectively inhibit inflammatory responses by inhibiting MAVS activity.

[0058] According to one embodiment of this application, the above-mentioned peptide can inhibit the expression, production, activity, etc. of inflammatory cytokines or inflammasomes, or inhibit the proliferation of inflammatory cells. Specifically, the above-mentioned inflammatory cytokines can be one or more selected from the group consisting of IFN-β, IL-1β, IL-6 and TNF-α, as long as they are substances known in the art that cause inflammatory responses, and are not limited thereto.

[0059] The term "inflammasome" as used throughout this specification refers to the substance that induces the maturation of inflammatory cytokines such as IL-1 associated with innate immune defenses such as infection or stress in cells. As a caspase-1-activating protein complex, it consists of 1) NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) as a sensor protein, 2) an adapter protein containing a caspase-recruitment domain, an adapter protein apoptosis-associated spec-like protein (ASC) as an adapter protein, and 3) an inactive form of caspase-1 as an effector protein. The components of the aforementioned inflammasome assemble upon infection by microorganisms such as bacteria or viruses, or upon tissue damage. In the cytoplasm, the assembled activated inflammasome converts the inactive form of caspase-1 into the active form. The converted, active caspase-1 is known to cleave precursor forms of IL-1β or IL-18 to generate activated IL-1β or IL-18, which is then secreted extracellularly to perform the host's innate immune defense functions.

[0060] According to one embodiment of this application, it was confirmed that the anti-inflammatory peptide of this application inhibits the expression levels of various inflammatory cytokines and inflammasomes. Therefore, the anti-inflammatory peptide of this application can effectively inhibit inflammatory responses.

[0061] According to one example of this application, the above-mentioned peptide can inhibit the mechanism of inflammation / immune activation response. The mechanism may be caspase-1, interferon regulatory factor 3 (IRF3), or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), as long as it is a mechanism of inflammation / immune response known in the art.

[0062] According to one embodiment of this application, it was confirmed that the anti-inflammatory peptide of this application inhibits the activity of the NF-κB signaling mechanism by inhibiting the phosphorylation of NF-κB. Therefore, the anti-inflammatory peptide of this application can effectively inhibit the inflammatory response.

[0063] According to one embodiment of this application, the above-mentioned peptide can inhibit inflammation caused by bacteria or viruses, specifically, it can inhibit inflammation induced by pathogen-associated molecular patterns (PAMPs) derived from bacteria and viruses. Furthermore, it can inhibit inflammation induced by damage-associated molecular patterns (DAMPs) secreted extracellularly due to cell damage caused by bacterial or viral infection.

[0064] The term "pathogen-associated molecular pattern (PAMP)" as used throughout this specification refers to a molecule derived from a pathogen that elicits an immune response. The immune system possesses pattern recognition receptors that respond to specific molecular patterns, eliminating the infectious agent through phagocytosis or inducing antibody formation. Therefore, common molecular patterns of infectious agents that elicit a response to pattern recognition receptors can be termed pathogen-associated molecular patterns. These pathogen-associated molecular patterns can include endotoxins, exotoxins, lipopolysaccharides (LPS), lipoteichoic acid (LTA), muramyldipeptide (MDP), nigericin, dsRNA (polyIC, etc.), dsDNA (polydAdT, etc.), outer membrane vesicles (OMV), and flagellin, as long as they are substances known in the art to originate from pathogens and can elicit an inflammatory / immune response. This is not an limitation.

[0065] The term "danger-associated molecular pattern (DAMP)" as used throughout this specification refers to a molecule that, as an intracellular metabolite or protein, is secreted extracellularly through cell damage and elicits an immune response in surrounding cells. This damage-associated molecular pattern can include, but is not limited to, ATP, histones, high mobility group box 1 (HMGB1), mitochondrial DNA (mtDNA), uric acid, etc., as long as it is a substance known in the art to be intracellular in origin and capable of inducing an inflammatory / immune response.

[0066] According to one example of this application, the above-mentioned peptide can inhibit inflammatory responses or treat or alleviate symptoms of diseases induced by inflammatory responses. Specifically, the above-mentioned peptide can be included in various compositions such as anti-inflammatory drug compositions, food compositions, cosmetic compositions, health functional food compositions, and feed compositions.

[0067] According to a second aspect of this application, a polynucleotide is provided that encodes the anti-inflammatory peptide of this application. The same applies to the polynucleotide of the second aspect as to the first aspect.

[0068] The term "polynucleotide" as used throughout this manual refers to a high molecular weight substance bound to nucleotides, specifically DNA that encodes genetic information.

[0069] According to one embodiment of this application, the polynucleotide may comprise one or more base sequences encoding an amino acid sequence of SEQ ID NO.1 to SEQ ID NO.63.

[0070] According to one example of this application, the base sequence encoding the aforementioned anti-inflammatory peptide not only includes the base sequence encoding the amino acid described by each sequence number, but also includes base sequences that exhibit 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 98% or more, and most specifically 99% or more homology with the aforementioned sequences. It may, without limitation, include base sequences encoding proteins that substantially exhibit the same or corresponding effects as the aforementioned peptides. Furthermore, it is obvious that sequences homologous to the aforementioned sequences, as long as they are amino acid sequences substantially having the same or corresponding biological activity as the peptides with the described sequence numbers, include cases where amino acid sequences have partial deletions, modifications, substitutions, or additions. Moreover, considering the degeneracy of codons and the preferred codons in the organism to express the aforementioned peptides, the polynucleotide encoding the aforementioned peptides can be modified in various ways in the coding region without altering the amino acid sequence of the peptide expressed from the coding region. Therefore, the aforementioned polynucleotide may, without limitation, include polynucleotide sequences encoding each peptide. Furthermore, it may include probes that can be prepared from known sequences, for example, sequences that, under stringent conditions, hybridize to all or part of the complementary sequence of the aforementioned polynucleotide sequence and encode proteins having the activity of the aforementioned peptides.

[0071] The term "strict conditions" as used throughout this specification refers to conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (e.g., see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). Examples include, for instance, conditions that enable hybridization between genes with high homology, such as genes with more than 40%, specifically more than 90%, more specifically more than 95%, more specifically more than 97%, and especially more specifically more than 99%, while preventing hybridization between genes with lower homology; or conditions equivalent to washing once, specifically two to three times, at a salt concentration and temperature of 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, as in conventional Southern hybridization. Although mismatches between bases are possible depending on the stringency of hybridization, hybridization requires two polynucleotides to have complementary sequences. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, this application may include, in addition to substantially similar polynucleotide sequences, separate polynucleotide fragments complementary to the entire sequence.

