Peptides and use thereof

Abstract

To provide novel bioactive peptides present in human blood.SOLUTION: Provided are an anti-obesity drug containing a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2 or a mutant peptide thereof as an active ingredient; an expression inducer of inflammatory cytokines, chemokines, cell growth factors, or angiogenesis-promoting factors through the activation of NF-κB signaling in vascular smooth muscle cells, vascular endothelial cells, or macrophage cells, which contains a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 or 4, or a mutant peptide thereof as an active ingredient; and an arteriosclerosis-promoting agent containing a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5 or a mutant peptide thereof as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to peptides and uses thereof. [Background technology]

[0002] There are a huge amount of high molecular weight proteins in blood. Therefore, it was impossible to identify the bioactive peptides present in blood by conventional mass spectrometry alone. In the past 20 years, almost no bioactive peptides have been discovered in humans.

[0003] The inventors previously developed a plasma peptidome analysis technique, which had previously been considered difficult, and succeeded in determining the structures of the large number of small molecular weight peptides present in human plasma (see Non-Patent Documents 1 and 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kawashima Y., et al., High-yield peptide-extraction method for the discovery of subnanomolar biomarkers from small serum samples, J Proteome Res, 9 (4), 1694-1705, 2010. [Non-Patent Document 2] Saito T., et al., Establishment and application of a high-quality comparative analysis strategy for the discovery and small-scale validation of low-abundance biomarker peptides in serum based on an optimized novel peptide extraction method, J Electrophoresis, 57, 1, 1-9, 2013. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a novel physiologically active peptide present in human blood. More specifically, the present invention aims to provide an anti-obesity drug; an inducer of expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoters through activation of NF-κB signaling in vascular smooth muscle cells, vascular endothelial cells or macrophage cells; a method for screening a therapeutic agent for inflammatory diseases or tumors; a therapeutic agent for inflammatory diseases or tumors; and an arteriosclerosis promoter and therapeutic agent for arteriosclerosis. [Means for solving the problem]

[0006] The present invention includes the following aspects. [1] An anti-obesity drug comprising as an active ingredient: (i) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (ii) a peptide consisting of an amino acid sequence in which one to three amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1, and which has activity to suppress eating or drinking when administered to a mammal; (iii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2; or (iv) a peptide consisting of an amino acid sequence in which one to three amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2, and which has activity to suppress eating or drinking when administered to a mammal. [2] (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3, and having an activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (viii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4. An agent for inducing the expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling in vascular smooth muscle cells, vascular endothelial cells or macrophage cells, comprising as an active ingredient a peptide having an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted or added, and having activity of inducing the expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells. [3] (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3, and which has an activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (viii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4. a peptide having an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted or added, and having activity of inducing expression of an inflammatory cytokine, a chemokine, a cell growth factor or an angiogenesis promoting factor through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; and a screening step of identifying an inhibitor of the receptor, wherein the inhibitor is a therapeutic agent for an inflammatory disease or tumor. [4] (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence in which one to three amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3, and having an activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (viii) a peptide consisting of an amino acid sequence in which one to three amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 4, and The method for screening a therapeutic agent for an inflammatory disease or tumor includes a step of culturing vascular smooth muscle cells, vascular endothelial cells, or macrophage cells in the presence of a peptide having activity of inducing expression of an inflammatory cytokine, a chemokine, a cell growth factor, or an angiogenesis promoting factor through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells, or macrophage cells, and measuring the expression level of the inflammatory cytokine, chemokine, cell growth factor, or angiogenesis promoting factor in the cells, wherein a significant decrease in the expression level compared to that in the absence of the test substance indicates that the test substance is a therapeutic agent for an inflammatory disease or tumor. [5] A therapeutic agent for inflammatory diseases or tumors, comprising as an active ingredient an antibody or a fragment thereof against: (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 in which one to three amino acids are deleted, substituted or added, and which has activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (viii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which one to three amino acids are deleted, substituted or added, and which has activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells. [6] (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, or (x) an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which 1 to 3 amino acids have been deleted, substituted or added, and which is an ApoE - / - An arteriosclerosis promoter comprising, as an active ingredient, a peptide that has the activity of promoting arteriosclerosis when administered to mice. [7] (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, or (x) an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which 1 to 3 amino acids have been deleted, substituted or added, and which is an ApoE - / - A method for screening an arteriosclerosis therapeutic agent, comprising the steps of: identifying a receptor for a peptide having an activity of promoting arteriosclerosis when administered to a mouse; and screening a test substance to identify an inhibitor of the receptor, wherein the inhibitor is an arteriosclerosis therapeutic agent. [8] (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, or (x) an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which 1 to 3 amino acids have been deleted, substituted or added, and which is an ApoE - / -A method for screening an arteriosclerosis therapeutic agent, comprising the steps of culturing vascular smooth muscle cells, vascular endothelial cells or macrophage cells in the presence of a peptide having an activity of promoting arteriosclerosis when administered to a mouse and a test substance, and measuring the expression level of inflammatory cytokines or arteriosclerosis-promoting factors in the cells, wherein a significant decrease in the expression level compared to that in the absence of the test substance indicates that the test substance is an arteriosclerosis therapeutic agent. [9] (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, or (x) an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which 1 to 3 amino acids have been deleted, substituted or added, and which is an ApoE - / - An arteriosclerosis treatment agent comprising, as an active ingredient, an antibody or a fragment thereof against a peptide having activity of promoting arteriosclerosis when administered to a mouse. Effect of the Invention

[0007] According to the present invention, it is possible to provide novel physiologically active peptides present in human blood. More specifically, it is possible to provide an anti-obesity drug, an inducer of expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling in vascular smooth muscle cells, vascular endothelial cells and macrophage cells, a method for screening a therapeutic agent for inflammatory diseases or tumors, a therapeutic agent for inflammatory diseases or tumors, an arteriosclerosis promoter and an arteriosclerosis therapeutic agent. [Brief description of the drawings]

[0008] [Figure 1] 14(a) and (b) are graphs showing the results of Experimental Example 2. 14(c) to (f) are confocal microscope photographs showing the results of Experimental Example 2. [Diagram 2] 6(a) and 6(b) are graphs showing the results of Experimental Example 3. [Diagram 3] 6(a) and (b) are photographs showing the results of Western blotting in Experimental Example 4. 6(c) to (e) are fluorescent micrographs showing the results of immunostaining in Experimental Example 4. [Figure 4]6(a) and 6(b) are graphs showing the results of Experimental Example 5. [Diagram 5] 6(a) to 6(f) are graphs showing the results of Experimental Example 6. [Figure 6] Fluorescence micrographs showing the results of immunostaining in Experimental Example 8, (a) to (e), (a') to (e'), and (a'') to (e''). [Figure 7] 13(a) to (f) are confocal microscope photographs showing the results of Experimental Example 9. [Figure 8] 13(a) to 13(c) are graphs showing the results of Experimental Example 10. [Figure 9] 6(a) to 6(c) are graphs showing the results of Experimental Example 11. [Figure 10] 13(a) to (d) are confocal microscope photographs showing the results of Experimental Example 12. [Figure 11] 6(a) to 6(d) are micrographs showing the results of Experimental Example 13. [Figure 12] 13(a) to 13(d) are macroscopic tissue specimens showing the results of Experimental Example 14. 13(e) to 13(l) are micrographs showing the results of Experimental Example 14. [Figure 13] 6(a) and 6(b) are graphs showing the results of Experimental Example 14. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] [Anti-obesity drugs] In one embodiment, the present invention provides an anti-obesity drug containing as an active ingredient: (i) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (ii) a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 1 in which 1 to 3 amino acids have been deleted, substituted or added, and which has activity to suppress eating or drinking when administered to a mammal; (iii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2; or (iv) a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 2 in which 1 to 3 amino acids have been deleted, substituted or added, and which has activity to suppress eating or drinking when administered to a mammal.

