Novel peptide and uses thereof

Abstract

The present invention relates to a novel inflammation-regulating peptide having an inflammation-modulating function and uses thereof. The peptide of the present invention can be advantageously utilized for the treatment of inflammation, various immune-related diseases, or cancers by regulating the activity of immune cells.

Description

Novel peptides and their uses

[0001] This patent application claims priority to patent application No. 10-2024-0082914, filed with the Korean Intellectual Property Office on June 25, 2024, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a novel peptide, a pharmaceutical composition for preventing or treating inflammatory diseases or cancer, and an anti-inflammatory cosmetic composition comprising the same.

[0003] Cells receive various information transmitted from the outside through receptors present on the cell membrane and reflect it in the physiological activity or function of the cell through the signal transduction system. Cell membrane receptors exist in various forms. G protein-coupled receptors (GPCRs), also called 7-transmembrane receptors (7TMR), are receptors that have a structure that crosses the membrane seven times across the inside and outside of the cell. It is known that more than 800 GPCRs exist in the body, and they are largely classified into six types (Class A to F) according to their shape. Their functions are very wide, including detecting light, smell, taste, hormones, biological substances, and infectious agents. Receptors with unknown functions are referred to as orphan GPCRs. The substance that binds to the receptor is called a ligand, and is divided into agonists that promote receptor signaling and antagonists that inhibit it.

[0004] The receptors of the FPR (N-Formyl-Peptide Receptor) family are Class A type G protein-coupled receptors that are mainly present in immune cells and mediate various cellular functions through lower signal transduction systems. The name FPR was given when it was known that they are receptors that bind to N-formyl peptides, which are mainly present in mitochondria, which are intracellular organelles, or bacteria. In humans, FPR1 to 3 isotypes are known, and among them, FPR2 / ALX is known to be a receptor that binds to a biolipid substance called Lipoxin A4, and is written as 'FPR2 / ALX'. Functionally, FPR1 primarily binds to bacterial-derived formyl peptides to induce acute inflammatory responses and defense against infections, while FPR2 / ALX binds to various forms of biological ligands, including peptides, proteins, and lipids, and mediates anti-inflammatory effects as a receptor for inflammatory response regulators, such as LXA4, AnxA1, Ac2-26, and RvD1.

[0005] Numerous studies using gene-deleted mice or ligands have elucidated the function of FPR2 in diseases arising in various tissues, including the heart, lungs, liver, intestines, nervous tissue (brain and spinal cord), skin, and eyes. Furthermore, FPR2 activation has been shown to improve disease outcomes. FPR2 has recently emerged as a key target for therapeutic development. In particular, active efforts are being made to develop FPR2-targeted therapeutics for inflammatory diseases. The target diseases are inflammatory diseases that appear in various tissues such as the skin, respiratory system, digestive system, eye, musculoskeletal system, and circulatory system due to local or systemic immune response abnormalities, such as atopic dermatitis, psoriasis, asthma, lung injury, lung inflammation, conjunctivitis, keratitis, dry eye syndrome, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, and systemic sclerosis. In particular, clinical trials of peptides, proteins, or small molecule compounds targeting FPR2 are in progress for atopic dermatitis, ocular inflammation, dry eye, heart failure, myocardial infarction, cardiovascular thrombosis, and Crohn's disease (Non-patent Document 1).

[0006] FPR2 plays a crucial role in promoting anti-inflammatory action, thereby regenerating damaged tissues and restoring structural and functional homeostasis. Therefore, activating FPR2 can suppress tissue injury caused by various stimuli, such as inflammatory responses, drug toxicity, exposure to toxic substances, infections, and wounds, and accelerate the recovery of normal tissue structure and function. It is also effective in preventing or improving fibrosis, which occurs during persistent and excessive inflammatory responses or incomplete healing after injury. In this regard, FPR2 activation has been proven to be effective in suppressing tissue damage and improving fibrosis in various tissues, including the lungs, liver, kidneys, heart, small intestine, skin, and brain, and related research is actively underway. Furthermore, new drugs targeting FPR2 are being developed to treat tissue damage or fibrosis.

[0007] Meanwhile, FPR2 not only regulates inflammatory responses but also plays a role in regulating immune cell activity, which may influence cancer progression and the inflammatory response of the cancer microenvironment. Chronic inflammation is widely known as a major risk factor for cancer development, and inflammation in the cancer microenvironment caused by rapid cancer growth or anticancer drugs is closely related to the responsiveness and prognosis of anticancer drugs. In particular, the inflammatory response can promote cancer cell growth, survival, proliferation, invasion, and metastasis. In this regard, regulating the inflammatory response through FPR2 activation can suppress chronic inflammation or inflammation in the cancer microenvironment, thereby inhibiting cancer development and progression. Furthermore, FPR2 activation can enhance the immune response against cancer by modulating the activity of immune cells, and in particular, it can promote phagocytosis of cancer cells and contribute to the elimination of cancer cells through cytotoxic reactions (Non-patent Document 2).