[0072] Specifically, homologous polynucleotides are hybridized at a Tm value of 55°C using hybridization conditions including a hybridization step, and can be detected using the conditions described above. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited to these values; those skilled in the art can make appropriate adjustments according to their purpose. The appropriate stringency of the hybridized polynucleotides depends on the length and complementarity of the polynucleotides, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0073] As another aspect of the second aspect of this application, an expression vector comprising the aforementioned polynucleotides is provided.

[0074] The term “expression vector” as used throughout this specification refers to a recombinant vector that is introduced into a suitable host cell and is capable of expressing a target protein, and is a gene construct containing the necessary regulatory elements that are operatively linked in a manner that enables the gene insert to be expressed.

[0075] The term "operably linked" as used throughout this specification refers to the functional ligation of a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein to perform a general function. Operable ligation with a recombinant vector can be prepared using gene recombination techniques well known in the art, and site-specific DNA cutting and ligation can be readily performed using enzymes or other methods known in the art.

[0076] Suitable expression vectors of this application may include, in addition to expression regulatory elements such as promoters, start codons, stop codons, polyadenylation signals, and enhancers, signal sequences for membrane targeting or secretion. Start and stop codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein, must function in the individual when the gene construct is administered, and must be in-frame with the coding sequence. Promoters can typically be constitutive or inducible. For prokaryotic cells, there are lac, tac, T3, and T7 promoters; for eukaryotes, there are simian vacuolating virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, and human immunodeficiency virus (HIV) promoters, including not only HIV long terminal repeat (LTR) promoters, but also promoters for Moroni virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Raul's sarcoma virus (RSV), as well as promoters derived from β-actin, human hemoglobin, human muscle creatine, and human metallothionein, but are not limited to these.

[0077] Furthermore, the aforementioned expression vector may include selective markers for selecting host cells containing the vector. These selective markers, used to select cells transformed into the vector, can be markers that confer selectable phenotypes, such as drug resistance, auxotrophic phenotypes, resistance to cytotoxic agents, or expression of surface proteins. In an environment treated with a selective agent, transformed cells can be screened because only cells expressing the selective marker can survive. Additionally, when the vector is a reproducible expression vector, it may include a replication origin, which is a specific nucleic acid sequence in which replication begins.

[0078] As recombinant expression vectors for inserting foreign genes, various forms of vectors such as plasmids, viruses, and granules can be used. There are no particular restrictions on the type of recombinant vector, as long as it can perform the function of expressing the required gene and producing the required protein in various host cells of prokaryotic and eukaryotic cells. Specifically, vectors that can exhibit strong promoter activity and can produce large quantities of foreign proteins in a form similar to that in the natural state while maintaining strong expression ability can be used.

[0079] To express the anti-inflammatory peptide according to this application, various combinations of host and vector can be used. Expression vectors suitable for eukaryotic hosts may include, but are not limited to, SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and expression regulatory sequences derived from retroviruses. Expression vectors suitable for bacterial hosts may include, but are not limited to, bacterial plasmids obtained from *Escherichia coli*, including pET, pRSET, pBluescript, pGEX2T, pUC, col E1, pCR1, pBR322, pMB9, or derivatives thereof; plasmids with a broader host range, such as RP4; bacteriophage DNA, exemplified by phage lambda derivatives such as λgt10, λgt11, or NM989; and other DNA phages, such as M13 and filamentous single-stranded DNA phages. Plasmids of type 2C or their derivatives may be used in yeast cells, and pVL941 may be used in insect cells.

[0080] As another aspect of the second aspect of this application, a transformation other than that of a person containing the aforementioned expression vector is provided.

[0081] The term "transformer" as used throughout this specification can refer to a host cell capable of receiving the aforementioned expression vector. Specifically, the transformant in this application can be a non-human transformant, but is not limited thereto.

[0082] Suitable host cells for introducing the aforementioned vectors can be prokaryotic cells, such as *Escherichia coli*, *Bacillus subtilis*, *Streptomyces* sp., *Pseudomonas* sp., *Proteus mirabilis*, or *Staphylococcus* sp. Alternatively, they can be fungi, such as *Aspergillus*; yeasts, such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces* sp., or *Neurosporacrassa*; other lower eukaryotic cells; or higher eukaryotic cells, such as plant or insect cells. In addition, mammalian cells can be used, specifically monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, or HEK293 cells, but are not limited to these.

[0083] The transformation methods described in this application include any method for introducing nucleic acids into an organism, cell, tissue, or organ, and can be performed using appropriate standard techniques based on the host cells known in the art. Specifically, these include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate, liposomes, and drying / inhibition-mediated transformation methods.

[0084] As another aspect of the second aspect of this application, a method for preparing an anti-inflammatory peptide is provided, which includes the step of culturing the above-mentioned transformant.

[0085] The method for preparing the anti-inflammatory peptide includes the step of culturing the transformant of this application. Specifically, it may include: the step of preparing an expression vector by inserting a polynucleotide sequence encoding the anti-inflammatory peptide into a vector; the step of preparing a transformant by introducing the expression vector into a host cell; the step of culturing the transformant; and the step of isolating and purifying the anti-inflammatory peptide from the cultured transformant.

[0086] More specifically, peptides can be produced in large quantities by culturing the above-mentioned transformants in a nutrient medium. The medium and culture conditions can be appropriately selected and used according to the host cell. During culture, conditions such as temperature, pH of the medium, and culture time can be appropriately adjusted to suit cell growth and the large-scale production of proteins.

[0087] As described above, the recombinant peptides or proteins can be recovered from the culture medium or cell lysates. In the case of membrane-bound peptides, they can be released from the membrane using a suitable surfactant solution (e.g., Triton X-100) or by enzymatic cleavage. Cells expressing anti-Oscar antibodies or fragments thereof can be disrupted by various substances or chemical means, such as freeze-thaw acclimation, sonication, mechanical disruption, or cell lysis agents, and can be separated and purified using conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd Ed., ColdSpring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182, Academic Press, Inc., San Diego, CA (1990)). Electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunosorbent chromatography, size exclusion chromatography, etc.), isoelectric focusing, and various variations and combinations thereof can be used, but are not limited to these methods.

[0088] According to a third aspect of this application, an anti-inflammatory composition is provided, comprising the anti-inflammatory peptide of this application as an active ingredient. The content repeated in the first and second aspects also applies to the composition of the third aspect.