[0010] As described later in the Examples, the inventors have demonstrated that a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 (a peptide consisting of amino acids 279 to 295 in the amino acid sequence of human suprabasin; hereinafter sometimes referred to as "SBSN_HUMAN[279-295]") and a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 (a peptide consisting of amino acids 448 to 462 in the amino acid sequence of human proangiotensinogen; hereinafter sometimes referred to as "ANGT_HUMAN[448-462]") have strong feeding suppressant activity in experiments using mice.

[0011] These peptides have a strong appetite suppressing effect far superior to that of GLP-1 agonists, which have become the second-line drug for type 2 diabetes worldwide. Therefore, they can be used as anti-obesity drugs, which have long been desired internationally, to treat type 2 diabetes and other conditions.

[0012] Furthermore, since these peptides are naturally present in human blood, they are safe to administer to humans.

[0013] As long as the peptides have the activity of this embodiment, the peptides SBSN_HUMAN[279-295] (SEQ ID NO: 1) and ANGT_HUMAN[448-462] (SEQ ID NO: 2) may have one to three amino acid deletions, substitutions, or additions. Furthermore, these peptides may be in the form of pharma- ceutically acceptable salts or solvates. Examples of salts include trifluoroacetic acid (TFA) salts, acetate salts, and hydrochloride salts. Examples of solvates include hydrates and organic solvent solvates.

[0014] [Inducers of expression of inflammatory cytokines, chemokines, cell growth factors, or angiogenesis promoting factors] In one embodiment, the present invention relates to (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3, and having activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (viii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4. The present invention provides an agent for inducing the expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling in vascular smooth muscle cells, vascular endothelial cells or macrophage cells, the active ingredient of which is a peptide consisting of an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted or added, and which has activity of inducing the expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells.

[0015] As described later in the Examples, the inventors have revealed that when a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 (a peptide consisting of amino acids at positions 225 to 237 in the amino acid sequence of human suprabasin; hereinafter sometimes referred to as "SBSN_HUMAN[225-237]") and a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 (a peptide consisting of amino acids at positions 243 to 259 in the amino acid sequence of human suprabasin; hereinafter sometimes referred to as "SBSN_HUMAN[243-259]") are contacted with vascular smooth muscle cells, vascular endothelial cells, or macrophage cells, the peptide induces the expression of inflammatory cytokines, chemokines, cell growth factors, and angiogenesis promoting factors through the activation of NF-κB signaling.

[0016] Therefore, these peptides can be used as research reagents, etc., as agents for inducing the expression of inflammatory cytokines, chemokines, cell growth factors, or angiogenesis-promoting factors through activation of NF-κB signaling in vascular smooth muscle cells, vascular endothelial cells, or macrophage cells.

[0017] As long as the peptides have the activity of this embodiment, the peptides SBSN_HUMAN[225-237] (SEQ ID NO: 3) and SBSN_HUMAN[243-259] (SEQ ID NO: 4) may have one to three amino acid deletions, substitutions, or additions. Furthermore, these peptides may be in the form of pharma- ceutically acceptable salts or solvates. Examples of salts include trifluoroacetic acid (TFA) salts, acetate salts, and hydrochloride salts. Examples of solvates include hydrates and organic solvent solvates.

[0018] [Method of screening therapeutic agents for inflammatory diseases or tumors] (First embodiment) In one embodiment, the present invention relates to (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3, and having activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (viii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4. The present invention provides a method for screening a therapeutic agent for an inflammatory disease or tumor, comprising the steps of: identifying a receptor for a peptide, the peptide consisting of an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted or added, and which has activity of inducing expression of an inflammatory cytokine, a chemokine, a cell growth factor or an angiogenesis promoting factor through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; and screening a test substance to identify an inhibitor of the receptor, wherein the inhibitor is a therapeutic agent for an inflammatory disease or tumor.

[0019] As described later in the Examples, the inventors have demonstrated that SBSN_HUMAN[225-237] (SEQ ID NO: 3) and SBSN_HUMAN[243-259] (SEQ ID NO: 4) bind to vascular smooth muscle cells. Thus, these peptides have receptors.

[0020] The above-mentioned SBSN_HUMAN[225-237] (sequence number 3), SBSN_HUMAN[243-259] (sequence number 4), and inhibitors of the receptors of these mutant peptides can be used as therapeutic agents for diseases caused by inflammatory cytokines, chemokines, cell growth factors, or angiogenesis promoting factors, i.e., inflammatory diseases such as cytokine storms such as rheumatoid arthritis and severe COVID-19 pneumonia; tumors, etc.

[0021] Therefore, in one embodiment, the present invention provides a method for screening a therapeutic agent for an inflammatory disease or tumor, comprising the steps of identifying a receptor for the above-mentioned peptide, and screening a test substance to identify an inhibitor of the receptor, wherein the inhibitor is a therapeutic agent for an inflammatory disease or tumor.

[0022] The test substance is not particularly limited, and examples thereof include natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, antibody libraries, and the like.

[0023] Receptors can be identified, for example, as follows. First, membrane proteins from vascular smooth muscle cells are extracted and mixed with liposomes to produce liposomes with embedded membrane proteins. Next, the liposomes are passed through a membrane, and liposomes with a diameter of, for example, 200 nm or less are collected. These are used as a membrane protein library, and liposomes that exhibit binding to the above peptides are collected, and the receptor proteins can be identified by SDS polyacrylamide gel electrophoresis (SDS-PAGE), band excision, trypsin digestion-liquid chromatography (LC)-mass spectrometry (MS) / MS, etc.

[0024] Examples of receptor inhibitors include substances that inhibit the binding between the receptor and the above-mentioned peptide, and substances that inhibit signal transduction after the receptor and the above-mentioned peptide bind to each other.

[0025] Substances that inhibit the binding between the receptor and the above-mentioned peptide include decoy receptors, fragment peptides of the above-mentioned peptides, antibodies against the above-mentioned peptides or fragments thereof, etc. Examples of decoy receptors include solubilized receptors, and more specifically, fusion proteins of receptors and antibody constant regions, etc.

[0026] Second embodiment In one embodiment, the present invention relates to (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 3 in which one to three amino acids are deleted, substituted or added, and which has activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (viii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which one to three amino acids are deleted, substituted or added. The present invention provides a method for screening a therapeutic agent for an inflammatory disease or tumor, comprising the steps of culturing vascular smooth muscle cells, vascular endothelial cells, or macrophage cells in the presence of a test substance and a peptide having activity of inducing expression of an inflammatory cytokine, chemokine, cell growth factor, or an angiogenesis promoting factor through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells, or macrophage cells, and measuring the expression level of the inflammatory cytokine, chemokine, cell growth factor, or angiogenesis promoting factor in the cells, wherein a significant decrease in the expression level compared to that in the absence of the test substance indicates that the test substance is a therapeutic agent for an inflammatory disease or tumor.