[0008] In prior research, the inventors of the present invention developed a peptide with antibacterial activity and secured a patent (Patent Document 1) and also developed a peptide with excellent antibacterial and immune function-modulating capabilities and secured a patent (Patent Document 2). In addition to these prior patents, the inventors of the present invention have continued research and development on peptides targeting FPR2, and have invented a novel peptide ligand that can be useful in the treatment of inflammation, various immune diseases, tissue damage, fibrotic diseases, and cancer by activating FPR2 and suppressing inflammatory responses and modulating immune cell activity.

[0009] Prior art documents for the present invention include Patent Document 1: KR Registered Patent No. 10-1855170, Patent Document 2: KR Registered Patent No. 10-2170236, Non-Patent Document 1: N-Formyl Peptide Receptor 2 (FMLP Related Receptor I or Formyl Peptide Receptor Like 1 or HM63 or Lipoxin A4 Receptor or RFP or FPR2) - Pipeline Review, H2 2019, Non-Patent Document 2: 2018 1. JEM_Resolvins suppress tumor growth and enhance cancer therapy.

[0010] The present invention aims to provide a novel peptide having an inflammatory response control function and an inflammatory disease treatment effect by activating FPR2.

[0011] In addition, the present invention aims to provide various uses of the peptide, such as a pharmaceutical composition for preventing or treating inflammatory diseases or cancer and an anti-inflammatory cosmetic composition.

[0012] To achieve the above purpose, the present invention provides a peptide composed of an amino acid sequence represented by the following general formula 1:

[0013] [General Formula 1]

[0014] X1-X2-X3-X4-X5-X6-m

[0015] In the above general formula, X1 is proline (P) or histidine (H), X2 is isoleucine (I), X3 is arginine (R) or lysine (K), X4 is tyrosine (Y), X5 is lysine (K), X6 is valine (V) or proline (P), and m is D-type methionine.

[0016] In one embodiment of the present invention, a pharmaceutical composition for preventing or treating an inflammatory disease is provided, comprising the peptide as an active ingredient.

[0017] In another embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer is provided, comprising the peptide as an active ingredient.

[0018] In another embodiment of the present invention, an anti-inflammatory cosmetic composition comprising the peptide as an active ingredient is provided.

[0019] The novel peptide according to the present invention has the function of controlling inflammatory response and regulating immune cell activity due to FPR2 activation, and therefore can be utilized as a pharmaceutical composition for treating inflammatory diseases, immune diseases, or cancer, or an anti-inflammatory cosmetic composition containing the peptide.

[0020] Figure 1 shows an analysis of the FPR2 activation ability of the peptide.

[0021] The present invention relates to a peptide comprising an amino acid sequence represented by the following general formula 1:

[0022] [General Formula 1]

[0023] X1-X2-X3-X4-X5-X6-m

[0024] In the above general formula, X1 is proline (P) or histidine (H), X2 is isoleucine (I), X3 is arginine (R) or lysine (K), X4 is tyrosine (Y), X5 is lysine (K), X6 is valine (V) or proline (P), and m is D-type methionine.

[0025] Throughout this specification, whenever a part is said to “include” a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0026] Additionally, when a numerical range is disclosed herein, such range is continuous and includes all values ​​from the minimum value to the maximum value inclusive, unless otherwise specified.

[0027] Throughout this specification, “%” used to indicate the concentration of a particular substance is (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise noted.

[0028] Hereinafter, the present invention will be described in detail.

[0029] The present invention provides a novel peptide that binds to the FPR2 receptor and performs the function of controlling an inflammatory response.

[0030] In one aspect of the present invention, a peptide is provided comprising an amino acid sequence represented by the following general formula 1:

[0031] [General Formula 1]

[0032] X1-X2-X3-X4-X5-X6-m

[0033] In the above general formula, X1 is proline (P) or histidine (H), X2 is isoleucine (I), X3 is arginine (R) or lysine (K), X4 is tyrosine (Y), X5 is lysine (K), X6 is valine (V) or proline (P), and m is D-type methionine.

[0034] The term "peptide" as used herein refers to a polymer composed of one or more amino acids linked by amide bonds (or peptide bonds), and includes peptides as well as pharmaceutically acceptable salts of peptides. Typically, the amino terminus (or N-terminus) of a peptide is indicated at the left end of the described amino acid sequence of the peptide, and the carboxy terminus (or C-terminus) is indicated at the right end of the described amino acid sequence. The amino acid sequence of a peptide may be indicated by a single letter symbol to indicate amino acids covalently linked by peptide bonds. If the D-conformation is not specifically indicated, the L-conformation may be assumed, and the amino acid residues constituting the peptide may be natural or non-natural amino acid residues.

[0035] The peptide of the present invention can be obtained using various methods widely known in the art. For example, the peptide can be produced by extracting a protein from a living body and then treating it with a protease to reduce its molecular weight, by biologically producing it using genetic recombination and a protein expression system, by in vitro synthesis through chemical synthesis such as peptide synthesis, or by cell-free protein synthesis.

[0036] In the present invention, one or more amino acids of the peptide may be chemically modified to obtain better chemical stability, enhanced pharmacological properties (e.g., half-life, absorbability, potency, efficacy, etc.), altered specificity (e.g., a broad spectrum of biological activity), and reduced antigenicity. The term "stability" is used to mean not only in vivo stability, which protects the peptide of the present invention from attack by in vivo protein-cleaving enzymes, but also storage stability (e.g., room temperature storage stability).