[0089] According to one example of this application, the above-mentioned anti-inflammatory composition can be used as a drug, quasi-pharmaceutical, cosmetic, food, and feed composition.

[0090] According to one embodiment of this application, the above composition may further include pharmaceutically acceptable salts, specifically, examples of hydrochloride, sulfate, phosphate, acetate, citrate, tartrate, succinate, lactate, maleate, fumarate, oxalate, methanesulfonate, or p-toluenesulfonate.

[0091] According to one example of this application, the above-mentioned pharmaceutical composition, in addition to the active ingredient, also includes a pharmaceutically acceptable carrier, and can be prepared into an oral or parenteral dosage form using conventional methods known in the art and depending on the route of administration. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient while not exhibiting toxicity beyond what the intended user can tolerate.

[0092] According to one example of this application, the above-mentioned pharmaceutical composition can be formulated into oral dosage forms, external dosage forms, suppositories, or sterile injections, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, using conventional methods, but is not limited thereto.

[0093] According to one example of this application, when formulating the above-mentioned pharmaceutical composition, a diluent or excipient can be used, such as commonly used fillers, extenders, binders, wetting agents, disintegrants or surfactants, but not limited thereto.

[0094] According to one example of this application, when the above-described pharmaceutical composition is prepared into an oral dosage form, it can be prepared into powder, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, suspensions, wafers, etc., together with a suitable carrier according to methods known in the art. Examples of pharmaceutically acceptable suitable carriers include: sugars such as lactose, glucose, sucrose, dextran, sorbitol, mannitol, xylitol, etc.; starches such as corn starch, potato starch, wheat starch, etc.; celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, etc.; polyvinylpyrrolidone; water; methylparaben; propylparaben; magnesium stearate; mineral oil; malt; gelatin; talc; polyols; vegetable oils, etc. In the case of formulation, diluents and / or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be included as needed for formulation.

[0095] According to one example of this application, when the above-described pharmaceutical composition is prepared into a parenteral dosage form, it can be formulated into an injection, transdermal drug delivery system, nasal inhaler, or suppository form using methods known in the art, along with a suitable carrier. When formulated into an injection, suitable carriers include sterile water, ethanol, glycerol, or polyols such as propylene glycol, or mixtures thereof. Preferably, isotonic solutions such as Riger's solution, phosphate-buffered saline (PBS) containing triethanolamine, or sterile water for injection, 5% dextran, etc., can be used. When formulated into a transdermal drug delivery system, it can be formulated into an ointment, cream, emulsion, gel, topical solution, paste, liniment, aerosol, etc. For nasal inhalers, suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide can be used to formulate them into aerosol sprays. When formulated into suppositories, semi-synthetic fatty acid esters (Witepsol), Tween 61, polyethylene glycols, cocoa butter, laurate, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, and sorbitan fatty acid esters can be used as the main agent.

[0096] According to one embodiment of this application, the pharmaceutical composition described above can be administered at a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an adequate dose for treating or preventing disease in a reasonable benefit / risk ratio suitable for medical treatment or prevention. The effective dose level can be determined based on the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the timing of administration of the composition of the present invention, the route of administration and excretion rate, the duration of treatment, factors including drugs to be used in combination with or concurrently with the composition of this application, and other factors well-known in the medical field. The pharmaceutical composition of this application can be administered alone or in combination with well-known ingredients known to have therapeutic effects on intestinal diseases. Taking all the above factors into account, it is important to administer the dose to achieve the maximum effect with the minimum amount without side effects.

[0097] According to one example of this application, those skilled in the art can determine the dosage of the above-mentioned pharmaceutical composition by considering factors such as the intended use, the degree of addiction to the disease, the patient's age, weight, sex, medical history, or the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention can be administered at a dose of about 0.1 ng to about 1000 mg / kg per adult, preferably at a dose of 1 ng to about 100 mg / kg per adult. The frequency of administration of the composition of the present invention is not particularly limited thereto; it can be administered once daily or in multiple divided doses. The above-mentioned dosage or frequency of administration, in any respect, does not limit the scope of this application.

[0098] According to a fourth aspect of this application, a pharmaceutical composition for the prevention or treatment of inflammatory diseases is provided, comprising the anti-inflammatory peptide of this application as an active ingredient. The content repeated in the first through third aspects also applies to the composition of the fourth aspect.

[0099] The term "treatment" as used throughout the description of this application refers to all actions that improve or alleviate the symptoms of an inflammatory disease by administering the composition of this application.

[0100] The term “prevention” as used throughout the description of this application refers to all actions that suppress or delay inflammatory diseases or the likelihood of their onset by administering the composition of this application.

[0101] The term "inflammatory disease" as used throughout this application refers to a local or systemic biological defense response to external physical or chemical stimuli or external infectious agents such as bacteria, fungi, viruses, and various allergens, or to autoimmune responses. It can be defined as pathological symptoms caused by a specific inflammatory response. Such inflammatory responses are accompanied by a series of complex physiological reactions, such as the activation of various inflammatory mediators and immune cell-related enzymes (e.g., iNOS, COX-2), the secretion of inflammatory mediators (e.g., NO, TNF-α, IL-6), fluid infiltration, cell migration, and tissue destruction, manifesting externally through symptoms such as erythema, pain, edema, fever, and a decline or loss of specific bodily functions. The aforementioned inflammatory diseases can be acute, chronic, ulcerative, allergic, or necrotic; therefore, any disease included in the definition of inflammatory disease as described above, regardless of whether it is acute, chronic, ulcerative, allergic, or necrotic, is included therein.

[0102] According to one example of this application, the aforementioned inflammatory diseases may be selected from 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 (IPF), irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, myofascial diseases, viral infections (e.g., hepatitis C infection), bacterial infections, fungal infections, burns, surgical or dental trauma, prostaglandin E overdose syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis, atopic dermatitis, eczema, systemic lupus erythematosus (SLE). One or more of the following groups: lupus erythematosus (SLE) and multiple sclerosis, provided that the disease is induced by an inflammatory response, specifically, a disease induced by an inflammatory response caused by bacterial or viral infection, but not limited thereto.

[0103] According to a fifth aspect of this application, a method for the prevention or treatment of inflammation or an inflammatory disease is provided, comprising the step of administering to an individual an anti-inflammatory composition of this application or a pharmaceutical composition for the prevention or treatment of an inflammatory disease. The content repeated in the first through fourth aspects also applies to the method of the fifth aspect.

[0104] The term "individual" as used throughout the description of this application may include, but is not limited to, mammals such as rats, livestock, and humans that are at risk of developing or experiencing inflammatory reactions or diseases, as well as farmed fish.

