Peptides with mesenchymal stem cell mobilization activity

Abstract

The present inventors have synthesized a plurality of peptides each comprising a partial sequence of an artificial sequence peptide "1r10" ("1r10 fragment peptides") that has been discovered from their own studies in the past and have examined as to the presence or absence of an activity to mobilize a mesenchymal stem cell into peripheral blood. As a result, it is found that a 1r10 fragment peptide having a specific amino acid sequence has this activity. On the basis of this finding, a peptide is provided which is considered to mobilize a mesenchymal stem cell into peripheral blood to exhibit the therapeutic effect thereof on various diseases.

Description

[Technical field]

[0001] The present application relates to a peptide having mesenchymal stem cell mobilizing activity, a composition for mobilizing mesenchymal stem cells, and a therapeutic agent for diseases based on the mobilization of mesenchymal stem cells. [Background technology]

[0002] Mesenchymal stem cells contained in bone marrow fluid have the ability (multipotency) to differentiate into various tissues such as bone, cartilage, fat, muscle, nerve, and epithelium. In recent years, there have been many attempts to perform regenerative medicine (cell transplantation therapy) using bone marrow-derived mesenchymal stem cells grown by culture. However, the collection of bone marrow blood containing mesenchymal stem cells is performed by an invasive technique in which a thick needle is repeatedly inserted into the iliac bone, which places a heavy burden on the donor. In addition, mesenchymal stem cells gradually lose their proliferation ability and multipotency when subcultured outside the body. Furthermore, the cultivation of mesenchymal stem cells based on high-quality control to ensure the safety of in vivo transplantation requires special culture facilities such as a cell processing center (CPC), so the current situation is that it can only be performed at a limited number of universities and companies. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] PNAS 2011 Apr 19;108(16):6609-14 [Patent documents]

[0004] [Patent Document 1] WO2008 / 053892 [Patent Document 2] WO2009 / 133939 [Patent Document 3] WO2012 / 147470 Summary of the Invention [Problem to be solved by the invention]

[0005] The present application aims to develop a new regenerative medicine technology that can overcome the problems associated with cell transplantation therapy. [Means for solving the problem]

[0006] Based on their own research results, the present inventors have previously discovered an artificial sequence peptide "1r10" that has the activity of mobilizing mesenchymal stem cells into peripheral blood, and have filed a patent application for the peptide (PCT / JP2019 / 039232). In this study, the present inventors synthesized multiple peptides consisting of partial sequences of the artificial sequence peptide "1r10" (hereinafter also referred to as "1r10 fragment peptides") and investigated whether they have the activity of mobilizing mesenchymal stem cells into peripheral blood. As a result, they found that a 1r10 fragment peptide having a specific amino acid sequence exhibits the activity. Based on these findings, the present application provides peptides that are believed to have therapeutic effects on various diseases through the mobilization of mesenchymal stem cells into peripheral blood.

[0007] That is, the present application provides the following: [1] A composition for use in mobilizing mesenchymal stem cells into peripheral blood, comprising a peptide selected from the following: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [2] A composition for use in treating a disease or pathological condition in a subject by mobilizing mesenchymal stem cells into peripheral blood, comprising a peptide selected from: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [3] The composition according to [2], wherein the treatment of a disease or pathological condition is selected from anti-inflammatory treatment, immunomodulatory treatment, tissue regeneration-inducing treatment, and tissue fibrosis-inhibiting treatment. [4] The composition according to [2], wherein the disease or pathological condition is selected from inflammatory diseases, autoimmune diseases, diseases involving tissue damage, ischemia or necrosis, and fibrotic diseases. [5] The composition according to [2], wherein the disease or pathological condition is inflammatory bowel disease. [6] The composition according to [2], wherein the disease or pathological condition is ulcerative colitis. [7] The composition according to [2], wherein the disease or pathological condition is atopic dermatitis. [8] The composition according to [2], wherein the disease or pathological condition is cerebral infarction. [9] A composition for use in treating a disease selected from inflammatory bowel disease, atopic dermatitis and cerebral infarction, comprising a peptide selected from the following: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19.

[10] A peptide selected from: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [A1] A method for mobilizing mesenchymal stem cells into peripheral blood, comprising administering to a subject an effective amount of a peptide selected from the following: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [A2] 1. A method of treating a disease or pathological condition in a subject by mobilizing mesenchymal stem cells into peripheral blood, comprising administering to the subject an effective amount of a peptide selected from: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [A3] The method according to [A2], wherein the treatment of a disease or pathological condition is selected from anti-inflammatory treatment, immunomodulatory treatment, tissue regeneration-inducing treatment, and tissue fibrosis-inhibiting treatment. [A4] The method according to [A2], wherein the disease or pathological condition is selected from inflammatory diseases, autoimmune diseases, diseases involving tissue damage, ischemia or necrosis, and fibrotic diseases. [A5] The method according to [A2], wherein the disease or pathological condition is inflammatory bowel disease. [A6] The method according to [A2], wherein the disease or pathological condition is ulcerative colitis. [A7] The method according to [A2], wherein the disease or pathological condition is atopic dermatitis. [A8] The method according to [A2], wherein the disease or pathological condition is cerebral infarction. [A9] A method for treating a disease in a subject selected from inflammatory bowel disease, atopic dermatitis and cerebral infarction, comprising administering to the subject an effective amount of a peptide selected from: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [B1] 1. For use in mobilizing mesenchymal stem cells into peripheral blood, a peptide selected from: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [B2] 1. For use in treating a disease or pathological condition in a subject by mobilizing mesenchymal stem cells into peripheral blood, a peptide selected from: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [B3] The peptide according to [B2], wherein the treatment of a disease or pathological condition is selected from anti-inflammatory treatment, immunomodulatory treatment, tissue regeneration-inducing treatment, and tissue fibrosis-inhibiting treatment. [B4] The peptide according to [B2], wherein the disease or pathological condition is selected from inflammatory diseases, autoimmune diseases, diseases involving tissue damage, ischemia or necrosis, and fibrotic diseases. [B5] The peptide according to [B2], wherein the disease or pathological condition is inflammatory bowel disease. [B6] The peptide according to [B2], wherein the disease or pathological condition is ulcerative colitis. [B7] The peptide according to [B2], wherein the disease or pathological condition is atopic dermatitis. [B8] The peptide according to [B2], wherein the disease or pathological condition is cerebral infarction. [B9] 1. A peptide selected from the following for use in treating a disease selected from inflammatory bowel disease, atopic dermatitis and cerebral infarction: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [C1] Use of a peptide selected from the following in the manufacture of a medicament or reagent for mobilizing mesenchymal stem cells into peripheral blood: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [C2] 2. Use of a peptide selected from the following in the manufacture of a medicament for the treatment of a disease or pathological condition in a subject by mobilizing mesenchymal stem cells into peripheral blood: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [C3] The use described in [C2], wherein the treatment of a disease or pathological condition is selected from anti-inflammatory treatment, immunomodulatory treatment, tissue regeneration-inducing treatment, and tissue fibrosis-inhibiting treatment. [C4] The use described in [C2], wherein the disease or pathological condition is selected from inflammatory diseases, autoimmune diseases, diseases involving tissue damage, ischemia or necrosis, and fibrotic diseases. [C5] The use described in [C2], wherein the disease or pathological condition is inflammatory bowel disease. [C6] The use described in [C2], wherein the disease or pathological condition is ulcerative colitis. [C7] The use described in [C2], wherein the disease or pathological condition is atopic dermatitis. [C8] The use described in [C2], wherein the disease or pathological condition is cerebral infarction. [C9] Use of a peptide selected from the following in the manufacture of a medicament for the treatment of a disease selected from inflammatory bowel disease, atopic dermatitis and cerebral infarction: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19; (b) a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, and which contains the amino acid sequence of SEQ ID NO: 17 or 19; and (c) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, said peptide including the amino acid sequence of SEQ ID NO: 17 or 19. [Brief description of the drawings]