[0037] In one embodiment of the present invention, the peptide may have high stability in vivo, but is not limited thereto. High stability in vivo may, for example, mean that the peptide remains in an active form in plasma for a relatively long time when dissolved in plasma. The organism may include any mammal, such as humans and primates, as well as livestock such as cows, pigs, sheep, horses, dogs, and cats, without limitation, but may preferably be humans.

[0038] For example, a protecting group may be added to the N-terminus or C-terminus of the peptide of the present invention. Preferably, the protecting group may be an acetyl group, a butanoyl group (butyryl group), a hexanoyl group, an octanoyl group, a fluorenylmethoxycarbonyl group, a palmitoyl group, a myristoyl group, a stearyl group, a butoxycarbonyl group, an aryloxycarbonyl group, or polyethylene glycol (PEG), but any component that can modify the peptide, particularly enhance the stability of the peptide, may be included without limitation.

[0039] In the present invention, the N-terminal amino acid of the peptide may be alkylated or acylated at the N-terminal amino group (H2N-). For example, the N-terminal amino group of the peptide may be acylated to include an aliphatic acyl group (i.e., including an acetyl group, a myristoyl group, a butanoyl group, etc.), a carboxylic acid, a benzoic acid, a trifluoroacetic acid, or a sulfonic acid. As another example, the N-terminal amino group of the peptide may be alkylated by an aliphatic alkyl, a halide, or an aliphatic alkynesulfonic acid ester.

[0040] In the present invention, the C-terminal carboxyl group (-COOH) of the peptide may be amidated or esterified. For example, the C-terminal carboxyl group may be chemically modified by forming an amide with an amine. In another example, the C-terminal carboxyl group may be chemically modified by forming an ester with an alcohol.

[0041] Additionally, in the present invention, the side chains of the amino acids of the peptide may be chemically modified. For example, the phenyl group in tyrosine may be substituted with, but is not limited to, an aliphatic alkyl group, an aliphatic carboxyl group, or an alkoxy group. The epsilon amino group in lysine may be chemically modified, for example, by amide formation with an aliphatic carboxylic acid group, a benzoic acid group, or an amino acid group. Furthermore, the epsilon amino group in lysine may be chemically modified by, but is not limited to, alkylation of one or two C1 to C4 aliphatic alkyl groups.

[0042] In one embodiment of the present invention, any one selected from the group consisting of an acetyl group, a butanoyl group, a hexanoyl group, and an octanoyl group may be added to the N-terminus of the peptide.

[0043] In one embodiment of the present invention, the C-terminus of the peptide may be amidated.

[0044] In one embodiment of the present invention, the peptide may be composed of any one of the amino acid sequences of SEQ ID NOs: 1 to 32.

[0045] In the present specification, the peptide can act as an immunomodulatory peptide, such as directly or indirectly regulating the inflammatory response of immune cells, immune cell migration, and regulating the inflammatory response of epidermal or endothelial cells. In the present invention, the inflammation-regulating peptide can induce an inflammation-resolution response by activating formyl peptide receptor 2 (FPR2) expressed in immune cells such as phagocytic cells such as neutrophils and monocytes, antigen-presenting cells such as dendritic cells, eosinophils, basophils, mast cells, microglia, and neurons, epidermal cells, endothelial cells, etc., thereby exhibiting the functions of inflammation regulation, tissue regeneration, and homeostasis recovery.

[0046] As confirmed in the experimental examples below, the peptide according to the present invention showed high FPR2 activation efficacy even at low concentrations, and was also confirmed to have high selectivity compared to FPR1.

[0047] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising the peptide as an active ingredient, a use of the peptide for preventing or treating an inflammatory disease, and a method for preventing or treating an inflammatory disease comprising administering the peptide to a subject.

[0048] As used herein, "inflammatory disease" refers to any condition in which an inflammatory response serves as the primary pathological mechanism of a disease or plays a significant role in its progression. Inflammation is a process in which the body's immune system is activated in response to stimuli such as infection or injury, and can be classified as acute or chronic. Furthermore, inflammation can induce tissue remodeling due to immune activation and result in the replacement of normal tissue with connective tissue. Therefore, inflammatory disease encompasses not only infectious, autoimmune, allergic, or non-infectious inflammatory diseases in which inflammation itself is the core of the disease, but also tissue damage and fibrosis diseases in which inflammation persists for a long time, resulting in structural and functional changes in tissues.

[0049] In one embodiment of the present invention, the inflammatory disease is atopic dermatitis, psoriasis, infantile eczema, sepsis, conjunctivitis, keratitis, ocular inflammatory disease, dry eye syndrome, asthma, lung injury, lung inflammation, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, peritonitis, systemic sclerosis, heart failure, myocardial infarction, or cardiovascular thrombosis, neuroinflammation, Parkinson's disease, Alzheimer's disease, cirrhosis, intestinal fibrosis, renal fibrosis, glomerulonephritis, cardiac hypertrophy, cardiac fibrosis, pulmonary fibrosis, acute liver injury, subarachnoid hemorrhage, intestinal mucosal wound, acute kidney injury, myocardial ischemia-reperfusion injury,This may include, but is not limited to, acute lung injury or lung ischemia-reperfusion injury.