[0027] As described later in the Examples, the inventors have demonstrated that SBSN_HUMAN[225-237] (SEQ ID NO: 3) and SBSN_HUMAN[243-259] (SEQ ID NO: 4) act on vascular smooth muscle cells, vascular endothelial cells, or macrophage cells to induce the expression of inflammatory cytokines, chemokines, cell growth factors, and angiogenesis promoting factors through the activation of NF-κB signaling. Therefore, a test substance that suppresses the induction of the expression of these factors can be used as a therapeutic agent for inflammatory diseases or tumors.

[0028] That is, the present invention provides a method for screening a therapeutic agent for an inflammatory disease or tumor, comprising the steps of culturing vascular smooth muscle cells, vascular endothelial cells or macrophage cells in the presence of the above-mentioned SBSN_HUMAN[225-237] (SEQ ID NO: 3), SBSN_HUMAN[243-259] (SEQ ID NO: 4) or mutant peptides thereof, and a test substance, and measuring the expression levels of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors in the cells, wherein a significant decrease in the expression levels compared to that in the absence of the test substance indicates that the test substance is a therapeutic agent for an inflammatory disease or tumor. The method of the second embodiment can also be used to screen therapeutic agents for an inflammatory disease or tumor.

[0029] The test substances are the same as those described above. Examples of inflammatory cytokines, chemokines, cell growth factors, or angiogenesis promoting factors include vascular endothelial growth factor a (VEGFa), Dickkopf-related protein 1 (DKK1), Endoglin, urokinase plasminogen activator receptor (uPAR), granulocyte macrophage colony-stimulating factor (GM-CSF), vascular cell adhesion molecule 1 (VCAM1), cystatin C, hepatocyte growth factor (HGF), osteopontin (OPN), Interleukin-6 (IL-6), and Angiogenin.

[0030] [Treatment agent for inflammatory diseases or tumors] In one embodiment, the present invention provides a therapeutic agent for inflammatory diseases or tumors, comprising as an active ingredient an antibody or a fragment thereof against (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 3 in which one to three amino acids are deleted, substituted or added, and which has activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (viii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which one to three amino acids are deleted, substituted or added, and which has activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells.

[0031] As described later in the Examples, the inventors have demonstrated that SBSN_HUMAN[225-237] (sequence number 3) and SBSN_HUMAN[243-259] (sequence number 4) act on vascular smooth muscle cells, vascular endothelial cells or macrophage cells to induce the expression of inflammatory cytokines, chemokines, cell growth factors and angiogenesis promoting factors through the activation of NF-κB signaling.

[0032] Therefore, an antibody or a fragment thereof against SBSN_HUMAN[225-237] (SEQ ID NO: 3), SBSN_HUMAN[243-259] (SEQ ID NO: 4) or a mutant peptide thereof inhibits the binding of these peptides to their receptors, and inhibits the induction of expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling in vascular smooth muscle cells, vascular endothelial cells or macrophage cells. Examples of antibody fragments include F(ab')2, Fab', Fab, Fv, scFv, etc. Such an antibody or a fragment thereof can be used as a therapeutic agent for inflammatory diseases or tumors.

[0033] The antibody is preferably a human antibody. Examples of human antibodies include chimeric antibodies, humanized antibodies, and fully human antibodies. Here, the term "chimeric antibody" refers to an antibody whose variable region is derived from a non-human animal and whose constant region is at least partially derived from a human. The term "humanized antibody" refers to an antibody whose heavy and light chain complementarity determining regions are derived from a non-human animal and whose constant region and framework region are derived from a human. The term "fully human antibody" refers to an antibody whose entirety, including the complementarity determining regions, is derived from a human.

[0034] If the antibody or a fragment thereof is a human antibody or a fragment thereof, it has low immunogenicity when administered to humans, and therefore side effects such as anaphylactic shock can be suppressed.

[0035] [Arteriosclerosis promoter] In one embodiment, the present invention relates to (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, or (x) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which 1 to 3 amino acids have been deleted, substituted or added, and which is ApoE - / - The present invention provides an arteriosclerosis promoter, which contains as an active ingredient a peptide that has activity of promoting arteriosclerosis when administered to mice.

[0036] As described later in the Examples, the inventors administered a peptide consisting of the amino acid sequence set forth in SEQ ID NO:5 (a peptide consisting of the 22nd to 51st amino acids in the amino acid sequence of human glucose-dependent insulinotropic polypeptide (GIP), hereinafter sometimes referred to as "GIP_HUMAN[22-51]") to ApoE, a disease model mouse of atherosclerosis. - / - It was revealed that when administered to mice, it significantly promoted arteriosclerosis.

[0037] Therefore, GIP_HUMAN[22-51] (SEQ ID NO: 5) can be used as an arteriosclerosis promoter in research reagents and the like. As long as it has the activity of this embodiment, the peptide of GIP_HUMAN[22-51] (SEQ ID NO: 5) may have deletion, substitution, or addition of 1 to 3 amino acids. Furthermore, these peptides may be in the form of pharma-ceutically acceptable salts or solvates. Examples of salts include trifluoroacetic acid (TFA) salts, acetate salts, and hydrochlorides. Examples of solvates include hydrates and organic solvent solvates.

[0038] [Method of screening for arteriosclerosis treatment agents] (First embodiment) In one embodiment, the present invention relates to (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, or (x) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which 1 to 3 amino acids have been deleted, substituted or added, and which is ApoE - / -The present invention provides a method for screening an arteriosclerosis therapeutic agent, comprising the steps of: identifying a receptor for a peptide having an activity of promoting arteriosclerosis when administered to a mouse; and screening a test substance to identify an inhibitor of the receptor, wherein the inhibitor is an arteriosclerosis therapeutic agent.

[0039] As described later in the Examples, the inventors administered GIP_HUMAN[22-51] (SEQ ID NO: 5) to a mouse model of atherosclerosis, ApoE - / - It was revealed that administration of this compound to mice significantly promoted arteriosclerosis. Therefore, an inhibitor of the receptor for GIP_HUMAN[22-51] (SEQ ID NO: 5) or its mutant peptide can be used as a therapeutic agent for arteriosclerosis.

[0040] That is, the present invention provides a method for screening an arteriosclerosis therapeutic agent, comprising the steps of identifying a receptor of GIP_HUMAN[22-51] (SEQ ID NO: 5) and screening a test substance to identify an inhibitor of the receptor, the inhibitor being an arteriosclerosis therapeutic agent. The test substance, the identification of the receptor, and the receptor inhibitor are the same as those described above.

[0041] Second embodiment In one embodiment, the present invention relates to (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, or (x) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which 1 to 3 amino acids have been deleted, substituted or added, and which is ApoE - / - The present invention provides a method for screening an arteriosclerosis therapeutic agent, comprising the steps of culturing vascular endothelial cells or macrophage cells in the presence of a peptide having an activity of promoting arteriosclerosis when administered to a mouse and a test substance, and measuring the expression level of an inflammatory cytokine or an arteriosclerosis-promoting factor in the cells, wherein a significant decrease in the expression level compared to that in the absence of the test substance indicates that the test substance is an arteriosclerosis therapeutic agent.