[0008] [Figure 1] This is a graph plotting the number of colonies obtained by culturing peripheral blood 16 hours after administration of saline, 1% DMSO, or peptide. In the graph, "Saline" and "DMSO" represent the control groups, while the others represent the peptide-administered groups. The number of colonies is shown per volume of peripheral blood (800 μL) taken from one mouse. In the error bars, the long horizontal bar represents the average value, and the short horizontal bar represents the standard deviation. The dotted line represents the threshold (number of colonies: 10) for determining the presence or absence of activity. [Diagram 2] This is a graph plotting the number of colonies obtained by culturing peripheral blood 16 hours after administration of saline or peptide. In the graph, "Saline" indicates the control group, and the others indicate the peptide-administered groups. The number of colonies is shown per volume of peripheral blood (800 μL) taken from one mouse. In the error bars, the long horizontal bar indicates the average value, and the short horizontal bar indicates the standard deviation. The dotted line indicates the threshold (number of colonies: 10) for determining the presence or absence of activity. [Diagram 3]This is a graph showing changes in mouse body weight (mean ± standard error (SEM); * p<0.05 (Student's t test)). In the graph, "saline" indicates the control group, and "1r10" indicates the peptide 1r10 administration group. The horizontal axis indicates the number of days after starting to drink dextran sodium sulfate (DSS) aqueous solution. [Figure 4] 1 is a graph showing the stool scores of mice (mean ± standard error (SEM)). In the graph, "saline" indicates the control group, and "1r10" indicates the peptide 1r10 administration group. The horizontal axis indicates the number of days after starting to drink dextran sodium sulfate (DSS) aqueous solution. [Diagram 5] 1 is a graph showing the DAI scores of mice (mean ± standard error (SEM); * p<0.05 (Mann Whitney U test)). In the graph, "saline" indicates the control group, and "1r10" indicates the peptide 1r10 administration group. The horizontal axis indicates the number of days after starting to drink dextran sodium sulfate (DSS) aqueous solution. [Figure 6] 1 is a graph showing colon length 10 days after starting to drink dextran sodium sulfate (DSS) aqueous solution (mean ± standard error (SEM)). In the graph, "saline" indicates the control group and "1r10" indicates the peptide 1r10 administration group. [Figure 7] This is a photograph showing the ear of a mouse four days after the start of peptide administration. The arrow indicates the edge of the ear where desquamation is likely to be observed. [Figure 8] This is a graph showing the change in ear thickness during the peptide administration period (the vertical axis is the relative value with the day of peptide administration starting as 100%). Each point shows the mean and standard error (*p<0.05; Repeated one way ANOVA test; post hoc Tukey-Kramer test). [Figure 9] Representative photographs showing HE stained images of auricular skin sections taken 4 days after the start of peptide administration. The arrows in the photographs indicate examples of measured dermal thickness. [Figure 10]This is a graph (Box-Whisker plot) showing the dermal thickness of each group on the 4th day after the start of peptide administration. The central box and whisker indicate the median, quartile, and maximum / minimum values, respectively. The diamond in the box indicates the average value. (**p<0.01 Mann Whitney U-test; 7 mice per group x 9 measurements per mouse.) [Figure 11] This is a photograph showing a multi-fluorescence immunostained image of an ear skin section on the 4th day after the start of peptide administration (Laminin (green)-CD45 (red)-DAPI (blue)). Laminin was used to label intra-tissue structures (blood vessels, basement membrane, and nerves), and DAPI was used as a counterstain for the DNA of each cell. [Figure 12] This is a graph (Box-Whisker plot) showing the number of CD45+ immune cells per unit area in high power field (HPF (400x)) in ear skin sections in each group on the 4th day after the start of peptide administration. The central box and whisker indicate the median, quartile, and maximum / minimum values, respectively. Diamonds within the boxes indicate the mean values. x indicates outliers. (**p<0.01 Mann Whitney U-test; 7 mice per group x 9 measurements per mouse. In both the control and peptide-administered groups, an outlier occurred in 1 out of 7 mice (approximately 14%), so the mean value of the other 6 mice without outliers was substituted for each group.) [Figure 13] This is a graph showing the cerebral infarct volume ratio 48 hours after right MCA occlusion (mean ± standard error). **p<0.01, Mann-Whitney U test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present inventors have discovered a substance that activates stem cells in vivo or mobilizes them to damaged tissues via peripheral circulation, and believe that this substance is promising as a new type of medicine that can overcome the weaknesses of cell therapy. Specifically, the present inventors have discovered that high mobility group box 1 (HMGB1) released from necrotic tissue mobilizes platelet-derived growth factor receptor α (PDGFRα)-positive cells (presumably mesenchymal stem cells: MSCs), which play a role in inducing tissue regeneration in vivo, to necrotic tissues via peripheral circulation, thereby suppressing inflammation in necrotic tissues and promoting tissue regeneration. The present inventors have also discovered that a fragment peptide of HMGB1 exhibits peripheral blood mobilization activity and tissue regeneration induction activity for mesenchymal stem cells. In this study, the inventors synthesized multiple peptides (hereinafter also referred to as "1r10 fragment peptides") consisting of partial sequences of the artificial sequence peptide "1r10" previously discovered, and examined whether they had the activity of mobilizing MSCs to the blood.As a result, it was found that 1r10 fragment peptides containing a specific amino acid sequence exhibit the activity, and that there are core regions at each of the N-terminus and C-terminus of peptide 1r10 that exhibit the activity of mobilizing MSCs to the blood.

[0010] It is well known to those skilled in the art that mesenchymal stem cells exert anti-inflammatory, immunomodulatory, and antifibrotic effects. In addition, since mesenchymal stem cells have the pluripotency to differentiate into various tissues, it is also well known to those skilled in the art that they exert an effect of promoting the regeneration of damaged tissues. Therefore, when a 1r10 fragment peptide having an activity of mobilizing mesenchymal stem cells into peripheral blood, or a peptide having an activity of mobilizing mesenchymal stem cells into peripheral blood containing the amino acid sequence of the fragment peptide, is administered to a subject, mesenchymal stem cells are mobilized into the peripheral blood, and the anti-inflammatory, immunomodulatory, antifibrotic, and tissue regeneration promoting effects of the mesenchymal stem cells (due to the differentiation and / or inflammation suppression effects of the mesenchymal stem cells) are thought to bring about therapeutic effects against various diseases.

[0011] The artificial sequence peptide 1r10 previously discovered by the present inventors shows therapeutic effects against inflammatory bowel disease, atopic dermatitis, and cerebral infarction, which is believed to be an effect brought about through mobilization of mesenchymal stem cells into peripheral blood. Therefore, a 1r10 fragment peptide having activity of mobilizing mesenchymal stem cells into peripheral blood, or a peptide having activity of mobilizing mesenchymal stem cells into peripheral blood, which contains the amino acid sequence of the fragment peptide, is believed to show therapeutic effects against inflammatory bowel disease, atopic dermatitis, and cerebral infarction, similar to the artificial sequence peptide 1r10.