[0050] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of atopic dermatitis. Further details are disclosed in Baxendell et al. J Immunol May 1, 2018, 200 (1 Supplement) 105.4;

[0051] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of psoriasis. Further details are disclosed in Liu et al. Sci Rep. 2017 Aug 2;7(1):7100. doi: 10.1038 / s41598-017-07485-1.

[0052] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of infantile eczema. Further details are disclosed in Wu et al. Br J Dermatol. 2013 Jan;168(1):172-8. doi: 10.1111 / j.1365-2133.2012.11177.x.

[0053] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of sepsis. Further details are disclosed in Walker et al. SHOCK, Vol. 36, No. 4, pp. 410Y416, 2011.

[0054] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of conjunctivitis. Further details are disclosed in Hodges et al. Mucosal Immunol. 2017 Jan;10(1):46-57. doi: 10.1038 / mi.2016.33. Epub 2016 Apr 13.

[0055] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of keratitis. Further details are disclosed in Zhu et al. Int Immunopharmacol. 2021 Jul;96:107785. doi: 10.1016 / j.intimp.2021.107785. Epub 2021 May 24.

[0056] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of uveitis (ocular inflammation). Further details are disclosed in AU 2018203719 B2.

[0057] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of dry eye. Further details are disclosed in Gao et al. J Immunol. 2015 Oct 1;195(7):3086-99. doi: 10.4049 / jimmunol.1500610. Epub 2015 Aug 31.

[0058] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of asthma. Further details are disclosed in Barnig et al. Eur Respir Rev. 2015 Mar;24(135):141-53. doi: 10.1183 / 09059180.00012514.

[0059] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for preventing or treating lung damage. Further details are disclosed in Ba et al. J Thorac Dis. 2019 Aug;11(8):3599-3608. doi: 10.21037 / jtd.2019.08.86.

[0060] In the present invention, the inflammation-regulating peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of pulmonary inflammation. Further details are disclosed in Sekheri et al. Proc Natl Acad Sci US A. 2020 Apr 7;117(14):7971-7980. doi: 10.1073 / pnas.1920193117. Epub 2020 Mar 23.

[0061] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of rheumatoid arthritis. Further details are disclosed in Kao et al. Br J Pharmacol. 2014 Sep;171(17):4087-96. doi: 10.1111 / bph.12768.

[0062] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of ankylosing spondylitis. Further details are disclosed in Kao et al. Br J Pharmacol. 2014 Sep;171(17):4087-96. doi: 10.1111 / bph.12768.

[0063] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of ulcerative colitis or Crohn's disease. For further details, see Kim et al. Exp Mol Med. 2013 Sep 13;45(9):e40. doi: 10.1038 / emm.2013.77.

[0064] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of scleroderma. Further details are disclosed in Park et al. Front Immunol. 2019 Sep 3;10:2095. doi: 10.3389 / fimmu.2019.02095. eCollection 2019.

[0065] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for preventing or treating heart failure. Further details are disclosed in Perretti, Mauro, and Catherine Godson. British journal of pharmacology vol. 177,20 (2020): 4595-4600. doi:10.1111 / bph.15212.

[0066] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of myocardial infarction. Further details are disclosed in Garcia, Ricardo A et al. JACC. Basic to translational science vol. 6, 8 676-689. 23 Aug. 2021, doi:10.1016 / j.jacbts.2021.07.007.

[0067] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of thrombosis. Further details are disclosed in Senchenkova, Elena Y et al. Circulation vol. 140,4 (2019): 319-335. doi:10.1161 / CIRCULATIONAHA.118.039345.

[0068] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of neuroinflammation. Further details are disclosed in Ponce et al. Front Aging Neurosci. 2022 Feb 17;14:780811. doi: 10.3389 / fnagi.2022.780811. eCollection 2022.

[0069] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of Parkinson's disease. Further details are disclosed in Krashia et al. Nat Commun. 2019 Sep 2;10(1):3945. doi: 10.1038 / s41467-019-11928-w.

[0070] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of Alzheimer's disease. Further details are disclosed in Pamplona et al. Transl Psychiatry. 2022 Oct 10;12(1):439. doi: 10.1038 / s41398-022-02208-1.

[0071] In the present invention, the peptide can inhibit tissue damage caused by various stimuli and accelerate the recovery of normal tissue structure and function by enhancing the anti-inflammatory action through FPR2 receptor activation and promoting the biological healing action.

[0072] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating tissue damage and fibrotic diseases, comprising the peptide as an active ingredient, a use of the peptide for preventing or treating tissue damage and fibrotic diseases, and a method for preventing or treating tissue damage and fibrotic diseases, comprising administering the peptide to a subject.

[0073] In one embodiment of the present invention, the tissue damage and fibrotic disease may occur in, but is not limited to, the lung, liver, kidney, heart, intestine, skin, and brain.