[0105] According to one example of this application, the aforementioned individual may be an individual other than a human being.

[0106] According to one example of this application, the above method can administer a pharmaceutically effective amount of the composition to prevent or treat inflammation or inflammatory diseases, and may vary due to various factors, such as the extent of the inflammatory response or inflammatory disease, the patient's age, weight, the characteristics and severity of symptoms, the type of current treatment, the number of treatments, the form of administration, and the route of administration, which can be readily determined by an expert in the art. The composition of this application can be administered co-administered or sequentially with the above-mentioned pharmacological or physiological components. Furthermore, it can be administered in combination with additional existing therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents. Such administration can be a single or multiple doses. Taking all the above factors into account, it is important to administer the amount that achieves the maximum effect with the minimum amount without side effects, and this can be readily determined by a person skilled in the art.

[0107] The term "administration" as used throughout this application means the introduction of a predetermined substance into an individual by an appropriate method. The composition of this application can be administered via any conventional route, as long as it reaches the target tissue. Intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, and rectal administration methods can be used, but are not limited to these. However, when administered orally, since proteins are digestible, the composition for oral administration preferably encapsulates the active pharmaceutical ingredient or formulates it to prevent its breakdown in the stomach. Furthermore, the above-mentioned composition can be administered via any device capable of transporting the active substance to target cells.

[0108] The present invention will be described in more detail below through embodiments of this application. The following embodiments are only used to help understand this application, and the content of this application is not limited to the following embodiments.

[0109] Example 1

[0110] Example 1: Synthesis of novel peptides with anti-inflammatory effects

[0111] In order to develop anti-inflammatory peptides that alleviate inflammatory responses by inhibiting the aggregation of MAVS protein, a well-known major hub protein in the innate immune response mechanism, the sequence of an anti-inflammatory peptide that inhibits MAVS signaling was derived as follows.

[0112] Specifically, protein synthesis in vivo involves the formation of transcribed RNA (mRNA) from one side of the DNA structure (5'-3' direction) of a double-helix DNA, thereby synthesizing proteins. However, when a new protein is synthesized by transcribing a complementary DNA sequence from the other direction (3'-5') in the direction of the original protein synthesis, it exhibits properties opposite to those of the original protein, thus exhibiting a phenomenon known as hydropathic complementarity. Furthermore, MAVS proteins aggregate during activation upon external stimulation. Based on the aforementioned hydropathic complementarity theory, a portion of the domain of the MAVS protein, which may play a key role in this stage, can be synthesized into novel peptides (SEQ ID NO. 1 to SEQ ID NO. 4, SEQ ID NO. 48 to SEQ ID NO. 52) complementary to this amino acid sequence.

[0113] Example 2: Confirmation of the MAVS aggregation inhibitory efficacy of the novel peptide

[0114] To confirm whether the novel peptide prepared in Example 1 above has the effect of inhibiting MAVS protein aggregation, the following experiment was conducted.

[0115] Specifically, to induce MAVS protein aggregation, it was stimulated using pathogen-associated molecular patterns (PAMPs), and as an example of these PAMPs, endotoxin (lipopolysaccharide, LPS) and nigericin were used. 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 endotoxin, and 1×10⁶ cells were cultured. 5A mouse peritoneal macrophage cell line (IC21) was treated for 4 hours. Subsequently, the cell lines were further treated for 1 hour in serum-free cell culture medium containing 5 mM nigrain, with the addition of 5 mM MQP-15, MQP-23, MQP-31, or MQP-37. Next, mitochondria from the macrophage line were isolated using hypotonic buffer and centrifugation. To confirm MAVS aggregation results by Western blotting, the isolated mitochondria were sampled using a semi-denaturing detergent and subjected to electrophoresis. The mitochondrial proteins separated by electrophoresis were transferred to a polyvinylidene fluoride (PVDF) membrane and further processed with a primary antibody recognizing MAVS proteins and a secondary antibody recognizing the primary antibody. Subsequently, the aggregation of MAVS protein was confirmed by inducing a chemiluminescence reaction in the secondary antibody.

[0116] The results confirmed that the MAVS protein aggregation observed in the control groups treated with endotoxin and nigericin was reduced in the experimental groups treated with MQP-15, MQP-23, MQP-31, or MQP-37. Figure 2 Therefore, it can be seen that the MQP-15, MQP-23, MQP-31 or MQP-37 prepared in this application effectively inhibit the aggregation of MAVS protein.

[0117] Example 3: Confirmation of the antiviral response-inhibiting efficacy of the novel peptide

[0118] To confirm whether the novel peptide prepared in Example 1 above has the effect of inhibiting the antiviral response caused by viral infection, the following experiment was conducted.

[0119] Specifically, to induce a viral infection response, polyinosinic-polycytidylic acid (polyIC) was used. PolyIC is known for its association with viral infection in pathogen-associated molecular models and acts as a promoter of interferon production in the synthesis of RNA; it is an RNA virus gene analog. First, 1×10 5Mouse lung epithelial cell line (MLE-12) was treated for 16 hours with serum-free cell culture medium supplemented with polyinosinic acid (5 mg / ml) and MQP-15, MQP-23, MQP-31, MQP-37, MQP-Y9, or MQP-T234. The supernatant was then collected, and the expression level of interferon-β (IFN-β) was confirmed using enzyme-linked immunosorbent assay (ELISA).

[0120] The results confirmed that, compared with the control group treated only with polyinosine:polycytidylic acid, the expression level of IFN-β was reduced when treated with MQP-15, MQP-23, MQP-31, MQP-37, MQP-Y9, or MQP-T234. In particular, it was confirmed that the expression level of IFN-β was significantly reduced in the case of MQP-37 compared with other peptides. Figure 3a (A). Furthermore, results from the same experiments performed at various concentrations of the highly effective MQP-37 confirmed that IFN-β expression levels decreased in a concentration-dependent manner. Figure 3a The results of the same experiments as above, performed on MQP-Y9 peptide at various concentrations, confirmed that the expression level of IFN-β decreased in a concentration-dependent manner. Figure 3b (C), and in the same experiment treating the MQP-T234 peptide, the expression level of IFN-β was also reduced (C). Figure 3b Based on the above results, it can be seen that MQP-15, MQP-23, MQP-31, MQP-37, MQP-Y9 or MQP-T234 prepared in this application effectively inhibit the antiviral response caused by viral infection.