[0012] In the present application, the term "artificial sequence peptide" refers to a peptide having an amino acid sequence that does not exist in nature. In the present application, the term "artificial sequence peptide" is also simply referred to as "artificial peptide."

[0013] In the present application, the term "1r10 fragment peptide" refers to a peptide consisting of a part of the amino acid sequence of the artificial sequence peptide 1r10 (SEQ ID NO: 1).

[0014] The present application provides a composition for use in mobilizing mesenchymal stem cells into peripheral blood, which contains a specific 1r10 fragment peptide or a peptide including the amino acid sequence of the fragment peptide.

[0015] The composition for use in the mobilization of mesenchymal stem cells into peripheral blood of the present application can be used as a pharmaceutical composition or a reagent composition. In the present application, the term "pharmaceutical composition" is used interchangeably with "medicine", "drug" or "pharmaceutical composition", and the term "reagent composition" is used interchangeably with "reagent".

[0016] The compositions for use in mobilizing mesenchymal stem cells into peripheral blood of the present application can be used to treat a disease or pathological condition in a subject, for example, by mobilizing mesenchymal stem cells into peripheral blood.

[0017] Mesenchymal stem cells mobilized into peripheral blood using the composition for mobilizing mesenchymal stem cells into peripheral blood of the present application can be collected ex vivo, concentrated, and used to treat a disease or pathological condition in a subject. The present application also provides the use of a specific 1r10 fragment peptide or a peptide containing the amino acid sequence of the fragment peptide in the manufacture of a medicine or reagent for collecting mesenchymal stem cells ex vivo.

[0018] The composition for use in mobilizing mesenchymal stem cells to peripheral blood of the present application can also be used, for example, in basic research, clinical research, etc. Examples of basic research and clinical research include, but are not limited to, research on mesenchymal stem cell mobilization in a test tube and research on mesenchymal stem cell mobilization in experimental animals. The present application also provides the use of a specific 1r10 fragment peptide or a peptide containing the amino acid sequence of the fragment peptide (hereinafter, these peptides are also referred to simply as "peptides") in the manufacture of medicines or reagents for basic research or clinical research.

[0019] The compositions for use in mobilizing mesenchymal stem cells into peripheral blood herein can include one or more peptides.

[0020] In the present application, the term "mesenchymal stem cell" refers to a cell that has the ability to differentiate into mesenchymal tissues / cells such as bone, cartilage, fat, and muscle, and the ability to self-replicate. In one embodiment, the mesenchymal stem cell also has the ability to differentiate into epithelial tissue and neural tissue. Mesenchymal stem cells with self-replicating ability can be identified, for example, by using as an indicator that they have colony-forming ability. In one embodiment, the mesenchymal stem cell is a cell that has colony-forming ability. Mesenchymal stem cells can be collected from bone marrow or other tissues (blood, for example, umbilical cord blood, and skin, fat, dental pulp, etc.), and can be cultured and grown as cells attached to a solid phase, for example, a culture dish. The mesenchymal stem cell in the present application may exist as a heterogeneous cell population that includes not only stem cells in the narrow sense (cells that have the ability to self-replicate and differentiate) but also precursor cells, and may include differentiated cells in addition to stem cells in the narrow sense, or stem cells and precursor cells in the narrow sense under culture conditions. In one embodiment, the mesenchymal stem cell may be composed only of stem cells in the narrow sense.

[0021] In the present application, progenitor cells are defined as cells that have the ability to differentiate unidirectionally into specific tissue cells other than blood cells, and include cells that have the ability to differentiate into mesenchymal tissue, epithelial tissue, neural tissue, parenchymal organs, and vascular endothelium.

[0022] In the present application, mesenchymal stem cells include, but are not limited to, bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells. "Bone marrow mesenchymal stem cells" are mesenchymal stem cells present in bone marrow. In one embodiment, bone marrow mesenchymal stem cells also have the ability to differentiate into epithelial tissues and neural tissues. In one embodiment, bone marrow mesenchymal stem cells are cells with colony-forming ability. In the present application, the term "bone marrow mesenchymal stem cells" is used interchangeably with "bone marrow mesenchymal stromal cells", "bone marrow pluripotent stem cells" or "bone marrow pluripotent stromal cells".

[0023] In addition, "bone marrow-derived mesenchymal stem cells" refer to mesenchymal stem cells mobilized from bone marrow to outside the bone marrow. Bone marrow-derived mesenchymal stem cells can be obtained by peripheral blood collection, or by collection from mesenchymal tissues such as fat, epithelial tissues such as skin, and neural tissues such as the brain. In this application, the term "bone marrow-derived mesenchymal stem cells" is used interchangeably with "bone marrow-derived mesenchymal stromal cells," "bone marrow-derived pluripotent stem cells," or "bone marrow-derived pluripotent stromal cells."

[0024] In one embodiment, bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells can also be capable of differentiating into epithelial tissues such as keratinocytes that make up the skin, or nervous system tissues that make up the brain, by administering the cells directly after collection, or by administering the cells to an injured area of ​​the body after first attaching them to a culture dish.

[0025] Bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells can be capable of differentiating into osteoblasts (which can be identified by observing calcium deposition when differentiation is induced), chondrocytes (which can be identified by positive Alcian blue staining, positive Safranin-O staining, etc.), and adipocytes (which can be identified by positive Sudan III staining, etc.), as well as mesenchymal cells such as fibroblasts, smooth muscle cells, skeletal muscle cells, stromal cells, and tendon cells, nerve cells, pigment cells, epidermal cells, hair follicle cells (which express the cytokeratin family, hair keratin family, etc.), epithelial cells (for example, epidermal keratinocytes and intestinal epithelial cells express the cytokeratin family, etc.), endothelial cells, and even parenchymal organ cells such as liver, kidney, and pancreas, although the differentiated cells are not limited to the above-mentioned cells.

[0026] Examples of markers for human mesenchymal stem cells include all or some of the following, but are not limited to: PDGFRα positive, PDGFRβ positive, Lin negative, CD45 negative, CD44 positive, CD90 positive, CD29 positive, Flk-1 negative, CD105 positive, CD73 positive, CD90 positive, CD71 positive, Stro-1 positive, CD106 positive, CD166 positive, CD31 negative, CD271 positive, and CD11b negative.

[0027] Examples of mouse mesenchymal stem cell markers include all or some of the following, but are not limited to: CD44 positive, PDGFRα positive, PDGFRβ positive, CD45 negative, Lin negative, Sca-1 positive, c-kit negative, CD90 positive, CD105 positive, CD29 positive, Flk-1 negative, CD271 positive, and CD11b negative.

[0028] Examples of markers for rat mesenchymal stem cells include all or some of the following, but are not limited to: PDGFRα positive, CD44 positive, CD54 positive, CD73 positive, CD90 positive, CD105 positive, CD29 positive, CD271 positive, CD31 negative, and CD45 negative.

[0029] In the present application, mesenchymal stem cells include, but are not limited to, PDGFRα-positive mesenchymal stem cells, PDGFRα-positive bone marrow-derived mesenchymal stem cells, and PDGFRα-positive bone marrow-derived cells obtained as adherent cells by cell culture of mononuclear cell fractions in blood obtained by bone marrow collection (bone marrow cell collection) or peripheral blood collection. Examples of PDGFRα-positive mesenchymal stem cells include cells that are positive for PDGFRα and CD44, cells that are positive for PDGFRα and CD90, cells that are positive for PDGFRα and CD105, and cells that are positive for PDGFRα and CD29. In one embodiment, the PDGFRα-positive mesenchymal stem cells may be cells that are negative for CD44.