[0074] In one embodiment of the present invention, the tissue damage and fibrosis disease may be, but is not limited to, cirrhosis, intestinal fibrosis, renal fibrosis, glomerulonephritis, cardiac hypertrophy, cardiac fibrosis, pulmonary fibrosis, systemic sclerosis, acute liver injury, subarachnoid hemorrhage, intestinal mucosal wound, acute kidney injury, myocardial ischemia-reperfusion injury, acute lung injury, or lung ischemia-reperfusion injury.

[0075] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of liver cirrhosis. Further details are disclosed in Kurtoðlu EL et al. Turk J Gastroenterol. 2019 Aug;30(8):745-757. doi: 10.5152 / tjg.2019.18276.

[0076] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of intestinal fibrosis. Further details are disclosed in Xu, C et al. TurkSci Rep7, 16351 (2017). https: / doi.org / 10.1038 / s41598-017-16753-z.

[0077] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of renal fibrosis. Further details are disclosed in Neymeyer H et al. Acta Physiol (Oxf). 2015 Nov;215(3):144-58. doi: 10.1111 / apha.12586. Epub 2015 Sep 22.

[0078] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of glomerulonephritis. Further details are disclosed in Labes R et al. Front Physiol. 2022 Oct 12;13:984362. doi: 10.3389 / fphys.2022.984362.

[0079] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of cardiac hypertrophy or cardiac fibrosis. Further details are disclosed in Wang M et al. Drug Des Devel Ther. 2023 Oct 11;17:3073-3083. doi: 10.2147 / DDDT.S421894.

[0080] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of pulmonary fibrosis. Further details are disclosed in Martins V et al. J Immunol. 2009 May 1;182(9):5374-81. doi: 10.4049 / jimmunol.0802259.

[0081] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of scleroderma. Further details are disclosed in Park et al. Front Immunol. 2019 Sep 3;10:2095. doi: 10.3389 / fimmu.2019.02095. eCollection 2019.

[0082] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for preventing or treating acute liver damage. Further details are disclosed in El-Agamy DS et al. J Physiol Biochem. 2014 Mar;70(1):141-9. doi: 10.1007 / s13105-013-0288-x. Epub 2013 Sep 14.

[0083] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of subarachnoid hemorrhage. Further details are disclosed in Guo Z et al. Stroke. 2016 Feb;47(2):490-7. doi: 10.1161 / STROKEAHA.115.011223. Epub 2016 Jan 5.

[0084] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for preventing or treating intestinal mucosal damage. Further details are disclosed in Birkl D et al. FASEB J. 2019 Dec;33(12):13632-13643. doi: 10.1096 / fj.201901163R. Epub 2019 Oct 4.

[0085] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of acute kidney injury. Further details are disclosed in Luan H et al. Front Physiol. 2020 Apr 3;11:285. doi: 10.3389 / fphys.2020.00285.

[0086] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of myocardial ischemia-reperfusion injury. Further details are disclosed in Qin, C., May, L et al. Nat Commun 8, 14232 (2017). https: / doi.org / 10.1038 / ncomms14232.

[0087] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of acute lung injury or pulmonary ischemia-reperfusion injury. Further details are disclosed in Tang H et al. J Immunol. 2014 Oct 1;193(7):3769-78. doi: 10.4049 / jimmunol.1400942. Epub 2014 Aug 29.

[0088] In the present invention, the peptide can act as an anti-inflammatory factor that binds to the FPR2 receptor. Activation of FPR2 can modulate immune cell activity, thereby enhancing the immune response against cancer. In particular, it can promote phagocytosis of cancer cells and contribute to the elimination of cancer cells through cytotoxicity.

[0089] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the peptide as an active ingredient, a use of the peptide for preventing or treating cancer, and a method for preventing or treating cancer comprising administering the peptide to a subject.

[0090] In one embodiment of the present invention, the cancer may be a solid cancer or a non-solid cancer, and may be gastric cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, colorectal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, It could be urethral cancer, prostate cancer, bronchogenic cancer, bone marrow tumor, or lymphoma.

[0091] Preferably, the cancer may be, but is not limited to, breast cancer, colon cancer, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, melanoma, or lymphoma.

[0092] In the present invention, the peptide acts as a ligand that binds to the FPR2 receptor and can be used as a pharmaceutical composition for the prevention or treatment of cancer. For further details, see Kim et al. PLoS One. 2012;7(1):e30522. doi: 10.1371 / journal.pone.0030522. Epub 2012 Jan 25; Wang et al. Antioxidants (Basel). 2021 Sep 14;10(9):1459. doi: 10.3390 / antiox10091459.; Liu et al. Cancer Res. 2013 Jan 15;73(2):550-60. doi: 10.1158 / 0008-5472.CAN-12-2290. Epub 2012 Nov 8; Gilligan et al. Proc Natl Acad Sci U S A. 2019 Mar 26;116(13):6292-6297. doi: 10.1073 / pnas.1804000116. Epub 2019 Mar 12; Du et al. Ann Transl Med. 2021 May;9(9):802. doi: 10.21037 / atm-21-1873. Erratum in: Ann Transl Med. 2022 Apr;10(7):428; Sulciner et al. J Exp Med. 2018 Jan 2;215(1):115-140. doi: 10.1084 / jem.20170681. Epub 2017 Nov 30; Zhang et al. Front Immunol. 2017 Feb 2;8:71. doi: 10.3389 / fimmu.2017.00071; Liotti et al. Cancers (Basel). 2022 Jul 8;14(14):3333. doi: 10.3390 / cancers14143333.