[0042] As described later in the Examples, the inventors have demonstrated that GIP_HUMAN[22-51] (SEQ ID NO: 5) acts on vascular smooth muscle cells, vascular endothelial cells, or macrophage cells to induce the expression of inflammatory cytokines and atherosclerosis-promoting factors through activation of NF-κB signaling. Therefore, a test substance that inhibits the induction of the expression of these factors can be used as a therapeutic agent for atherosclerosis.

[0043] That is, the present invention provides a method for screening therapeutic agents for arteriosclerosis, comprising the steps of culturing vascular smooth muscle cells, vascular endothelial cells or macrophage cells in the presence of GIP_HUMAN[22-51] (SEQ ID NO: 5) or a mutant peptide thereof and a test substance, and measuring the expression levels of inflammatory cytokines or arteriosclerosis-promoting factors in the cells, wherein a significant decrease in the expression levels compared to that in the absence of the test substance indicates that the test substance is a therapeutic agent for arteriosclerosis. The method of the second embodiment can also be used to screen therapeutic agents for arteriosclerosis.

[0044] The test substances are the same as those described above. Examples of inflammatory cytokines, chemokines, and arteriosclerosis-promoting factors include angiopoietin 2 (Angpt2), angiopoietin 3 (Angpt3), serum amyloid P (SAP), CXCL16, PCSK9, fetuin A, cystatin C, low density lipoprotein receptor (LDLR), MMP-3, IGF binding protein (IGFBP), vascular cell adhesion molecule-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), and endostatin.

[0045] [Arteriosclerosis treatment] In one embodiment, the present invention relates to (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, or (x) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which 1 to 3 amino acids have been deleted, substituted or added, and which is ApoE - / -The present invention provides a therapeutic agent for arteriosclerosis, which contains as an active ingredient an antibody or a fragment thereof against a peptide that has activity of promoting arteriosclerosis when administered to mice.

[0046] As described later in the Examples, the inventors administered an antibody against GIP_HUMAN[22-51] (SEQ ID NO: 5) to a mouse model of atherosclerosis, ApoE - / - It was revealed that when administered to mice, arteriosclerosis was significantly suppressed.

[0047] Therefore, the above-mentioned antibody or a fragment thereof can be used as a therapeutic agent for arteriosclerosis. The antibody is preferably a human antibody. The antibody fragment and the human antibody are the same as those described above.

[0048] [Other embodiments] (Treatment method for type 2 diabetes) In one embodiment, the present invention provides a method for treating type 2 diabetes, comprising administering to a patient in need of treatment an effective amount of (i) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, (ii) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1 and having activity of suppressing food intake or water drinking when administered to a mammal, (iii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, or (iv) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2 and having activity of suppressing food intake or water drinking when administered to a mammal. The treatment method of this embodiment can also be said to be a method of suppressing food intake or water drinking in a patient.

[0049] In one embodiment, the present invention provides: (i) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (ii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which one to three amino acids have been deleted, substituted or added, and which has activity to suppress eating or drinking when administered to a mammal; (iii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2; or (iv) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which one to three amino acids have been deleted, substituted or added, and which has activity to suppress eating or drinking when administered to a mammal, all of which are used for the treatment of type 2 diabetes.

[0050] In one embodiment, the present invention provides use of (i) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (ii) a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 1 in which one to three amino acids have been deleted, substituted or added, and which has activity to suppress eating or drinking when administered to a mammal; (iii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2; or (iv) a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 2 in which one to three amino acids have been deleted, substituted or added, and which has activity to suppress eating or drinking when administered to a mammal, for the manufacture of a therapeutic agent for type 2 diabetes.

[0051] (Method of treating inflammatory disease or tumor) In one embodiment, the present invention provides a method for treating an inflammatory disease or tumor, comprising administering to a patient in need of treatment an effective amount of an antibody or a fragment thereof against (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 3 in which one to three amino acids are deleted, substituted or added, and which has activity of inducing expression of an inflammatory cytokine, a chemokine, a cell growth factor or an angiogenesis promoting factor through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells, or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (viii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which one to three amino acids are deleted, substituted or added, and which has activity of inducing expression of an inflammatory cytokine, a chemokine, a cell growth factor or angiogenesis promoting factor through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells, or macrophage cells.

[0052] Inflammatory diseases include rheumatoid arthritis, cytokine storms such as COVID-19 severe pneumonia, etc. The tumor is not particularly limited as long as it is a tumor accompanied by angiogenesis.

[0053] In one embodiment, the present invention provides an antibody or a fragment thereof for use in the treatment of an inflammatory disease or tumor: (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 in which one to three amino acids are deleted, substituted or added, and which has activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (viii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which one to three amino acids are deleted, substituted or added, and which has activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells.

[0054] In one embodiment, the present invention provides use of an antibody or a fragment thereof against (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 3 in which one to three amino acids are deleted, substituted or added, and which has activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (viii) a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 4 in which one to three amino acids are deleted, substituted or added, and which has activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells, for the manufacture of a therapeutic agent for an inflammatory disease or tumor.

[0055] (Treatment method for arteriosclerosis) In one embodiment, the present invention relates to (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, or (x) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which 1 to 3 amino acids have been deleted, substituted or added, and which is ApoE - / - The present invention provides a method for treating arteriosclerosis, which comprises administering to a patient in need of treatment an effective amount of an antibody or a fragment thereof against a peptide that has activity of promoting arteriosclerosis when administered to a mouse.

[0056] In one embodiment, the present invention relates to a peptide for use in the treatment of arteriosclerosis, which comprises (ix) the amino acid sequence set forth in SEQ ID NO: 5, or (x) the amino acid sequence set forth in SEQ ID NO: 5 in which 1 to 3 amino acids have been deleted, substituted or added, and which is an ApoE - / - The present invention provides an antibody or a fragment thereof against a peptide that has an activity of promoting arteriosclerosis when administered to mice.

[0057] In one embodiment, the present invention relates to a peptide for producing a medicament for arteriosclerosis, comprising (ix) the amino acid sequence of SEQ ID NO: 5, or (x) the amino acid sequence of SEQ ID NO: 5 in which 1 to 3 amino acids have been deleted, substituted or added, and which is an ApoE - / - The present invention provides use of an antibody or a fragment thereof against a peptide having an activity of promoting arteriosclerosis when administered to a mouse. EXAMPLES

[0058] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0059] [Experimental Example 1] The inventors have developed an improved differential solubilization method (see Non-Patent Document 2) to identify many peptides present in human plasma. Among them, we focused on the peptides having the amino acid sequences of SEQ ID NOs: 1 to 5 and carried out the following experiments.

[0060] <Examination of SBSN_HUMAN[225 - 237] (SEQ ID NO: 3), SBSN_HUMAN[243 - 259] (SEQ ID NO: 4), and SBSN_HUMAN[279 - 295] (SEQ ID NO: 1)> [Experimental Example 2] (Examination of the binding of peptides to vascular smooth muscle cells) The binding of SBSN_HUMAN[225 - 237] (SEQ ID NO: 3), SBSN_HUMAN[243 - 259] (SEQ ID NO: 4), and SBSN_HUMAN[279 - 295] (SEQ ID NO: 1) to vascular smooth muscle cells was examined.