[0030] The present application provides a composition for use in treating a disease or pathological condition in a subject by mobilizing mesenchymal stem cells into peripheral blood, the composition containing a specific 1r10 fragment peptide or a peptide comprising the amino acid sequence of the fragment peptide.

[0031] The composition for use in treating a disease or pathological condition in a subject by mobilizing mesenchymal stem cells into peripheral blood according to the present application can be utilized as a pharmaceutical composition.

[0032] The subject in the present application is not particularly limited, and may include mammals, birds, fish, etc. Mammals include humans and non-human animals, such as, but not limited to, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, whales, etc. In the present application, the term "subject" is used interchangeably with "patient," "individual," or "animal."

[0033] The compositions for use in treating a disease or pathological condition in a subject by mobilizing mesenchymal stem cells into the peripheral blood of the present application can include one or more peptides.

[0034] In the present application, the treatment of a disease or pathological condition is selected from, for example, but not limited to, an anti-inflammatory treatment, an immunomodulatory treatment, a treatment for inducing tissue regeneration, and a treatment for inhibiting tissue fibrosis.

[0035] In this application, the disease or pathological condition is selected from, but is not limited to, for example, inflammatory diseases, autoimmune diseases, diseases involving tissue damage, ischemia or necrosis, and fibrotic diseases.

[0036] In the present application, inflammatory diseases include, but are not limited to, inflammatory bowel disease and atopic dermatitis. Autoimmune diseases include, but are not limited to, inflammatory bowel disease. Fibrotic diseases include, but are not limited to, pulmonary fibrosis. Diseases involving tissue damage, ischemia or necrosis include, but are not limited to, inflammatory bowel disease and cerebral infarction. Inflammatory bowel diseases include, but are not limited to, ulcerative colitis and Crohn's disease.

[0037] In this application, the term "activity of mobilizing mesenchymal stem cells into peripheral blood" is used interchangeably with "activity of increasing the abundance of mesenchymal stem cells in peripheral blood" and "MSC blood mobilization activity."

[0038] The MSC blood mobilization activity of the peptides in the present application can be evaluated, for example, by i) collecting peripheral blood from individuals administered with the peptide and individuals not administered with the peptide, seeding the blood in a culture dish, culturing the blood (several days to 10 days), and counting the number of colonies formed, and ii) confirming that the cells that formed the colonies have the ability to adhere to a solid phase and have the ability to differentiate into osteoblasts, chondrocytes, and adipocytes. In the above method i), red blood cells may be removed from the peripheral blood by a desired method before seeding the collected peripheral blood in the culture dish. In the above method ii), instead of confirming the differentiation ability, surface marker analysis, transcriptome analysis, etc. may be performed on the cells that formed the colonies to confirm that the cells have properties characteristic of mesenchymal stem cells.

[0039] The peptides in the present application can be obtained as recombinants by inserting DNA encoding the peptides into an appropriate expression system, or can be artificially synthesized. Thus, the peptides in the present application include peptides produced using cells and artificially synthesized peptides (so-called synthetic peptides).

[0040] In order to obtain the peptide of the present application by genetic engineering techniques, DNA encoding the peptide may be incorporated into an appropriate expression system and expressed.

[0041] Hosts applicable to the present application include, but are not limited to, prokaryotic cells and eukaryotic cells. Hosts applicable to the present application also include, but are not limited to, bacteria (e.g., E. coli), yeast, animal cells (e.g., mammalian cells such as HEK293 cells and CHO cells, insect cells such as silkworm cells), and plant cells.

[0042] An example of a host / vector system applicable to the present application is the expression vector pGEX and E. coli. pGEX can express a foreign gene as a fusion protein with glutathione S-transferase (GST) (Gene, 67:31-40, 1988). Therefore, pGEX incorporating DNA encoding the peptide of the present application is introduced into an E. coli strain such as BL21 by heat shock, and after an appropriate incubation period, isopropylthio-β-D-galactoside (IPTG) is added to induce expression of the GST fusion peptide. Since the GST in the present application is adsorbed to glutathione Sepharose 4B, the expression product can be easily separated and purified by affinity chromatography.

[0043] Other host / vector systems that can be used to obtain recombinant proteins of the peptide of the present application include the following. First, when bacteria are used as hosts, expression vectors for fusion proteins using tags or the like are commercially available. Furthermore, recombinant proteins of the present application also include those to which tags or part of the peptides are added.

[0044] The tag added to the peptide of the present application is not particularly limited, so long as it does not affect the activity of the peptide of the present application, and examples include histidine tags (e.g., 6xHis, 10xHis), HA tags, FLAG tags, GST tags, T7 tags, HSV tags, E tags, lck tags, B tags, and the like.

[0045] It is known that yeasts of the genus Pichia are effective in expressing proteins with sugar chains. In terms of the addition of sugar chains, an expression system using a baculovirus vector with an insect cell as a host is also useful (Bio / Technology, 6:47-55, 1988). Furthermore, vectors using promoters such as CMV, RSV, or SV40 have been transfected using mammalian cells, and any of these host / vector systems can be used as an expression system for the peptide of the present application. Genes can also be introduced using viral vectors such as plasmid vectors, retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, Sendai virus envelope vectors, and papilloma virus vectors, but are not limited to these. The vector may contain a promoter DNA sequence that effectively induces gene expression, a factor that controls gene expression, and a molecule required to maintain DNA stability.

[0046] The obtained peptide of the present application can be isolated from inside or outside the host cell (such as the medium) and purified as a substantially pure and homogeneous peptide. Separation and purification of the peptide can be performed using any separation and purification method that is commonly used in the purification of peptides, and is not limited in any way. For example, the peptide can be separated and purified by appropriately selecting and combining a chromatography column, a filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and the like.

[0047] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Marshak et al., Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Cold Spring Harbor Laboratory Press, 1996). These chromatographies can be performed using liquid phase chromatography, such as HPLC and FPLC.

[0048] In addition, the peptide of the present application is preferably a substantially purified peptide. Here, "substantially purified" means that the degree of purification of the peptide of the present application (the proportion of the peptide of the present application in the entire protein component) is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 100% or close to 100%. The upper limit close to 100% depends on the purification technique and analysis technique of the skilled artisan, but is, for example, 99.999%, 99.99%, 99.9%, 99%, etc.

[0049] Furthermore, any method of purification that has the above-mentioned degree of purification is included in the substantially purified peptide. For example, the substantially purified peptide can be obtained by appropriately selecting or combining the above-mentioned chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc., but is not limited thereto.