[0121] Example 4: Confirmation of the cytotoxicity of the novel peptide

[0122] To confirm whether the anti-inflammatory activity of the novel peptide prepared in Example 1 above was a result of the peptide's own cytotoxicity, the following experiment was conducted.

[0123] Specifically, to confirm the cytotoxicity of the aforementioned peptides, the XTT assay, which measures intracellular mitochondrial activity, was used. First, mouse peritoneal macrophage lines were treated with serum-free cell culture medium supplemented with 5 mM MQP-15, MQP-23, MQP-31, or MQP-37 for 16 hours. Then, they were treated with XTT for 1 hour, and the amount of water-soluble formazan produced by reduction by dehydrogenases in the mitochondria was measured.

[0124] The results confirmed that the cytotoxicity of the novel peptide of this application did not occur within the concentration range used in the cell experiments. Figure 4 In particular, in the case of MQP-37 and MQP-Y9, peptides that have been shown to be very effective in inhibiting the inflammatory response induced by polyIC, no cytotoxicity was confirmed even at concentrations of 10 mM. Figure 4 (B and C). Therefore, it can be seen that the above-mentioned inflammatory response suppression effect is not due to its own cytotoxicity, and even if the above peptides are used as pharmaceuticals, they are harmless to individuals.

[0125] Example 5: Confirmation of the inflammasome response inhibitory efficacy of the novel peptide

[0126] To confirm the inhibitory effect of the novel peptide prepared in Example 1 above on the inflammasome response induced by bacterial infection, the following experiment was conducted.

[0127] Specifically, to induce an inflammasome response induced by bacterial infection, lipopolysaccharide (LPS) and outer membrane vesicles (OMV) derived from Gram-negative bacteria were used as endotoxins. First, serum-free cell culture medium containing 0.1 mg / ml LPS or OMV was supplemented with 5 mM MQP-15, MQP-23, MQP-31, or MQP-37, respectively, and 1×10⁻⁶ cells were added. 5 Mouse peritoneal macrophage cells were treated for 6 hours. Additionally, mouse peritoneal macrophage cells were treated for 5 hours (LPS treatment - 4 hours; Nigerian macrophage treatment - 1 hour) with serum-free cell culture medium supplemented with MQP-37 or MQP-Y9 containing 1 mg / ml LPS and 5 mM Nigerian macrophage. Afterwards, the supernatant was collected, and the expression level of interleukin-1β (IL-1β) was confirmed by ELISA. Cells were lysed for SDS-PAGE, and inflammasomes were blotted for proteomics.

[0128] The results confirmed that, compared with the control group treated with LPS or OMV alone, the expression level of IL-1b was reduced when treated with the novel peptide of this application. In particular, it was confirmed that, compared with other peptides, the expression level of IL-1b was significantly reduced in the case of MQP-37. Figure 5a A and Figure 5b Furthermore, results from the same experiments performed at various concentrations of the highly effective MQP-37 confirmed that the expression level of IL-1b decreased in a concentration-dependent manner after LPS treatment. Figure 5a (B). Treatment with LPS and Nigerian mycin confirmed that IL-1b production was inhibited in a concentration-dependent manner by MQP-Y9. Figure 5bFurthermore, it was confirmed that MQP-37 treatment inhibited caspase-1 activation. Figure 5c Based on the above results, it can be seen that the novel peptide prepared in this application effectively inhibits the inflammatory response caused by bacterial infection.

[0129] Example 6: Confirmation of the anti-inflammatory effect of the novel peptide

[0130] To confirm the inflammatory response-inhibiting effect of the novel peptide prepared in Example 1 above, the following experiments were conducted.

[0131] Specifically, LPS was used to induce an inflammatory response caused by bacterial infection. First, MQP-15, MQP-23, MQP-31, MQP-37, or MQP-Y9 were added to serum-free cell culture medium containing 0.1 mg / ml LPS, and then 1×10⁻⁶ cells were cultured. 5 Mouse peritoneal macrophage cells were treated for 6 hours. The supernatant was then collected, and the expression levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were confirmed using ELISA.

[0132] The results confirmed that, compared with the control group treated with LPS only, MQP-37 in the novel peptide of this application significantly reduced the expression levels of IL-6 and TNF-α. Figure 6a A and Figure 6b Furthermore, results from the same experiments performed at various concentrations of the highly effective MQP-37 confirmed that the expression level of IL-6 decreased in a concentration-dependent manner after LPS treatment. Figure 6a (B). Results from the same experiments performed with various concentrations of MQP-Y9 confirmed that the expression level of IL-6 decreased in a concentration-dependent manner after LPS treatment. Figure 6b (D). Based on the above results, it can be seen that the novel peptide prepared in this application effectively inhibits the inflammatory response.

[0133] Example 7: Confirmation of the inhibitory efficacy of the novel peptide on the immune activation mechanism

[0134] To confirm the inhibitory efficacy of the novel peptide prepared in Example 1 above on the immune activation mechanism, the following experiments were conducted.

[0135] Specifically, LPS was used to induce an immune activation response induced by bacterial infection, and nuclear factor κB phosphorylation was confirmed to establish 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 × 10⁻⁶ ions were added. 6 A mouse peritoneal macrophage cell line was treated for 6 hours. Afterwards, the cells were washed with PBS, lysed with lysis buffer (RIPA buffer), and the supernatant was separated. To determine changes in the phosphorylation level of nuclear factor κB protein using the separated supernatant, the same Western blotting procedure as in Example 2 was performed.

[0136] The results confirmed that, compared with the control group treated with LPS only, MQP-37 and MQP-Y9 significantly reduced the phosphorylation level of NF-κB in the novel peptides of this application. Figure 7 Based on the above results, it can be seen that the novel peptide prepared in this application effectively inhibits the immune activation response.

[0137] Example 8: Confirmation of improved proteolytic enzyme resistance in novel D-type peptides

[0138] To improve the resistance of the novel peptide MQP-37, whose anti-inflammatory activity was confirmed by experiments in Examples 2 to 7 above, to proteolytic enzymes, it was synthesized into D-type amino acids, and the following experiments were conducted to confirm the resistance to proteolytic enzymes.

[0139] Specifically, LPS was used to induce an inflammatory response induced by bacterial infection. First, 0.1 mg / ml of LPS was mixed into serum-free cell culture medium or 10% serum (fetal bovine serum, FBS) cell culture medium. Then, 5 mM of L-type MQP-37 peptide and 5 mM of D-type MQP-37 peptide were added to each cell culture medium containing LPS, and a 1×10⁻⁶ solution was applied. 5 Mouse peritoneal macrophage cells were treated for 6 hours. The supernatant was then collected, and the expression levels of interleukin-6 and tumor necrosis factor-α were confirmed using ELISA.