[0061] First, each peptide was chemically synthesized. A 5 - carboxyfluorescein (FAM) label was attached to the N - terminus of each peptide. Subsequently, each peptide was added to the medium of HAoSMC, a human vascular smooth muscle cell line, to a final concentration of 1 μM, and the presence or absence of binding to the cell surface was evaluated using a plate reader and a confocal microscope.

[0062] Figure 1(a) is a graph showing the results of measuring the binding of SBSN_HUMAN[225 - 237] (SEQ ID NO: 3) to vascular smooth muscle cells by detecting the fluorescence of FAM using a plate reader.

[0063] Figure 1(b) is a graph showing the results of measuring the binding of SBSN_HUMAN[243 - 259] (SEQ ID NO: 4) to vascular smooth muscle cells by detecting the fluorescence of FAM using a plate reader.

[0064] As a result, it became clear that for SBSN_HUMAN[225 - 237] (SEQ ID NO: 3) and SBSN_HUMAN[243 - 259] (SEQ ID NO: 4), the fluorescence intensity increased rapidly within 30 minutes after addition to the cell medium, and the binding was then maintained. On the other hand, it became clear that SBSN_HUMAN[279 - 295] (SEQ ID NO: 1) did not show binding to vascular smooth muscle cells.

[0065] Figures 1(c) to (f) are confocal micrographs taken. Figure 1(c) is a control where no peptide was added, Figure 1(d) is the result where SBSN_HUMAN[225-237] (SEQ ID NO: 3) was added to the medium, Figure 1(e) is the result where SBSN_HUMAN[243-259] (SEQ ID NO: 4) was added to the medium, and Figure 1(f) is the result where SBSN_HUMAN[279-295] (SEQ ID NO: 1) was added to the medium. As a result, FAM fluorescence was observed in Figures 1(d) and (e), but no fluorescence was observed in Figure 1(f).

[0066] These results demonstrate that SBSN_HUMAN[225-237] (sequence number 3) and SBSN_HUMAN[243-259] (sequence number 4) bind to vascular smooth muscle cells, whereas SBSN_HUMAN[279-295] (sequence number 1) does not bind to vascular smooth muscle cells.

[0067] [Experimental Example 3] (Examination of peptide functions) HAoSMC, a human vascular smooth muscle cell line, was cultured until confluent and then further cultured in serum-free medium for 16 hours. Then, SBSN_HUMAN[225-237] (SEQ ID NO: 3) or SBSN_HUMAN[243-259] (SEQ ID NO: 4) was added to the medium to a final concentration of 0.1 μM, and cultured for 24 hours. Next, the secretion amounts of cytokines, chemokines, cell growth factors, and cell death-related factors expressed in the culture supernatant were evaluated using a commercially available kit (product name: "Proteome Profiler Human XL Cytokine Arrays", R&D Systems).

[0068] FIG. 2(a) shows the results when SBSN_HUMAN[225-237] (SEQ ID NO: 3) was added to the medium. The table in FIG. 2(a) shows the relative increase in secretion amount for the top 5 factors with increased secretion amount. FIG. 2(b) shows the results when SBSN_HUMAN[243-259] (SEQ ID NO: 4) was added to the medium. The table in FIG. 2(b) shows the relative increase in secretion amount for the top 5 factors with increased secretion amount.

[0069] As a result, it was revealed that SBSN_HUMAN[225-237] (sequence number 3) significantly promoted the secretion of potent cytokines, chemokines, growth factors, angiogenesis-promoting factors, and apoptosis-regulating factors, including VEGFa, DKK1, Endoglin, uPAR, and cystatin C.

[0070] In addition, it was revealed that SBSN_HUMAN[243-259] (SEQ ID NO: 4) promoted the secretion of HGF, VEGFa, DKK1, OPN, Interleukin-6 (IL-6), and the like.

[0071] From the above results, it was revealed that SBSN_HUMAN[225-237] (sequence number 3) and SBSN_HUMAN[243-259] (sequence number 4) have strong inflammation-inducing, cell proliferation-promoting, and arteriosclerosis-promoting effects on vascular smooth muscle cells.

[0072] [Experimental Example 4] (Study of peptide-mediated signal transduction mechanisms) SBSN_HUMAN[225-237] (SEQ ID NO: 3) or SBSN_HUMAN[243-259] (SEQ ID NO: 4) was added to the culture medium of HAoSMC, a human vascular smooth muscle cell line, and the degradation of IκB-α and the localization of NF-κB p65 were examined. The degradation of IκB-α was examined by Western blotting. The localization of NF-κB p65 was examined by immunostaining.

[0073] Figure 3(a) is a photograph showing the results of Western blotting to detect the presence of IκB-α 0, 5, 10, 15, 30, and 60 minutes after adding SBSN_HUMAN[225-237] (SEQ ID NO: 3) to the medium of HAoSMCs, and also shows the results of detecting β-actin as a loading control.

[0074] Figure 3(b) is a photograph showing the results of Western blotting to detect the presence of IκB-α 0, 5, 10, 15, 30, and 60 minutes after adding SBSN_HUMAN[243-259] (SEQ ID NO: 4) to the medium of HAoSMCs, and also shows the results of detecting β-actin as a loading control.

[0075] Figures 3(c) to (e) are fluorescent micrographs showing the results of detecting NF-κB p65 by immunostaining. Figure 3(c) is a photograph taken immediately (0 min) after peptides were added to the HAoSMC medium. Figure 3(b) is a photograph taken 120 min after SBSN_HUMAN[225-237] (SEQ ID NO: 3) was added to the HAoSMC medium. Figure 3(c) is a photograph taken 120 min after SBSN_HUMAN[243-259] (SEQ ID NO: 4) was added to the HAoSMC medium. In Figures 3(b) and (c), the arrowheads indicate the immunoreactivity of NF-κB p65 in the cell nucleus.

[0076] The results showed that addition of either peptide resulted in the breakdown of IκB-α and the translocation of NF-κB p65 into the nucleus.

[0077] From the above results, it was revealed that SBSN_HUMAN[225-237] (SEQ ID NO: 3) and SBSN_HUMAN[243-259] (SEQ ID NO: 4) exhibit an NF-κB activation promoting effect. This suggests that the activation of NF-κB is involved in the induction of various cytokines, chemokines, growth factors, angiogenesis control factors, and the like, as shown in Experimental Example 3.

[0078] [Experimental Example 5] (Study of peptide-mediated signal transduction mechanisms) In order to clarify whether NF-κB is directly involved in the induction of various cytokines, chemokines, growth factors, angiogenesis control factors, etc. by SBSN_HUMAN[225-237] (sequence number 3) and SBSN_HUMAN[243-259] (sequence number 4), the inhibitory effect of MG132 (CAS number: 133407-82-6), a proteasome inhibitor, on VEGF expression was evaluated.

[0079] HAoSMC, a human vascular smooth muscle cell line, was cultured until confluent and then further cultured in serum-free medium for 16 hours. Then, MG132 was added to the medium at a final concentration of 100 nM and pretreated for 30 minutes. Then, SBSN_HUMAN[225-237] (SEQ ID NO: 3) or SBSN_HUMAN[243-259] (SEQ ID NO: 4) was added to a final concentration of 0.1 μM and cultured for 8 hours. Then, the expression of the VEGF gene was evaluated by quantitative RT-PCR.

[0080] Figure 4(a) is a graph showing the results of quantitative RT-PCR when SBSN_HUMAN[225-237] (SEQ ID NO: 3) was added to the HAoSMC medium, and Figure 4(b) is a graph showing the results of quantitative RT-PCR when SBSN_HUMAN[243-259] (SEQ ID NO: 4) was added to the HAoSMC medium.