[0050] On the other hand, the peptides of the present application can also be artificially synthesized. In the peptide synthesis method of the present application, peptides can be chemically synthesized by methods such as peptide liquid phase synthesis and peptide solid phase synthesis. Peptide solid phase synthesis is one of the methods commonly used when chemically synthesizing peptides. Polystyrene polymer gel beads with a diameter of about 0.1 mm, whose surface is modified with amino groups, are used as the solid phase, from which the amino acid chain is extended one by one by a dehydration reaction. Once the desired peptide sequence has been completed, it is cut from the solid phase surface to obtain the desired substance. Solid phase synthesis can be used to synthesize ribosomal peptides, which are difficult to synthesize in bacteria, and to synthesize peptides containing D-isomers or stable isotopes ( 2 H, 13 C. 15 It is also possible to introduce unnatural amino acids such as selenoamino acids (e.g., N) substitutes, heavy atom substitutes (e.g., selenoamino acids such as selenomethionine), and modifications of peptide and protein backbones. When a long peptide chain exceeding 70 to 100 amino acids is synthesized by the solid phase method, it is possible to synthesize the peptide by binding two peptide chains using a native chemical ligation method. The peptide of the present application may be in the form of a pharma- ceutical acceptable salt of the peptide. Examples of pharma-ceutical acceptable salts include, but are not limited to, hydrochloride, acetate, and trifluoroacetate. The peptide of the present application may be in the form of a solvate of the peptide or a solvate of a pharma-ceutical acceptable salt of the peptide. A solvate refers to a solute molecule coordinated with any number of solvent molecules, and includes, but is not limited to, hydrates.

[0051] The amino acid length of the 1r10 fragment peptide herein can be, for example, in the range of 11 to 29 amino acids, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids.

[0052] In the present application, examples of 1r10 fragment peptides include peptides selected from the following: (1) A peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and containing the amino acid sequence of SEQ ID NO: 17 or 19; (2) a peptide consisting of the amino acid sequence from the 1st to the 27th amino acids of SEQ ID NO: 1 (1r10_N03; SEQ ID NO: 2); (3) a peptide consisting of the 1st to 26th amino acid sequence of SEQ ID NO: 1 (1r10_N04; SEQ ID NO: 3); (4) a peptide consisting of the 1st to 25th amino acid sequence of SEQ ID NO: 1 (1r10_N05; SEQ ID NO: 4); (5) a peptide consisting of the 1st to 24th amino acid sequence of SEQ ID NO: 1 (1r10_N06; SEQ ID NO: 5); (6) a peptide consisting of the 1st to 23rd amino acid sequence of SEQ ID NO: 1 (1r10_N07; SEQ ID NO: 6); (7) a peptide consisting of the 1st to 19th amino acid sequence of SEQ ID NO: 1 (1r10_N11; SEQ ID NO: 7); (8) a peptide consisting of the 1st to 18th amino acid sequence of SEQ ID NO: 1 (1r10_N12; SEQ ID NO: 8); (9) a peptide consisting of the 1st to 16th amino acid sequence of SEQ ID NO: 1 (1r10_N14; SEQ ID NO: 9); (10) a peptide consisting of the 6th to 30th amino acid sequence of SEQ ID NO: 1 (1r10_C05; SEQ ID NO: 11); (11) A peptide consisting of the amino acid sequence from the 11th to the 30th amino acids of SEQ ID NO: 1 (1r10_C10; SEQ ID NO: 12); (12) A peptide consisting of the amino acid sequence from the 12th to the 30th amino acids of SEQ ID NO: 1 (1r10_C11; SEQ ID NO: 13); (13) A peptide consisting of the amino acid sequence from the 13th to the 30th amino acids of SEQ ID NO: 1 (1r10_C12; SEQ ID NO: 14); (14) A peptide consisting of the amino acid sequence from the 16th to the 30th amino acids of SEQ ID NO: 1 (1r10_C15; SEQ ID NO: 15); (15) A peptide consisting of the amino acid sequence from the 17th to the 30th amino acids of SEQ ID NO: 1 (1r10_C16; SEQ ID NO: 16); (16) A peptide consisting of the 18th to 30th amino acid sequence of SEQ ID NO: 1 (1r10_C17; SEQ ID NO: 17); and (17) A peptide consisting of the 6th to 16th amino acids of SEQ ID NO: 1 (1r10_MF5; SEQ ID NO: 19).

[0053] Of the amino acid sequences described in (1) to (17) above, the "amino acid sequence from positions 18 to 30 of SEQ ID NO: 1" described in (16) above (amino acid sequence of SEQ ID NO: 17) and the "amino acid sequence from positions 6 to 16 of SEQ ID NO: 1" described in (17) above (amino acid sequence of SEQ ID NO: 19) are important core regions for the 1r10 fragment peptide to retain its MSC blood mobilization activity. If the 1r10 fragment peptide contains at least one of these two core regions, the MSC blood mobilization activity is retained.

[0054] In the above (1) "a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 and including the amino acid sequence of SEQ ID NO: 17 or 19," "a part of the amino acid sequence of SEQ ID NO: 1" may be any of the above (2) to (15), but is not limited thereto.

[0055] The peptides of the present application also include peptides selected from the following: (i) a peptide consisting of all or a part of the amino acid sequence of SEQ ID NO: 1 in which 1 to 5 amino acids have been substituted, deleted, inserted or added, the peptide including the amino acid sequence of SEQ ID NO: 17 or 19; and (ii) A peptide consisting of all or part of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, which includes the amino acid sequence of SEQ ID NO: 17 or 19. Such peptides can be prepared by methods well known to those skilled in the art.

[0056] The peptides selected from (i) and (ii) above may be, for example, peptides consisting of 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, or 11 or less amino acids. The peptides selected from (i) and (ii) above may be peptides other than the peptide consisting of the amino acid sequence of SEQ ID NO:1.

[0057] With respect to the peptides described in (i) above, "1 to 5" includes, for example, 5, 4, 3, 2, 1, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0058] With respect to the peptides described in (ii) above, "90% or more" can also be expressed as "90% or more but less than 100%", and examples of "90% or more" include, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0059] The 1r10 fragment peptide selected from the above (1) to (17) is a 1r10 fragment peptide having the activity of mobilizing mesenchymal stem cells into peripheral blood. The peptides selected from the above (i) and (ii) are also peptides having the activity of mobilizing mesenchymal stem cells into peripheral blood. Therefore, these peptides are considered to have the effect of mobilizing mesenchymal stem cells into peripheral blood, as well as the therapeutic effect on inflammatory diseases, autoimmune diseases, diseases accompanied by tissue damage, ischemia or necrosis, and fibrotic diseases.

[0060] The present application also provides the peptides disclosed herein, for example, a 1r10 fragment peptide selected from (1) to (17) above, and a peptide selected from (i) and (ii) above.

[0061] The amino acid sequence of SEQ ID NO:1 is the amino acid sequence of the artificial sequence peptide 1r10 of the present application. The amino acid sequences of SEQ ID NOs:2 to 19 are the amino acid sequences of the 1r10 fragment peptides of the present application. The regions of the amino acid sequence of SEQ ID NO:1 (amino acid sequence of 1r10) that correspond to the amino acid sequence of the 1r10 fragment peptide are shown in Table 1 below.

[0062] The base sequence set forth in SEQ ID NO: 20 is an example of the base sequence of DNA encoding the artificial sequence peptide 1r10 of the present application. The base sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22 are examples of the base sequences of DNA encoding the 1r10 fragment peptides 1r10_C17 and 1r10_MF5 of the present application, respectively. The regions in the base sequence set forth in SEQ ID NO: 20 (the exemplified base sequence of 1r10) that correspond to the base sequence of DNA encoding the 1r10 fragment peptide are shown in Table 1 below.

[0063] [Table 1]

[0064] Other examples of the base sequence of DNA encoding the artificial sequence peptide 1r10 or 1r10 fragment peptide of the present application will be obvious to those skilled in the art. The DNA can be prepared by a method (reverse translation) of converting the amino acid residues of the artificial sequence peptide 1r10 or 1r10 fragment peptide into the corresponding codons using a codon table known to those skilled in the art. Reverse translation can be performed, if desired, using various software (including programs, algorithms, etc.) developed for the analysis of amino acid and nucleic acid sequences.