[0140] The results confirmed that, compared with the control group treated with LPS only, both the novel L-type and D-type peptides of this application exhibited the same anti-inflammatory response in serum-free cell culture medium, which is a condition without proteolytic enzymes. However, in cell culture medium containing 10% serum, which is a condition in the presence of proteolytic enzymes, the novel L-type peptide of this application did not exhibit anti-inflammatory activity, while the novel D-type peptide still retained anti-inflammatory activity. Figure 8Based on the above results, it can be seen that the novel D-type peptides prepared in this application effectively inhibit inflammatory responses, regardless of the presence or absence of proteolytic enzymes.

[0141] Example 9: Confirmation of the improved anti-inflammatory effect of the novel alanine-substituted peptide

[0142] To confirm the key amino acids of the novel peptides MQP-37 and MQP-Y9, whose anti-inflammatory activity was confirmed by the experiments in Examples 2 to 8 above, peptides were synthesized with each sequence replaced by alanine (MQP-37 alanine-substituted peptide [MQP-37 derivative] - SEQ ID NO. 5 to SEQ ID NO. 11; MQP-Y9 alanine-substituted peptide [MQP-Y9 derivative] - SEQ ID NO. 53 to SEQ ID NO. 62). The following experiments were conducted to confirm the anti-inflammatory effect.

[0143] Specifically, LPS and ATP were used to induce an inflammasome response induced by bacterial infection, and polyIC was used to induce an inflammatory response induced by viral infection. First, 1 mg / ml of LPS and 5 mM or 1 mM of alanine-substituted novel peptides were mixed in serum-free cell culture medium, and then 1 × 10⁻⁶ ATP was added. 5 Mouse peritoneal macrophage cell lines were treated. Then, 5 mM ATP and 5 mM or 1 mM 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-1β was confirmed using ELISA. Additionally, 5 mg / ml PolyIC and 25 mM or 2 mM alanine-substituted novel peptide were mixed in serum-free cell culture medium and treated with 1×10⁻⁶ cells. 5 A mouse lung epithelial cell line was treated for 16 hours. The supernatant was then collected, and the expression level of interleukin-1β was confirmed using ELISA.

[0144] The results confirmed that, compared with the original MQP-37 peptide, MQP-37 with amino acids at positions 1 and 6 replaced by alanine (A1 and A6) significantly improved the inflammasome response induced by bacterial infection and the inflammatory response induced by viral infection. Figure 9a (A and B). Furthermore, it was confirmed that MQP-Y9 (A2, A7, and A9), with amino acids substituted at positions 2, 7, and 9 for alanine, exhibited superior anti-inflammatory responses to inflammasomes induced by bacterial infection compared to the original strain. Figure 9b Furthermore, it was confirmed that MQP-Y9(A10), with the 10th amino acid replaced by alanine, more effectively suppressed the inflammatory response induced by viral infection compared to the original formulation. Figure 9cBased on the above results, it can be seen that the alanine-substituted novel peptide prepared in this application effectively inhibits the inflammasome response caused by bacterial infection and the inflammatory response caused by viral infection (SEQ ID NO.5 to SEQ ID NO.11; SEQ ID NO.53 to SEQ ID NO.62).

[0145] Example 10: Confirmation of the anti-inflammatory properties of novel peptides with the first and sixth amino acid sequences replaced by other amino acids. Effect

[0146] The novel peptide MQP-37A6 (SEQ ID NO. 9), which was confirmed to have anti-inflammatory activity by experiments in Example 9, was synthesized by substituting the first position with 19 amino acids other than serine (MQP-37A1 derivatives - SEQ ID NO. 12 to SEQ ID NO. 29, SEQ ID NO. 63). Furthermore, MQP-37A6 was synthesized by substituting the sixth position with 18 amino acids other than aspartic acid (MQP-37A6 derivatives - SEQ ID NO. 30 to SEQ ID NO. 47), and the following experiments were conducted to confirm the anti-inflammatory effect of the above peptides.

[0147] Specifically, LPS and ATP were used to induce an inflammasome response induced by bacterial infection, and polyIC was used to induce an inflammatory response induced by viral infection. First, 1 mg / ml of LPS and 2 mM of a novel amino acid-substituted peptide were mixed in serum-free cell culture medium, and then 1 × 10⁻⁶ LPS was added to the culture medium. 5 A mouse peritoneal macrophage cell line was treated. Then, 5 mM ATP and 2 mM amino acid-substituted novel peptides were mixed in serum-free cell culture medium and treated for 30 minutes. The supernatant was then collected, and the expression level of interleukin-1β was confirmed using ELISA. Additionally, 5 mg / ml PolyIC and 2 mM amino acid-substituted novel peptides were mixed in serum-free cell culture medium and treated with 1×10⁻⁶ cells. 5 A mouse lung epithelial cell line was treated for 16 hours. The supernatant was then collected, and the expression level of interleukin-1β was confirmed using ELISA.

[0148] The results confirmed that MQP-37A1 with the first amino acid replaced by alanine most effectively inhibited the inflammatory response induced by bacterial infection, and that even substitutions with phenylalanine (F), leucine (L), arginine (R), and tyrosine (Y) effectively inhibited the inflammasome response induced by bacterial infection. Figure 10a(A). Furthermore, it was confirmed that when the sixth amino acid of MQP-37 is replaced with another amino acid, such as... Figure 10a As shown in Figure B, alanine (A), phenylalanine (F), histidine (H), leucine (L), and methionine (M) effectively inhibited the inflammasome response induced by bacterial infection, with the novel peptide substituted with alanine showing the most effective inhibition. It was also confirmed that in the inflammatory response induced by viral infection, compared with the original peptide, replacing the sixth amino acid of MQP-37 with tryptophan (W) or tyrosine (Y) effectively inhibited the inflammatory response induced by viral infection, even at low peptide concentrations. Figure 10b (C). Based on the above results, it can be seen that the novel amino acid-substituted peptides prepared in this application effectively inhibit inflammatory responses caused by bacterial infections and viral infections.

[0149] Example 11: Comparative confirmation of the binding affinity between the novel peptide MQP-37 and the alanine-substituted novel peptide MQP-37A6

[0150] To compare the binding affinity of the alanine-substituted novel peptide MQP-37A6, whose anti-inflammatory activity was confirmed by experiments in Examples 9 and 10 above, to MAVS protein with that of the original peptide MQP-37, the following experiments were conducted.