[0081] 4(a) and (b), "-" indicates that MG132, SBSN_HUMAN[225-237] (SEQ ID NO: 3), or SBSN_HUMAN[243-259] (SEQ ID NO: 4) was not added to the medium, and "+" indicates that MG132, SBSN_HUMAN[225-237] (SEQ ID NO: 3), or SBSN_HUMAN[243-259] (SEQ ID NO: 4) was added to the medium. Additionally, "**" indicates that there is a significant difference at p<0.01.

[0082] As a result, it was revealed that pretreatment with MG132 significantly suppressed the expression of the VEGF gene. This result indicates that NF-κB is directly involved in the induction of various cytokines, chemokines, growth factors, angiogenesis regulators, etc. by SBSN_HUMAN[225-237] (SEQ ID NO: 3) and SBSN_HUMAN[243-259] (SEQ ID NO: 4).

[0083] [Experimental Example 6] (Administration of peptides to mice) SBSN_HUMAN[279-295] (SEQ ID NO: 1) was administered to mice, and its function was examined. C57BL / 6 mice (adult males, Clea) were kept at 22-25°C under a 12-hour light-dark cycle, with free food and water intake. The mice were fed normal food, CE2. After habituation to intraperitoneal administration of saline for at least 7 days, SBSN_HUMAN[279-295] (SEQ ID NO: 1) was administered intraperitoneally 30 minutes before the start of the dark period, and the amount of food intake, water intake, and activity during the dark period were monitored over time using a food intake, water intake, and activity monitoring system (product name "ACTIMO-100M", Shin Factory) under free movement. The dose of SBSN_HUMAN[279-295] (SEQ ID NO: 1) was 10 pmol / mouse or 100 pmol / mouse.

[0084] Figures 5(a)-(f) are graphs showing the results of monitoring food intake, water intake, and activity. Figures 5(a)-(c) show the results of administering 10 pmol / mouse of SBSN_HUMAN[279-295] (SEQ ID NO: 1), and Figures 5(d)-(f) show the results of administering 100 pmol / mouse of SBSN_HUMAN[279-295] (SEQ ID NO: 1).

[0085] In addition, the vertical axis of Figures 5(a) and (d) indicates the amount of food intake (g), and the horizontal axis indicates the time (min). In Figures 5(b) and (e), the vertical axis indicates the amount of water consumed (mL), and the horizontal axis indicates the time (min). In Figures 5(c) and (f), the vertical axis indicates the amount of activity (counts), and the horizontal axis indicates the time (min). In Figures 5(a) to (f), "vehicle" indicates that only purified water not containing peptide was administered. Furthermore, "*" indicates that there is a significant difference at p<0.05, and "**" indicates that there is a significant difference at p<0.01.

[0086] As a result, a significant decrease in food intake and water intake was observed when only 10 pmol / mouse of SBSN_HUMAN[279-295] (SEQ ID NO: 1) was administered. This result indicates that SBSN_HUMAN[279-295] (SEQ ID NO: 1) is a potent endogenous factor that regulates food intake and water intake.

[0087] [Experimental Example 7] (Analysis of peptide expression in various major human organs) Rabbits were immunized with SBSN_HUMAN[225-237] (SEQ ID NO: 3), SBSN_HUMAN[243-259] (SEQ ID NO: 4), and SBSN_HUMAN[279-295] (SEQ ID NO: 1), and specific polyclonal antibodies against each peptide were produced.

[0088] Next, a commercially available human tissue array (catalog number "MNO341", US Biomax) was immunostained with each polyclonal antibody, and peptide expression in various major human organs was analyzed. The results are shown in Table 1 below. In Table 1, "-" indicates that no expression was observed, and "+" and "++" indicate that expression was observed. Furthermore, "++" indicates that the expression level was higher than "+".

[0089] [Table 1]

[0090] As a result, it was revealed that SBSN_HUMAN[243-259] (SEQ ID NO: 4) is not a peptide universally expressed in various tissues, but is specifically expressed in the liver and pancreas, whereas SBSN_HUMAN[225-237] (SEQ ID NO: 3) and SBSN_HUMAN[279-295] (SEQ ID NO: 1) are peptides universally expressed in many tissues.

[0091] [Experimental Example 8] (Analysis of peptide expression in various human cell lines) Peptide expression in various cultured human cells was analyzed by immunostaining using specific polyclonal antibodies against SBSN_HUMAN[225-237] (sequence number 3), SBSN_HUMAN[243-259] (sequence number 4), and SBSN_HUMAN[279-295] (sequence number 1).

[0092] 6(a)-(e), (a')-(e'), (a'')-(e'') are fluorescent micrographs showing the results of immunostaining. FIG. 6(a)-(e) show the results of staining with an antibody against SBSN_HUMAN[225-237] (sequence number 3), (a')-(e') show the results of staining with an antibody against SBSN_HUMAN[243-259] (sequence number 4), and (a'')-(e'') show the results of staining with an antibody against SBSN_HUMAN[279-295] (sequence number 4). The antibody dilution ratio was 1000 times in all cases.

[0093] In addition, Figure 6 (a), (a'), and (a'') show the staining results of HAoSMC, a human vascular smooth muscle cell line, Figure 6 (b), (b'), and (b'') show the staining results of human monocyte THP-1-derived macrophage cells, Figure 6 (c), (c'), and (c'') show the staining results of healthy human macrophage cells, Figure 6 (d), (d'), and (d'') show the staining results of HaCaT, a human epidermal keratinocyte cell line, and Figure 6 (e), (e'), and (e'') show the staining results of HepG2, a human liver cancer cell line. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI).

[0094] As a result, it was revealed that all three types of peptides were expressed in cancer cells and macrophage cells.

[0095] <II. Examination of ANGT_HUMAN[448-462] (SEQ ID NO: 2)> [Experimental Example 9] (Examination of the binding of the peptide to macrophage cells) The binding of ANGT_HUMAN[448-462] (SEQ ID NO: 2) to macrophage cells was examined. First, the peptide was chemically synthesized. The N-terminus of the peptide was labeled with 5-carboxyfluorescein (FAM). Subsequently, the peptide was added to the medium of THP1 cells, which is a human monocyte cell line, to a final concentration of 1 μM and incubated for 60 minutes, after which the presence or absence of binding to the cell surface was evaluated using a confocal microscope.

[0096] Also, for comparison, samples pretreated with β-casomorphin 5 at a final concentration of 10 μM or β-casomorphin 7 at a final concentration of 10 μM for 30 minutes were prepared. β-Casomorphin 5 and β-casomorphin 7 are known to be ligands of the β-chain of ATP synthase. At the same time, immunostaining of the α-chain and β-chain of ATP synthase was also performed.

[0097] Figures 7(a) to (f) are the photographed confocal microscope images. Figures 7(a) to (c) are the results of immunostaining of the α-chain of ATP synthase, and Figures 7(d) to (f) are the results of immunostaining of the β-chain of ATP synthase.

[0098] Figures 7(b) and (e) are the results of pretreatment with β-casomorphin 5, which is considered to show a partial inhibitory effect on the α-chain of ATP synthase before administration of FAM-labeled ANGT_HUMAN[448-462], and Figures 7(c) and (f) are the results of pretreatment with β-casomorphin 7, which is considered to show a competitive inhibitory effect on the α-chain of ATP synthase, and Figures 7(a) and (d) are the results without pretreatment.