[0065] An effective amount of the peptide of the present application or a pharmaceutical composition containing it (hereinafter referred to as "pharmaceutical composition, etc.") is administered to a subject for the treatment of a disease or condition described herein.

[0066] As used herein, an effective amount refers to an amount sufficient to treat a disease or pathological condition as described herein, including, but not limited to, alleviating, delaying, preventing, ameliorating, remission, curing, or eliminating the disease or condition.

[0067] There are no limitations on the site of administration of the pharmaceutical composition etc. of the present application, and the pharmaceutical composition etc. of the present application can exert its effects when administered to any site, including the site or vicinity of the site where symptoms of a disease or pathological condition appear, a site different from the site (a site other than the site), a site away from the site where symptoms of a disease or pathological condition appear, a site distal to the site where symptoms of a disease or pathological condition appear, or a site distal and ectopic to the site where symptoms of a disease or pathological condition appear.

[0068] Furthermore, the pharmaceutical composition etc. of the present application can exert its effect when administered to any tissue, such as a tissue different from the tissue in which symptoms of a disease or pathological condition appear, a tissue distant from the tissue in which symptoms of a disease or pathological condition appear, a tissue distal to the tissue in which symptoms of a disease or pathological condition appear, or a tissue distal and ectopic to the tissue in which symptoms of a disease or pathological condition appear.

[0069] Methods for administering the pharmaceutical composition etc. of the present application include oral administration or parenteral administration, and parenteral administration methods include, but are not limited to, intravascular administration (intraarterial administration, intravenous administration, etc.), intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, nasal administration, pulmonary administration, transdermal administration, etc. In addition, the pharmaceutical composition etc. of the present application can be administered systemically or locally (for example, subcutaneously, intradermally, on the skin surface, on the eyeball or palpebral conjunctiva, on the nasal mucosa, in the oral cavity and in the digestive tract mucosa, on the vagina or in the uterus mucosa, or at an injured site, etc.) by injection, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0070] Moreover, instead of the peptide of the present application, cells that secrete the peptide, a vector for gene therapy into which DNA encoding the peptide has been inserted, and a pharmaceutical composition containing these can also be used.

[0071] In addition, the administration method can be appropriately selected depending on the age and symptoms of the patient. When administering the pharmaceutical composition of the present application, the dosage can be selected, for example, in the range of 0.0000001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, in the range of 0.00001 to 100000 mg / body per patient. When administering cells secreting the peptide of the present application or a gene therapy vector into which DNA encoding the peptide is inserted, the amount of the peptide can be administered within the above range. However, the pharmaceutical composition of the present application is not limited to these dosages.

[0072] The pharmaceutical composition of the present application can be formulated according to a conventional method (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA), and may contain pharma- ceutically acceptable carriers and additives. Examples of such carriers include, but are not limited to, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. Other commonly used carriers can be used as appropriate. Specific examples of such carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, and inorganic salts.

[0073] All prior art documents cited in this specification are hereby incorporated by reference.

[0074] The present invention is further illustrated by the following non-limiting examples. EXAMPLES

[0075] Example 1 Mobilization of mesenchymal stem cells by 1r10 fragment peptide

[0076] (1) Materials and methods i) Production of peptides The peptides shown in the table below (1r10 and 1r10 fragment peptides) were chemically synthesized by the solid-phase method (the obtained peptides are all in the form of trifluoroacetic acid (TFA) salts).

[0077] [Table 2]

[0078] ii) Administration of peptides C57BL / 6J mice (8 weeks old, male, weighing 25g) were prepared and divided into a group administered with each peptide listed in the table above and a control group. Peptides were administered by injecting a solution of each peptide adjusted to a concentration of 1μg / μL using saline or 1% DMSO as a solvent into the tail vein at a volume of 100μL / mouse (4mg / kg of peptide administered) (the solvent for 1r10_N14 and 1r10_N15 was 1% DMSO, and the solvent for the other peptides was saline). For the control group, saline or 1% DMSO was injected into the tail vein at a volume of 100μL / mouse.

[0079] iii) Cell collection from peripheral blood Sixteen hours after administration of saline, 1% DMSO, or peptide, 800 μL of peripheral blood was collected from the heart of each mouse under general anesthesia (using a 1 mL syringe containing heparin). Peripheral blood collected from three mice for each peptide administration group and control group was pooled, and an equal amount of Hetasep (STEMCELL Technologies, Cat No. ST-07906) was added to the collected blood to remove red blood cells, centrifuged at 100G for 2 minutes, incubated at room temperature for 15 minutes, and the supernatant was collected. The supernatant was used in the next experiment as a sample containing nucleated cells in peripheral blood.

[0080] iv) Colony Assay The supernatant (peripheral blood-derived cell-containing sample) obtained by the above procedure was seeded on a 6-well plate (Corning, Cat No. 356400) coated with collagen I, and cultured for 10 days under conditions of 37°C, 5% CO2, and 5% O2 using a medium (all values ​​are final concentrations) containing 1% L-glutamine (Nacalai Tesque), 10 μM ROCK inhibitor (Y27632, Tocris Bioscience), and 1% penicillin / streptomycin (Nacalai Tesque) in the Expansion Medium prepared according to the manual of the MesenCult Expansion Kit (STEMCELL Technologies, Cat No. ST-05513). During the culture period, the medium was replaced with a fresh one twice a week. On day 10 of culture, the cells on the plate were stained using a Differential Quik Stain Kit (Sysmex Corporation, Cat No. 16920), and the number of colonies containing 50 or more cells was counted.

[0081] In experiments conducted by the inventors to date, it has been confirmed that all colonies obtained by culturing peripheral blood on a solid phase such as a dish or plate are not only adhesive to the solid phase and have the ability to self-replicate, but are also PDGFRα positive and have the ability to differentiate into osteoblasts, chondrocytes, adipocytes, and epithelial cells.

[0082] Furthermore, it has been confirmed that all colonies obtained by culturing peripheral blood on a solid phase after administration of a peptide consisting of amino acid residues 1-44 of the human HMGB1 protein (hereinafter referred to as HMGB1 peptide 1-44), which has the activity of mobilizing mesenchymal stem cells into peripheral blood, have the ability to adhere to the solid phase and self-replicate, and are PDGFRα positive. Furthermore, it has been confirmed that the colonies have a gene expression profile characteristic of mesenchymal stem cells, based on the results of clustering of transcriptome analysis data and gene ontology analysis.

[0083] Furthermore, it has already been confirmed that a greater number of colonies can be obtained by solid phase culture from peripheral blood following administration of HMGB1 peptide 1-44 than from peripheral blood following administration of physiological saline.

[0084] Therefore, the colonies obtained by culturing peripheral blood on a solid phase are mesenchymal stem cells, and an increase in the number of colonies detected by solid phase culture of peripheral blood is thought to indicate an increase in the number of mesenchymal stem cells in the peripheral blood.

[0085] Furthermore, since mesenchymal stem cells are usually hardly present in peripheral blood, it is believed that the increased number of mesenchymal stem cells were mobilized into peripheral blood from tissues other than peripheral blood (eg, bone marrow).

[0086] From the above, the number of colonies detected in solid phase culture of peripheral blood after administration of a test substance can be used as an index of the activity of the test substance to mobilize mesenchymal stem cells into peripheral blood.