[0093] The term "prevention" used in the present invention means any act of suppressing or delaying an immune disease, tissue damage, fibrotic disease or cancer by administering a pharmaceutical composition according to the present invention, and the term "treatment" means any act of improving or benefiting the symptoms of an immune disease, tissue damage, fibrotic disease or cancer by administering a pharmaceutical composition according to the present invention. The term "subject" in the present invention means a subject in need of prevention or treatment of a disease, and more specifically, any mammal in need of prevention or treatment of an immune disease, tissue damage, fibrotic disease or cancer, including, without limitation, humans and primates, as well as livestock such as cattle, pigs, sheep, horses, dogs and cats, but preferably humans.

[0094] The route of administration of the pharmaceutical composition comprising the above peptide as an active ingredient may be administered through any common route as long as the drug can reach the target tissue, and may be administered to the subject orally or parenterally. For example, the route may include intraperitoneal administration, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration (including skin application, drip administration, and inhalation), transdermal administration, intrathecal and epidural administration, intranasal administration, intraocular administration, intrapulmonary administration, rectal administration, vaginal administration, etc., but is not limited thereto. In addition, the pharmaceutical composition comprising the above peptide as an active ingredient may be administered in any convenient pharmaceutical product form, for example, tablets, powders, granules, capsules, oral liquids, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. However, since peptides are digested when administered orally, it is desirable to formulate oral compositions to coat the active agent or protect it from degradation in the stomach.

[0095] Pharmaceutical compositions comprising the peptides of the present invention may additionally include pharmaceutically and physiologically acceptable carriers, excipients, and diluents. As used herein, the term "pharmaceutically acceptable carriers, excipients, and diluents" refers to carriers, excipients, and diluents that do not stimulate organisms and do not inhibit the biological activity and properties of the administered compound. Examples of suitable carriers, excipients and diluents that may be included in such compositions include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, glycerol, ethanol, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, hypromellose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. When the composition is pharmaceutically active, it may additionally contain conventional dispersants, fillers, bulking agents, binders, disintegrants, surfactants, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, freeze-drying agents, etc.

[0096] In the present invention, the pharmaceutical composition may be administered in the form of an injection. The injection may be formulated as an intravenous or subcutaneous injection. Parenteral injections may include components typically found in injection compositions. For example, the injection composition may include a liquid carrier such as sterile water, water for injection, or physiological saline. Additionally, the composition may further include amino acids, sugars, lipids, vitamins, electrolytes, pH adjusters, stabilizers, osmotic pressure adjusters, or solubilizers.

[0097] In the present invention, when the pharmaceutical composition is administered topically, it may be formulated into a dosage form such as an ointment, gel, cream, or lotion. The form of topical administration is not limited thereto, but may include, for example, application to the skin, eye drops, transdermal penetration using microneedles, intradermal injection, etc. For example, it may be preferable to apply the composition or attach a patch formulation containing the composition to the skin. The composition may include, for example, a base, an excipient, a lubricant, and a preservative. For example, when the pharmaceutical composition is administered in the form of eye drops, it may further include a buffer, a viscosity modifier, an isotonic agent, a pH adjuster, and a solvent. In addition, when the pharmaceutical composition is administered in the form of an ointment for skin application, it may further include a gelling agent, a stabilizer, an emulsifier, and a suspending agent.

[0098] In addition, the pharmaceutical composition of the present invention can be applied differently depending on the purpose of administration and disease. The amount of active ingredient actually administered can be appropriately selected by those skilled in the art in consideration of various related factors, such as the disease to be treated, the severity of the disease, whether co-administered with other drugs, the activity of the drug, the sensitivity to the drug, the patient's age, sex, weight, food, administration time, administration route, and the administration ratio of the composition. The composition can be administered once a day or in 1-3 divided doses, although the dosage and administration route can be adjusted depending on the type and severity of the disease.

[0099] A pharmaceutical composition comprising the peptide of the present invention as an active ingredient may be administered at a dosage of 0.001 mg / kg to 1 g / kg, more preferably 0.1 mg / kg to 100 mg / kg. Meanwhile, the dosage may be appropriately adjusted depending on the patient's age, sex, and condition.

[0100] In addition, the present invention provides an anti-inflammatory cosmetic composition comprising the peptide as an active ingredient, a use of the peptide for suppressing inflammation, and a method for suppressing inflammation comprising applying the peptide to a subject.

[0101] The anti-inflammatory cosmetic composition comprising the peptide of the present invention can be prepared in the form of a general emulsified formulation or a solubilized formulation. For example, it can have the formulation of, but is not limited to, a solution, an external ointment, a toner such as an emollient toner or a nourishing toner, an emulsion such as a facial lotion or a body lotion, a cream such as a nourishing cream, a moisturizing cream or an eye cream, an essence, a cosmetic ointment, a spray, a gel, a pack, a sunscreen, a makeup base, a foundation such as a liquid type, a solid type or a spray type, a makeup remover such as a powder, a cleansing cream, a cleansing lotion, a cleansing oil, a cleanser such as a cleansing foam, a soap, a body wash, an oil, a patch, etc.