[0151] Specifically, to confirm the binding affinity to MAVS protein, recombinant MAVS protein and fluorescently labeled MQP-37 and MQP-37A6 peptides were used. First, 250 ng of recombinant MAVS protein was plated onto a 96-well ELISA plate and then blocked with 1% BSA / PBS solution. Afterward, various concentrations of fluorescently labeled peptides were added and reacted for 2 hours. The amount of peptide binding to MAVS protein was then confirmed by fluorescence assay.

[0152] The results showed that when MQP-37 was treated with different concentrations and its binding affinity to MAVS protein was measured, the measured binding affinity (K0) was significantly different. d The binding capacity was approximately 16 mM, while in the case of MQP-37A6, the measured binding capacity was approximately 0.75 mM, confirming that it has approximately 20 times stronger binding capacity. Figure 11 Based on the above results, it can be seen that the binding ability of the novel peptide MQP-37A6 prepared in this application to MAVS protein has been improved.

[0153] Example 12: Comparative confirmation of the physiological activities of novel peptide MQP-37 and alanine-substituted novel peptide MQP-37A6

[0154] 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 above, with that of the original peptide MQP-37, the following experiments were conducted.

[0155] Specifically, LPS was used to induce an inflammatory response caused by bacterial infection. First, MQP-37 or MQP-37A6 was added to serum-free cell culture medium containing 0.1 mg / ml LPS, and then... 5 Mouse peritoneal macrophage cells were treated for 6 hours. Afterwards, the supernatant was collected, and the expression level of interleukin-6 was confirmed using ELISA. Furthermore, to confirm the mechanism of immune activation, Western blotting was performed, and nuclear factor κB phosphorylation was confirmed. Next, LPS and ATP were used to induce an inflammasome response induced by bacterial infection. First, 1 mg / ml of LPS and L- or D-type MQP-37 and MQP-37A6 were mixed in serum-free cell culture medium, and then... 5 A mouse peritoneal macrophage cell line was processed. Then, 5 mM ATP and MQP-37 or MQP-37A6 were mixed in serum-free cell culture medium and treated for 30 minutes. The supernatant was collected, and the expression level of interleukin-1β was confirmed using ELISA.

[0156] The results confirmed that, compared with the original MQP-37, both L-type and D-type MQP-37A6, with the 6th amino acid replaced by alanine, more effectively inhibited the inflammatory response induced by bacterial infection. Figure 12a A) Inflammatory response ( Figure 12a (B and C) and immune activation responses (NF-κB phosphorylation, Figure 12b (D to F). Based on the above results, it can be seen that the alanine-substituted novel peptide (MQP-37A6) prepared in this application effectively inhibits the inflammatory response.

[0157] Example 13: Comparative confirmation of the novel peptide MQP-37 and amino acid-substituted novel peptides MQP-37D6Y, MQP-37D6W. Viral inflammatory response of MQP-37D6H

[0158] To compare the activity of the novel amino acid-substituted peptides MQP-37D6Y and MQP-37D6W, whose antiviral inflammatory response activity was confirmed by experiments in Example 10 above, with that of the original peptide MQP-37, the following experiments were conducted.

[0159] Specifically, polyIC was used to induce a viral infection response. First, 1×10 5Mouse lung epithelial cell lines were treated with serum-free cell culture medium supplemented with 5 mg / ml polyIC and MQP-37, MQP-37A6, MQP-37D6W, MQP-37D6Y, MQP-37D6H, MQP-37D6K, MQP-37D6R, or MQP-37D6V for 16 hours. Afterwards, the supernatant was collected, and the expression level of interferon-β was confirmed using enzyme-linked immunosorbent assay (ELISA).

[0160] The results confirmed that, compared with MQP-37 and MQP-37A6, MQP-37D6W and MQP-37D6Y showed a concentration-dependent superior inhibitory effect on the inflammatory response induced by viral infection. Figure 13a (A and B), and further, it was confirmed that MQP-37D6Y effectively inhibited polyIC-induced apoptosis. Figure 13b The E), and at the maximum concentration of MQP-37D6Y used in the experiment, did not show cytotoxicity (E), and did not exhibit cytotoxicity (E). Figure 13b Furthermore, it was confirmed that MQP-37D6H also exhibited concentration-dependent inhibition of the inflammatory response induced by viral infection. Figure 13a (C and D). Based on the above results, it can be seen that the novel amino acid-substituted peptides, including MQP-37D6Y, prepared in this application effectively inhibit the inflammatory response caused by viral infection.

[0161] Example 14: Confirmation of the efficacy of the novel peptide in a mouse sepsis model

[0162] To confirm the efficacy of D-type or L-type MQP-37 and MQP-37A6, which have been shown to inhibit the inflammatory response caused by bacterial infection in the above-described Examples 2 to 12, in a mouse sepsis animal model, the following experiments were conducted.

[0163] Specifically, 7-week-old C57BL / 6 wild-type mice were intraperitoneally administered LPS at doses of 2 mg / kg and 5 mg / kg every 6 hours. One hour later, the mice were also intraperitoneally administered novel peptides at doses of 0.4 mg / kg, 2 mg / kg, and 10 mg / kg. Mice were then checked for survival over 4 days.

[0164] The results confirmed that a survival rate of 40-80% was observed when administered 0.4 mg / kg, while 100% of mice survived when administered 2 mg / kg and 10 mg / kg. Figure 14 (A through C). Furthermore, it was confirmed that, compared to the control group, the level of IL-6, an inflammatory cytokine, in the blood was reduced first in the novel peptide treatment group ( Figure 14Based on the above results, it can be seen that type D or type L MQP-37 and MQP-37A6 have therapeutic effects on sepsis, which is an inflammatory disease.

[0165] The above description of this application is for illustrative purposes. Those skilled in the art will understand that it can be easily modified into other specific forms without altering the technical concept or essential features of this application. Therefore, it should be understood that the above embodiments are exemplary in all respects and not restrictive. For example, components described as a single type can be implemented separately, and similarly, components described as distributed can be implemented in combination.