[0087] After administration of the test substance, the timing (peak time) at which the number of mesenchymal stem cells in the peripheral blood reaches its maximum is thought to vary from individual to individual, and in the above-mentioned peripheral blood colony assay, cases often occur where the timing of blood collection (16 hours after administration of the test substance in the case of 1r10 and its fragment peptides) does not coincide with the peak time of the number of mesenchymal stem cells in the peripheral blood. Therefore, rather than averaging the results of multiple assays, we decided to evaluate test substances that caused colony formation exceeding a certain threshold even once as having MSC blood mobilization activity.

[0088] Furthermore, the results of numerous colony assays conducted by the present inventors have confirmed that the number of colonies formed in the negative control (saline or 1% DMSO administration group) was "less than 10" no matter how many times the assay was conducted. Therefore, in this example, the threshold number of colonies was set to "10," and a test substance that caused the formation of "10 or more" colonies in even one of four to six assays was determined to have the activity of mobilizing MSCs into the blood.

[0089] (2) Results The peptides listed in Table 2 were assayed 4 to 6 times. As a result, for the N-terminal fragment peptides, 10 or more colonies were formed in the groups administered with 1r10_N03-N07, N11-12, N14, and MF5, and the number of colonies formed in the group administered with 1r10_N15 was less than 10 (Figure 1). For the C-terminal fragment peptides, 10 or more colonies were formed in the groups administered with 1r10_C05, C10-12, and C15-C17, and the number of colonies formed in the group administered with 1r10_C18 was less than 10 (Figure 2). These results indicate that the amino acid sequences of 1r10_MF5 and 1r10_C17 are important core sequences for maintaining MSC blood mobilization activity.

[0090] Reference example 1 Efficacy of the artificial peptide 1r10 for inflammatory bowel disease

[0091] (1) Materials and methods i) Drugs Dextran sulfate sodium (DSS) (molecular weight 36,000-50,000, MP Biomedicals, product number 160110) was dissolved in purified water to prepare a 2.5% (w / v) DSS aqueous solution. Peptide 1r10 (TFA salt) consisting of the amino acid sequence of SEQ ID NO: 1 was chemically synthesized by solid phase method and used as a test substance.

[0092] ii) Creation of inflammatory bowel disease (IBD) model mice BALB / c mice (9 weeks old, male) were allowed to drink 2.5% DSS aqueous solution ad libitum for 7 days to induce colitis. After that, the drinking water was changed from DSS to tap water, and colon samples were taken 3 days later (10 days after the start of drinking DSS aqueous solution).

[0093] iii) Administration of peptides The IBD model mice prepared as described above were divided into a peptide 1r10 administration group (n=8) and a control group (n=8). The peptide was administered by injecting a solution of peptide 1r10 adjusted to a concentration of 0.5 mg / mL in saline into the tail vein at 10 mL / kg (5 mg / kg peptide dose) on days 1, 4, 7, 8, and 9 after the mice started drinking the DSS aqueous solution. For the control group, saline was injected into the tail vein at 10 mL / kg on days 1, 4, 7, 8, and 9 after the mice started drinking the DSS aqueous solution.

[0094] iv) Evaluation of the effect of peptide administration For 10 days after the start of drinking the DSS aqueous solution, the mice were weighed and observed for symptoms (feces condition and bleeding) every day except Saturdays, Sundays, and holidays, and the severity of symptoms was evaluated by calculating the DAI (disease activity index) score. The DAI score was calculated by scoring the weight loss rate, stool condition, and fecal bleeding as shown in the table below, and adding up the scores for these three items. The scoring criteria were partially based on the method of Li et al. (PLoS One. 2015 Dec 7; 10(12):e0144101).

[0095] [Table 3]

[0096] In addition, the length of the large intestine (from colon to rectum) collected 10 days after the start of DSS drinking was measured using a digital caliper.

[0097] (2) Results The changes in the body weight of the mice during the test period are shown in Figure 3 (see "saline" for the control group and "1r10" for the peptide-administered group). Both the control group and the peptide-administered group lost weight over time after starting to drink DSS water, but the degree of loss was slower in the peptide-administered group than in the control group, and the weight of the peptide-administered group showed a tendency to recover 10 days after starting to drink DSS aqueous solution.

[0098] The stool score (the sum of the "stool condition" score and the "bleeding" score, which were scored as shown in Table 3 above) and the DAI score 8 to 10 days after starting to drink the DSS aqueous solution are shown in Figures 4 and 5, respectively (see "saline" for the control group and "1r10" for the peptide administration group). Both the stool score and the DAI score were lower in the peptide administration group than in the control group.

[0099] Ten days after starting to drink the DSS aqueous solution, the colon length of the peptide-administered group was longer than that of the control group (FIG. 6; see "saline" for the control group and "1r10" for the peptide-administered group).

[0100] In IBD, inflammation occurs in the intestinal mucosa, leading to the formation of erosions and ulcers, and symptoms such as narrowing and shortening of the intestinal tract may occur. Another symptom of IBD is known to be weight loss, which is believed to be caused by inflammation in the intestinal mucosa and tissue damage (ulcers, etc.) that leads to a deterioration in nutritional status. It is also known that intravenous injection of mesenchymal stem cells in an animal IBD model improves various symptoms, including weight loss, epithelial damage in the intestine, and infiltration of inflammatory cells. The improvement of such symptoms is due to the suppression of inflammation in the intestinal mucosa by the anti-inflammatory effect of mesenchymal stem cells, and the resulting promotion of mucosal tissue regeneration.

[0101] Administration of the present artificial sequence peptide to IBD model mice inhibited weight loss, inhibited shortening of colon length, and improved DAI score. This is believed to be the result of mesenchymal stem cells being mobilized into peripheral blood by the action of the present artificial sequence peptide, and the cells exerting anti-inflammatory and tissue regenerative effects.

[0102] Reference example 2 Efficacy of artificial peptide 1r10 on atopic dermatitis

[0103] (1) Materials and methods i) Drugs MC903 (generic name: Calcipotriol) was used to induce atopic dermatitis (hereinafter also referred to as "AD") in mice. Peptide 1r10 (TFA salt) described in Reference Example 1 was used as a test substance.

[0104] ii) Creation of atopic dermatitis model mice C57BL / 6 mice (C57BL / 6JJcl, 7-8 weeks old, male, microbiological grade SPF) were obtained from CLEA Japan, and after acclimatization in an animal breeding room for more than 5 days, they were used in the following experiment. MC903 solution adjusted with ethanol was applied to the ear skin of the mice at an amount of 2.25 nmol MC903 / ear / once / day, 5 times a week for 2 weeks (total of 10 applications) to induce atopic dermatitis (AD). Such MC903-induced AD model is a model that can reproduce AD-like pathology in a short period (1-2 weeks) by artificially inducing TSLP secretion from epithelial cells that is actually seen in AD patients, thereby inducing a series of type 2 immune response cascades (Li M et al., Proc Natl Acad Sci US A. 2006 ;103 :11736-41.). Furthermore, as a preliminary test, the appearance and tissue of the ears of normal mice and MC903-induced AD mice (14 days after the start of induction) were observed, and it was confirmed that the AD-induced mice exhibited erythema due to inflammation, desquamation in the marginal area, lichenification of the keratin, acanthosis of the epidermis (thickening of the epidermis due to proliferation of spinous cells), localized barrier destruction, infiltration of immune cells into the dermis, increased thickness of the dermis, and spongiosis (spongiotic changes) in parts of the epidermis.