[0102] In the present invention, the cosmetic composition may further comprise one or more adjuvants and carriers commonly used in the field of cosmetics, such as pearl agents, thickeners, viscosity modifiers, pH modifiers, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents and chelating agents, preservatives, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic active agents, lipophilic active agents, lipid vesicles or any other ingredients commonly used in cosmetics.

[0103] Example: Preparation of FPR2 activating peptide

[0104] In the present invention, novel peptides with superior anti-inflammatory and immunomodulatory functions compared to the above peptides were synthesized using conventional amino acid synthesis methods (Umbarger, HE, Ann Rev Biochem., 47: 533-606, 1978), and these were used as candidate peptides for anti-inflammatory purposes. In addition, the inventors of the present invention chemically modified the N-terminus and / or C-terminus of the peptides synthesized above. The sequences of the synthesized peptides are shown in Table 1.

[0105] Sequence number sequence N-terminal X1 X2 X3 X4 X5 X6 mC-terminal 1-PIKYKVmNH 2 2 AcPIKYKVmNH 2 3 ButPIKYKVmNH 2 4 OctPIKYKVmNH 2 5-HIKYKVmNH 2 6 AcHIKYKVmNH 2 7 ButHIKYKVmNH 2 8 OctHIKYKVmNH 2 9-HIKYKPmNH 2 10 AcHIKYKPmNH 2 11 ButHIKYKPmNH 2 12 OctHIKYKPmNH 2 13-PIRYKPmNH 2 14 AcPIRYKPmNH 2 15 ButPIRYKPmNH 2 16 OctPIRYKPmNH 2 17-HIRYKPmNH 2 1 8AcHIRYKPmNH219ButHIRYKPmNH220OctHIRYKPmNH221-PIRYKVmNH222AcPIRYKVmNH223ButPIRYKVmNH224OctPIRYKVmNH225-HIRYKVmNH226AcHIRYKVmNH227B utHIRYKVmNH228OctHIRYKVmNH229-PIKYKPmNH230AcPIKYKPmNH231ButPIKYKPmNH232OctPIKYKPmNH2NCP112_0(33)-KFKWRYmNH2NCP112(34)OctKFKWRYmNH2

[0106] Experimental Example 1: Measurement of changes in calcium activity due to peptides

[0107] The inventors observed changes in calcium ion permeability to determine whether a candidate inflammation-modulating peptide, manufactured according to one embodiment of the present invention, activated the body's immune function. Specifically, to determine whether the peptide activated FPR2, the intracellular calcium ion concentration was measured.

[0108] 1) Cell and peptide preparation

[0109] The present inventors used cells that did not express FPR2 (Prarantal), cells that overexpressed FPR2 (FPR2 Overexpressed), or cells that overexpressed FPR1 (FPR1 Overexpressed), and used Fura-2 / AM, a dye with strong binding affinity to calcium, as a method for sensitively measuring the release of intracellular calcium ions.

[0110] Peptides dissolved in water or 50% DMSO at a concentration of 0.5 mM were prepared by diluting them to an appropriate concentration in HBSS (Sigma, #55037C) + 20 mM HEPES buffer.

[0111] 2) Culture conditions

[0112] In this experiment, cells were cultured in DMEM (Welgene, LM001-05) medium containing 10% fetal bovine serum (FBS; Gibco, 16000-044) and 1% Penicillin-Streptomycin at 37°C and 5% CO2 conditions.

[0113] 3) Experimental method

[0114] The day before measuring calcium activity, 3 x 10 per well were seeded in a 96-well flat clear bottom black plate (costar, #3603). 5Cells were prepared. The prepared cells were treated with the same volume of Fura-2 QBT calcium kit (component A; Molecular Devices, #8197) per well. After incubation at 37°C for 1 hour, the cells were incubated in the dark at room temperature for 30 minutes. The cells treated with Fura-2, the prepared peptides, and the dedicated tips were placed in each compartment of a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, #Flex3), and the calcium activity changes induced by the peptides were measured using SoftMax Pro 7 Software (Molecular Devices, #FLEX3-INSTALL-OS). As a control sequence, the calcium activity changes of the previously developed peptide, NCP112, were measured.

[0115] 4) Experimental results

[0116] As a result, all peptides showed a high level of FPR2 activation potency and excellent FPR2-selectivity with no or limited FPR1 activation potency. This suggests that the peptides according to the present invention can regulate the immune function of an individual through binding to and activating FPR2. The results of analyzing the FPR2 activation potency of the peptides were graded according to the values ​​and summarized in [Table 2] below. The criteria for each grade are described in [Table 3]. In addition, a graph comparing the FPR2 activation potency of the peptides represented by SEQ ID NOs: 1, 5, 9, 13, and 17 with that of the control sequence NCP112 (SEQ ID NO: 34) is shown in Fig. 1.