[0099] In Figures 7(a) to (f), the leftmost photograph is the result of staining the ATP synthase α chain (Figures 7(a) to (c)) or the ATP synthase β chain (Figures 7(d) to (f)), the second photograph from the left is the result of detecting fluorescence due to binding of FAM-labeled ANGT_HUMAN[448-462] (sequence number 2) to cells, the third photograph from the left is the result of staining the nucleus with DAPI, and the rightmost photograph is a composite of the first to third photographs from the left.

[0100] As a result, it was revealed that ANGT_HUMAN[448-462] (SEQ ID NO: 2) colocalizes with the ATP synthase α and β chains, which are known to act by binding to anti-feedant factors and anti-angiogenic factors. It was also revealed that the binding of ANGT_HUMAN[448-462] (SEQ ID NO: 2) to the cell surface ATP synthase α and β chains is competitively inhibited by β-casomorphin 5 and β-casomorphin 7.

[0101] [Experimental Example 10] (Administration of peptides to mice 1) ANGT_HUMAN[448-462] (SEQ ID NO: 2) was administered to mice, and its function was examined. C57BL / 6 mice (adult males, Claire) were kept at 22-25°C, with a 12-hour light-dark cycle, and with free food and water intake. The mice were fed normal food, CE2. After habituation to intraperitoneal administration of saline for at least 7 days, ANGT_HUMAN[448-462] (SEQ ID NO: 2) was administered intraperitoneally 30 minutes before the start of the dark period, and the amount of food intake, water intake, and activity during the dark period were monitored over time using a food intake, water intake, and activity monitoring system (product name "ACTIMO-100M", Shin Factory) under free movement. The dose of ANGT_HUMAN[448-462] (SEQ ID NO: 2) was 100 pmol / mouse.

[0102] Figures 8(a)-(c) are graphs showing the monitoring results of food intake, water intake, and activity. In Figure 8(a), the vertical axis shows food intake (g), and the horizontal axis shows time (min). In Figure 8(b), the vertical axis shows water intake (mL), and the horizontal axis shows time (min). In Figure 8(c), the vertical axis shows activity (counts), and the horizontal axis shows time (min). In Figure 8(a)-(c), "vehicle" indicates that only purified water not containing peptide was administered. Additionally, "*" indicates that there is a significant difference at p<0.05, and "**" indicates that there is a significant difference at p<0.01.

[0103] As a result, a significant decrease in food intake and water intake was observed when only 100 pmol / mouse of ANGT_HUMAN[448-462] (SEQ ID NO: 2) was administered, indicating that ANGT_HUMAN[448-462] (SEQ ID NO: 2) is a potent peripheral suppressor of food intake and water intake.

[0104] [Experimental Example 11] (Administration of peptides to mice 2) ANGT_HUMAN[448-462] (SEQ ID NO: 2) was administered to mice to examine its function. In Experiment 10, it was administered intraperitoneally, whereas in this experiment, it was administered intracerebroventricularly. C57BL / 6 mice (adult males, Claire) were kept at 22-25°C, with a 12-hour light-dark cycle, and with free food and water intake. The mice were fed with normal food, CE2. ANGT_HUMAN[448-462] (SEQ ID NO: 2) was administered intracerebroventricularly through an implanted intracerebroventricular catheter, and the amount of food intake, water intake, and activity during the dark period were monitored over time using a food intake, water intake, and activity monitoring system (product name "ACTIMO-100M", Synfactory) under free movement. The dose of ANGT_HUMAN[448-462] (SEQ ID NO: 2) was 1 pmol / mouse.

[0105] Figures 9(a) to (c) are graphs showing the monitoring results of food intake, water intake, and activity level. The vertical axis of Fig. 9(a) indicates food intake (g), and the horizontal axis indicates time (hours). The vertical axis of Fig. 9(b) indicates water intake (mL), and the horizontal axis indicates time (hours). The vertical axis of Fig. 9(c) indicates activity level (count), and the horizontal axis indicates time (hours). In Figs. 9(a) to (c), "vehicle" indicates administration of purified water without peptide. Also, "*" indicates the presence of a significant difference at p < 0.05.

[0106] As a result, a significant decrease in food intake and water intake was observed only by administering 1 pmol / mouse of ANGT_HUMAN[448-462] (SEQ ID NO: 2). This result further supports that ANGT_HUMAN[448-462] (SEQ ID NO: 2) is a potent central appetite and water intake inhibitor. Also, since ANGT_HUMAN[448-462] (SEQ ID NO: 2) exerts an effect at a very low concentration, it may have an effect of reducing food intake and water intake by oral administration.

[0107] <III. Examination of GIP_HUMAN[22-51] (SEQ ID NO: 5)> [Experimental Example 12] (Examination of the binding of the peptide to cells) The binding of GIP_HUMAN[22-51] (SEQ ID NO: 5) to vascular endothelial cells and macrophage cells was examined. First, the peptide was chemically synthesized. The N-terminus of the peptide was labeled with 5-carboxyfluorescein (FAM). Subsequently, the peptide was added to the medium of HAoEC, a human aortic endothelial cell line, at a final concentration of 1 μM and incubated for 30 minutes, and then the presence or absence of binding to the cell surface was evaluated by a confocal microscope. Also, the nuclei were stained with DAPI.

[0108] Figs. 10(a) and (b) are the photographed confocal microscope images. Fig. 10(a) is the result without reacting the peptide, and Fig. 10(b) is the result of reacting the peptide. As a result, it was revealed that GIP_HUMAN[22-51] (SEQ ID NO: 5) binds to vascular endothelial cells.

[0109] Next, FAM-labeled GIP_HUMAN[22-51] (SEQ ID NO: 5) was added to the medium of macrophage cells derived from THP1 cells, a human monocyte cell line, to a final concentration of 1 μM, and incubated for 5 minutes, after which the presence or absence of binding to the cell surface was evaluated using a confocal microscope. Nuclei were also stained with DAPI.

[0110] Figures 10(c) and (d) are confocal micrographs. Figure 10(c) shows the results without peptide reaction, and Figure 10(d) shows the results with peptide reaction. As a result, it was revealed that GIP_HUMAN[22-51] (SEQ ID NO: 5) binds to macrophage cells.

[0111] [Experimental Example 13] (Study of peptide-mediated signal transduction mechanisms) GIP_HUMAN[22-51] (SEQ ID NO: 5) was contacted with human aortic endothelial cells and human macrophage cells to examine the translocation of NF-κB p65 to the nucleus.

[0112] The peptides were added to the culture medium of HAoEC, a human aortic endothelial cell line, or macrophage cells derived from THP1 cells, a human monocyte cell line, at a final concentration of 1 μM and incubated for 60 minutes, after which NF-κB p65 was stained by immunostaining.

[0113] Figures 11(a) and (b) are photographs showing the results for aortic endothelial cells. Figure 11(a) shows the results when no peptide was added, and Figure 11(b) shows the results when GIP_HUMAN[22-51] (SEQ ID NO: 5) was added. As a result, it was revealed that when GIP_HUMAN[22-51] (SEQ ID NO: 5) was added to the culture medium of aortic endothelial cells, NF-κB was translocated to the nucleus.