[0166] The scope of this application is defined by the appended claims, rather than by the detailed description above. It should be understood that all alterations or modifications derived from the meaning and scope of the claims and their equivalents are included within the scope of this application. <110> Mitoquist Ltd. <120> Novel anti-inflammatory peptides and their uses <130> OP20210090KR <150> KR 10-2020-0064483 <151> 2020-05-28 <160> 63 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> MQP-15 <400> 1 Leu Lys Ser Leu Lys Thr Leu His 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <220> <223> MQP-23 <400> 2 Leu Gln His Leu Glu Asp Gly Met 1 5 <210> 3 <211> 6 <212> PRT <213> Artificial sequence <220> <223> MQP-31 <400> 3 Asp Gly Thr Glu Cys Arg 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37 <400> 4 Ser Leu Val Leu Ala Asp Ala Arg Trp 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37A1 <400> 5 Ala Leu Val Leu Ala Asp Ala Arg Trp 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37A2 <400> 6 Ser Ala Val Leu Ala Asp Ala Arg Trp 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37A3 <400> 7 Ser Leu Ala Leu Ala Asp Ala Arg Trp 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37A4 <400> 8 Ser Leu Val Ala Ala Asp Ala Arg Trp 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37A6 <400> 9 Ser Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37A8 <400> 10 Ser Leu Val Leu Ala Asp Ala Ala Trp 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37A9 <400> 11 Ser Leu Val Leu Ala Asp Ala Arg Ala 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1C <400> 12 Cys Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1D <400> 13 Asp Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1E <400> 14 Glu Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1F <400> 15 Phe Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1G <400> 16 Gly Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1H <400> 17 His Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1I <400> 18 Ile Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1K <400> 19 Lys Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1L <400> 20 Leu Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1M <400> twenty one Met Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> twenty two <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1N <400> twenty two Asn Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> twenty three <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1P <400> twenty three Pro Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> twenty four <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1Q <400> twenty four Gln Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1R <400> 25 Arg Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1T <400> 26 Thr Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1V <400> 27 Val Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1W <400> 28 Trp Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37S1Y <400> 29 Tyr Leu Val Leu Ala Ala Ala Arg Trp 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6C <400> 30 Ser Leu Val Leu Ala Cys Ala Arg Trp 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6E <400> 31 Ser Leu Val Leu Ala Glu Ala Arg Trp 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6F <400> 32 Ser Leu Val Leu Ala Phe Ala Arg Trp 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6G <400> 33 Ser Leu Val Leu Ala Gly Ala Arg Trp 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6H <400> 34 Ser Leu Val Leu Ala His Ala Arg Trp 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6I <400> 35 Ser Leu Val Leu Ala Ile Ala Arg Trp 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6K <400> 36 Ser Leu Val Leu Ala Lys Ala Arg Trp 1 5 <210> 37 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6L <400> 37 Ser Leu Val Leu Ala Leu Ala Arg Trp 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6M <400> 38 Ser Leu Val Leu Ala Met Ala Arg Trp 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6N <400> 39 Ser Leu Val Leu Ala Asn Ala Arg Trp 1 5 <210> 40 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6P <400> 40 Ser Leu Val Leu Ala Pro Ala Arg Trp 1 5 <210> 41 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6Q <400> 41 Ser Leu Val Leu Ala Gln Ala Arg Trp 1 5 <210> 42 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6R <400> 42 Ser Leu Val Leu Ala Arg Ala Arg Trp 1 5 <210> 43 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6S <400> 43 Ser Leu Val Leu Ala Ser Ala Arg Trp 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6T <400> 44 Ser Leu Val Leu Ala Thr Ala Arg Trp 1 5 <210> 45 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6V <400> 45 Ser Leu Val Leu Ala Val Ala Arg Trp 1 5 <210> 46 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6W <400> 46 Ser Leu Val Leu Ala Trp Ala Arg Trp 1 5 <210> 47 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MQP-37D6Y <400> 47 Ser Leu Val Leu Ala Tyr Ala Arg Trp 1 5 <210> 48 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-Y9 <400> 48 Lys Arg Leu Leu Phe Trp Ile Phe Ile Lys 1 5 10 <210> 49 <211> 7 <212> PRT <213> Artificial sequence <220> <223> MQP-91 <400> 49 Gln Met Val Ser Met Val Gly 1 5 <210> 50 <211> 12 <212> PRT <213> Artificial sequence <220> <223> MQP-T234 <400> 50 Lys Val Gly Asp Arg Ala Arg Trp Gly Ser Arg Ser 1 5 10 <210> 51 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-S442 <400> 51 Leu Glu Arg Gln Ser Arg Ser Trp Arg Asn 1 5 10 <210> 52 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-341 <400> 52 Glu Trp Leu Gly Arg Gly Arg Phe Asn Gly 1 5 10 <210> 53 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-Y9A1 <400> 53 Ala Arg Leu Leu Phe Trp Ile Phe Ile Lys 1 5 10 <210> 54 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-Y9A2 <400> 54 Lys Ala Leu Leu Phe Trp Ile Phe Ile Lys 1 5 10 <210> 55 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-Y9A3 <400> 55 Lys Arg Ala Leu Phe Trp Ile Phe Ile Lys 1 5 10 <210> 56 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-Y9A4 <400> 56 Lys Arg Leu Ala Phe Trp Ile Phe Ile Lys 1 5 10 <210> 57 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-Y9A5 <400> 57 Lys Arg Leu Leu Ala Trp Ile Phe Ile Lys 1 5 10 <210> 58 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-Y9A6 <400> 58 Lys Arg Leu Leu Phe Ala Ile Phe Ile Lys 1 5 10 <210> 59 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-Y9A7 <400> 59 Lys Arg Leu Leu Phe Trp Ala Phe Ile Lys 1 5 10 <210> 60 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-Y9A8 <400> 60 Lys Arg Leu Leu Phe Trp Ile Ala Ile Lys 1 5 10 <210> 61 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-Y9A9 <400> 61 Lys Arg Leu Leu Phe Trp Ile Phe Ala Lys 1 5 10 <210> 62 <211> 10 <212> PRT <213> Artificial sequence <220> <223> MQP-Y9A10 <400> 62 Lys Arg Leu Leu Phe Trp Ile Phe Ile Ala 1 5 10 <210> 63 <211> 9 <212> PRT <213> artificial sequence <220> <223> MQP‑37S1A <400> 63 Ala Leu Val Leu Ala Ala Ala Arg Trp 1 5

Claims

1. An anti-inflammatory peptide, characterized in that, the anti-inflammatory peptide consists of an amino acid sequence represented by X1LVLAX6ARW, wherein the amino acid X1 is S, and the amino acid X6 is D, A, F, G, H, I, L, M, Q, S, T or W, or, the amino acid X6 is A, and the amino acid X1 is A, C, F, H, I, L, M, R or Y.

2. A polynucleotide, comprising, a polynucleotide encoding the anti-inflammatory peptide of claim 1.

3. A pharmaceutical composition, characterized by, a pharmaceutical composition comprising the anti-inflammatory peptide of claim 1 as an active ingredient.

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