[0117] Analysis of the FPR2 and FPR1 activation potency of the peptide. Sequence number EC50 for [Ca 2+ ] intraFPR2 (nM)FPR1 (nM)1AE2AF3AE4AE5AF6AF7AE8BE9AF10AE11AE12AE13AF14AF15AE16AE17AE18AE19AE20BE21AE22AE25AE29AENCP112_0(33)CENCP112 (34)CE

[0118] Grade Table A< 10nMB11~100nMC101~1,000nMD1,001~10,000nME10,001~50,000nMF50,001<

[0119] As shown in the above [Table 2], the immunomodulatory peptide according to the present invention has an EC for FPR2. 50 While the values ​​are generally less than 10 nM, the EC for FPR1 50 The value was confirmed to be 10,000 nM or higher. Through this, it was confirmed that the peptide specifically binds to FPR2.

[0120] Experimental Example 2: Measurement of Plasma Stability of Peptides

[0121] To confirm the in vivo stability of an inflammation-modulating peptide candidate manufactured according to one embodiment of the present invention, the inventors measured the stability of the peptide in plasma. Specifically, the peptide was incubated in plasma for a specified period of time, and the residual amount was measured using LC-MS / MS.

[0122] 1) Preparation of plasma and peptides

[0123] The present inventors prepared the peptide dissolved in human plasma (Human plasma: Biochemed, 752PR-SC-PM, 3.8% sodium citrate) and DMSO at a concentration of 10 mM.

[0124] 2) Experimental method

[0125] 2 μl of the prepared 10 mM peptide was added to a tube containing 198 μl of plasma and incubated at 37°C for 0 or 30 minutes. After incubation, 20 μl of plasma was collected, added to the prepared tube with 180 μl of ACN (acetonitrile), and vortexed for 5 minutes to mix. After centrifugation (15,000 rpm, 4°C) for 5 minutes, the supernatant was collected. 2 μl of the collected supernatant was injected into an LC-MS / MS system (Applied biosystems, USA). The measured peaks were analyzed in MRM (multiple reaction monitoring) quantitative mode using analyst software, and each experiment was performed in duplicate. The results were calculated as the remaining amount (% Remaining) relative to the unincubated sample (0 minutes).

[0126] 3) Experimental results

[0127] As a result, it was confirmed that all peptides developed by the present invention exhibited a high level of plasma stability. This suggests that the peptides according to the present invention have high stability in vivo compared to the existing NCP112_0 or the similar sequences to which they are compared, which have low plasma stability. The results of analyzing the human plasma stability of the peptides were graded according to the numerical values ​​and summarized in [Table 4] below. The criteria for each grade are described in [Table 5].

[0128] Sequence numberSequence Blood stabilityX1X2X3X4X5X6mC-terminal1PIKYKVmNH2A5HIKYKVmNH2A9HIKYKPmNH2A13PIRYKPmNH2A17HIRYKPmNH2B21PIRYKVmNH2A25HIRYKVmNH2B29PIKYKPmNH2ANCP112_0(33)KFKWRYmNH2F35-IKYKVmNH2E36-IRYKVmNH2F37-IKYKPmNH2F38-IRYKPmNH2E

[0129] Grade A80% <B61~80%C41~60%D21~40%E1~20%F< 1%

[0130] The scope of protection of the present invention is not limited to the description of the embodiments explicitly described above. Furthermore, the scope of protection of the present invention cannot be limited by obvious modifications or substitutions within the technical field to which the present invention pertains.

Claims

1. A peptide consisting of an amino acid sequence represented by the following general formula 1: [General Formula 1] X1-X2-X3-X4-X5-X6-m In the above general formula, X1 is proline (P) or histidine (H), X2 is isoleucine (I), X3 is arginine (R) or lysine (K), X4 is tyrosine (Y), X5 is lysine (K), X6 is valine (V) or proline (P), and m is D-type methionine.

2. A peptide in which any one selected from the group consisting of an acetyl group, a butanoyl group, a hexanoyl group, and an octanoyl group is added to the N-terminus of the peptide of clause 1, or in which the C-terminus of the peptide is amidated.

3. In paragraph 2, A peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 32.

4. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising the peptide of any one of claims 1 to 3 as an active ingredient.

5. In paragraph 4, The above inflammatory diseases include atopic dermatitis, psoriasis, infantile eczema, sepsis, conjunctivitis, keratitis, ocular inflammatory disease, dry eye syndrome, asthma, lung injury, lung inflammation, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, peritonitis, systemic sclerosis, heart failure, myocardial infarction, cardiovascular thrombosis, neuroinflammation, Parkinson's disease, Alzheimer's disease. disease), cirrhosis, intestinal fibrosis, renal fibrosis, glomerulonephritis, cardiac hypertrophy, cardiac fibrosis, pulmonary fibrosis, acute liver injury, subarachnoid hemorrhage, intestinal mucosal wound, acute kidney injury, myocardial ischemia-reperfusion injury,A pharmaceutical composition for preventing or treating inflammatory diseases, such as acute lung injury or lung ischemia-reperfusion injury.

6. A pharmaceutical composition for preventing or treating cancer, comprising the peptide of any one of claims 1 to 3 as an active ingredient.

7. In paragraph 6, A pharmaceutical composition for preventing or treating cancer, wherein the cancer is breast cancer, colon cancer, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, melanoma, or lymphoma.

8. An anti-inflammatory cosmetic composition comprising the peptide of any one of claims 1 to 3 as an active ingredient.

Images