[0114] Figures 11(c) and (d) are photographs showing the results for macrophage cells. Figure 11(c) shows the results when no peptide was added, and Figure 11(d) shows the results when GIP_HUMAN[22-51] (SEQ ID NO: 5) was added. As a result, it was revealed that when GIP_HUMAN[22-51] (SEQ ID NO: 5) was added to the medium of macrophage cells, NF-κB was translocated to the nucleus.

[0115] [Experimental Example 14] (Study of effects on arteriosclerosis) The effect of GIP_HUMAN[22-51] (sequence number 5) on arteriosclerosis was examined. - / - Mice (17 weeks old) were administered GIP_HUMAN[22-51] (SEQ ID NO: 5) or saline for 4 weeks using an osmotic pump, and the progression of arteriosclerosis was evaluated. The effect of administering a polyclonal antibody against GIP_HUMAN[22-51] (SEQ ID NO: 5) was also examined. The polyclonal antibody was produced by immunizing rabbits with GIP_HUMAN[22-51] (SEQ ID NO: 5).

[0116] Figures 12(a)-(d) show macroscopic tissue specimens showing the results of the study, and (e)-(l) show microscopic photographs. Figures 12(a), (e), and (i) show the results of administering only physiological saline. Figures 12(b), (c), (f), (g), (j), and (k) show the results of administering GIP_HUMAN[22-51] (SEQ ID NO: 5) at a dose of 0.6 nM / kg / hour. Figures 12(c), (d), (g), (h), (k), and (l) show the results of administering an antibody against GIP_HUMAN[22-51] (SEQ ID NO: 5) at a dose of 1.4 μg / kg / hour.

[0117] Figures 12(a),(b),(c),(d) show the results of staining the surface of the aorta with Oil Red O. Figures 12(e),(f),(g),(h) show the results of staining the cross section of the aortic root with Oil Red O. Figures 12(i),(j),(k),(l) show the results of staining with anti-MOMA-2 antibody. Anti-MOMA-2 antibody is an antibody that binds to monocytes and macrophages.

[0118] As a result, it was revealed that administration of GIP_HUMAN[22-51] (sequence number 5) accelerates the progression of arteriosclerosis, and that administration of an antibody against GIP_HUMAN[22-51] (sequence number 5) can suppress this progression.

[0119] Subsequently, ApoE mice were administered GIP_HUMAN[22-51] (SEQ ID NO: 5) for 4 weeks. - / - Mice and ApoE mice administered GIP_HUMAN[22-51] (SEQ ID NO: 5) and an antibody against GIP_HUMAN[22-51] (SEQ ID NO: 5) for 4 weeks - / - Cytokines and chemokines in mouse serum were quantified using a commercially available antibody array (product name: Mouse XL Cytokine Kit, R&D Systems).

[0120] Figure 13(a) shows the relative abundance of cytokines and chemokines in the serum of mice administered GIP_HUMAN[22-51] (SEQ ID NO: 5). Figure 13(b) shows the relative abundance of cytokines and chemokines in the serum of mice administered GIP_HUMAN[22-51] (SEQ ID NO: 5) and an antibody against GIP_HUMAN[22-51] (SEQ ID NO: 5).

[0121] As a result, it was revealed that administration of GIP_HUMAN[22-51] (sequence number 5) increased serum levels of inflammatory cytokines and atherosclerosis-promoting factors such as angiopoietin 2 (Angpt2), serum amyloid P (SAP), CXCL16, PCSK9, fetuin A, and MMP-3.

[0122] On the other hand, it was revealed that administration of an antibody against GIP_HUMAN[22-51] (sequence number 5) reduced serum levels of ICAM-1, VCAM-1, fetuin A, PCSK9, CXCL16, SAP, and the like.

[0123] The above results indicate that GIP_HUMAN[22-51] (SEQ ID NO: 5) is an endogenous inducer of inflammatory cytokines, chemokines and atherosclerosis-promoting factors. [Industrial Applicability]

[0124] According to the present invention, it is possible to provide a novel physiologically active peptide present in human blood. More specifically, it is possible to provide an anti-obesity drug, an inducer of expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling in vascular smooth muscle cells, vascular endothelial cells or macrophage cells, a method for screening a therapeutic agent for inflammatory diseases or tumors, a therapeutic agent for inflammatory diseases or tumors, an arteriosclerosis promoter and an arteriosclerosis therapeutic agent.

Claims

1. (iii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, or (iv) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2, and having an activity of suppressing food intake or water drinking when administered to a mammal; An anti-obesity drug whose active ingredient is

2. (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3, and having an activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4, or (viii) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 4, and having an activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; An agent for inducing the expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signal transduction in vascular smooth muscle cells, vascular endothelial cells or macrophage cells, comprising the above-mentioned active ingredient.

3. (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3, and having an activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4, or (viii) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 4, and having an activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; identifying a receptor for screening test substances to identify inhibitors of the receptor; The inhibitor is a therapeutic agent for an inflammatory disease or a tumor. A method for screening a therapeutic agent for an inflammatory disease or tumor.

4. (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3, and having an activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4, or (viii) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 4, and having an activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; and The method includes a step of culturing vascular smooth muscle cells, vascular endothelial cells, or macrophage cells in the presence of a test substance, and measuring the expression level of an inflammatory cytokine, a chemokine, a cell growth factor, or an angiogenesis promoting factor in the cells; A significant decrease in the expression level compared to the absence of the test substance indicates that the test substance is a therapeutic agent for an inflammatory disease or a tumor. A method for screening a therapeutic agent for an inflammatory disease or tumor.

5. (v) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (vi) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3, and having an activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; (vii) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4, or (viii) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 4, and having an activity of inducing expression of inflammatory cytokines, chemokines, cell growth factors or angiogenesis promoting factors through activation of NF-κB signaling when contacted with vascular smooth muscle cells, vascular endothelial cells or macrophage cells; A therapeutic agent for inflammatory diseases or tumors, comprising as an active ingredient an antibody against the

6. (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO:5, or (x) an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 5, and ApoE －／－ A peptide having an activity of promoting arteriosclerosis when administered to mice, An arteriosclerosis promoter whose active ingredient is

7. (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO:5, or (x) an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 5, and ApoE －／－ A peptide having an activity of promoting arteriosclerosis when administered to mice, identifying a receptor for screening test substances to identify inhibitors of the receptor; The inhibitor is an arteriosclerosis treatment agent. A method for screening an agent for treating arteriosclerosis.

8. (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO:5, or (x) an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 5, and ApoE －／－ A peptide having an activity of promoting arteriosclerosis when administered to a mouse, and The method includes a step of culturing vascular smooth muscle cells, vascular endothelial cells, or macrophage cells in the presence of a test substance, and measuring the expression level of inflammatory cytokines or arteriosclerosis-promoting factors in the cells, A significant decrease in the expression level compared to that in the absence of the test substance indicates that the test substance is an agent for treating arteriosclerosis. A method for screening an agent for treating arteriosclerosis.

9. (ix) a peptide consisting of the amino acid sequence set forth in SEQ ID NO:5, or (x) an amino acid sequence in which 1 to 3 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 5, and ApoE －／－ A peptide having an activity of promoting arteriosclerosis when administered to mice, An agent for treating arteriosclerosis, comprising as an active ingredient an antibody against the

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