[0105] iii) Administration of peptides Two weeks after the start of MC903 application (Day 1), only those individuals in which AD was clearly induced were selected based on the main symptoms of scaling, erythema, and edema, and then, taking into account the difference in the severity of inflammation between individuals, the groups were averaged based on the ear thickness, and the animals were divided into a control group and a peptide-administered group (n=7 per group). The peptide was administered twice, once on the day of grouping (Day 15) and once during the following four days (Day 16 to Day 19), by injecting a solution of peptide 1r10 adjusted to 0.3 mg / mL with saline into the tail vein at a volume of 5 mL / kg (1.5 mg / kg as the peptide dose). In the control group, a solution of bovine serum albumin (BSA) adjusted to 0.3 mg / mL with saline was injected into the tail vein at a volume of 5 mL / kg (1.5 mg / kg BSA dose) twice: once on the day of grouping (Day 15) and once over the following 4 days (Days 16 to 19).

[0106] iv) Evaluation of the effect of peptide administration Assessment for edema and desquamation During the peptide administration period (Day 15 to Day 19), ear swelling was measured daily to assess edema, and scaling, a cardinal sign of AD, was assessed by photographing (three times a week) using a digital camera (E-M1 II; Olympus) placed on a fixed stand 13-14 cm above the ear.

[0107] Pathological analysis Four days after the start of peptide administration (Day 19), the mice were placed under isoflurane anesthesia, euthanized by cervical dislocation, and the skin of the inflamed area of ​​the ear and the surrounding area was quickly excised and collected on ice to prevent protein denaturation of the sample. The collected ear skin was removed of excess hair and skin with tweezers, immersed in 10% formalin, and fixed overnight at 4°C while shaking on ice with a shaker. The fixed skin was thoroughly washed with phosphate-buffered saline (PBS), trimmed, and then made into a paraffin block using an automatic embedding machine (Excelsior ES; Thermo Scientific), taking into consideration the polarity of the tissue intrinsic to the dorsal-ventral axis / left-right axis of the skin. The skin was then thinly sliced ​​(5 μm thick) using a rotary microtome (HM 325; Thermo Scientific), fixed on a slide glass, and subjected to various staining. For pathological analysis, the skin thickness and infiltration of immune cells were evaluated by histochemical staining and fluorescent immunostaining, respectively.

[0108] Measurement of dermal thickness To quantitatively evaluate the healing state of the inflammatory site, paraffin sections (5 μm thick) of the skin tissue prepared as described above were stained with hematoxylin and eosin (HE) and the dermal thickness was measured. In detail, three sections were prepared per animal, and one site each at the center of inflammation, head, and tail of each section was photographed under a microscope (Keyence BZ-X710) (i.e., a total of nine sites were photographed per animal). Next, perpendicular lines were drawn from the epidermis-dermis junction to the subcutaneous fat-cartilage junction on the photographed images, and only the longest perpendicular line in the field of view was selected and used as the measurement value of the dermal thickness.

[0109] Quantification of immune cells To quantitatively evaluate the number of immune cells at the inflammatory site, the paraffin sections (5 μm thick) prepared as described above were subjected to fluorescent immunostaining using anti-laminin antibody and anti-CD45 antibody. (To detect laminin, the primary antibody was anti-laminin (Sigma Aldrich; catalog number L9393), and the secondary antibodies were donkey anti-rabbit IgG (H+L) and Alexa Fluor 488 (Thermo Fisher Scientific; catalog number A-21206). To detect CD45, the primary antibody was goat anti-mouse CD45 (R&D Systems; catalog number AF114), and the secondary antibody was Cy3 AffiniPure Bovine Anti-Goat IgG (H+L) (Jackson ImmunoResearch; catalog number 805-165-180).) For the detailed quantification, three sections were prepared per animal, and high-power fields (HPF: 400x) were photographed with a microscope (Keyence BZ-X710) at one location each at the center of inflammation, head, and tail (i.e., a total of nine locations were photographed per animal). Next, the number of CD45-positive cells (detected by Cy3 fluorescence) in the field of view was counted for each photographed image, and used as the measurement value.

[0110] Statistical Processing The measured values ​​or calculated values ​​after correction obtained by the above analysis were analyzed and judged using statistical analysis software (statcel-3 (OMS Publishing) and Excel Statistics (BellCurve)) to calculate each statistical value. Specifically, (1) normality test (D'Agostino test for skewness / kurtosis coefficient; P>0.05 is considered to be normal distribution) and (2) rejection test (Grubbs test; P<0.05 is considered to be an outlier) were performed as pre-tests, and if the results obtained in (1) showed a normal distribution, a t-test was performed between groups. If a normal distribution was not shown, a Mann Whitney U-test was performed as a nonparametric test based on the ranks between groups, and a significant difference was judged to exist if p<0.05. For changes in body weight and ear thickness, which were mainly measured repeatedly over time, a Repeated One Way ANOVA test was performed, and a Tukey-Kramer test was also performed as a post hoc test. Regarding the treatment of outliers, (a) if the number of individuals in a group with P<0.05 in the Grubbs test was 10% or less of the total number of individuals in that group (n), the outlier was treated as a missing value, and (b) if the number of individuals with P<0.05 was more than 10% of n, the mean value of other individuals without outliers was imputed as a complement (so-called mean substitution).

[0111] (2) Results Desquamation After induction of the AD model, the control mice frequently showed typical AD signs of eczema and desquamation due to dryness at the ear margins (Figure 7, arrow in the left photo). On the other hand, the peptide-administered mice showed a reduction in desquamation at the ear margins (Figure 7, arrow in the right photo).

[0112] Ear thickness (edema) The change in ear thickness (edema) over time during peptide administration is shown in Figure 8 (see "Control" for the control group and "1r10" for the peptide administration group). A statistically significant decrease in ear thickness was observed in the peptide administration group compared to the control group.

[0113] Dermal thickness As a result of HE staining of skin tissue sections, an increase in dermal thickness (presumably due to cell infiltration and increased vascular permeability associated with the immune response caused by AD) was observed in the control group, which was not observed in non-AD-induced mice (Figure 9; see "Control" for the control group and "1r10" for the peptide administration group). On the other hand, a statistically significant decrease in dermal thickness was observed in the peptide administration group compared to the control group (Figure 10; see "Control" for the control group and "1r10" for the peptide administration group). In addition, when peptide 1r10 was administered at a dose of 0.5 mg / kg, a statistically significant decrease in dermal thickness was observed compared to the control group (data not shown).

[0114] Immune cell infiltration Fluorescent immunostaining of skin tissue sections showed that in the control group, infiltration of CD45-positive immune cells into tissues associated with the immune response caused by AD was observed throughout the skin, including around blood vessels (labeled with laminin) (arrows in Figure 11; see "Control" for the control group and "1r10" for the peptide administration group). On the other hand, a statistically significant decrease in the number of CD45-positive cells was observed in the peptide administration group compared to the control group (Figure 12; see "Control" for the control group and "1r10" for the peptide administration group).

[0115] Furthermore, there was no significant difference in the weight changes of the mice during the study period (from the start of administration (Day 15) to the time of skin collection (Day 19)) among any of the groups, and no statistically significant changes were observed between the groups (p>0.05, Repeated one-way ANOVA).

[0116] In this reference example, administration of the artificial sequence peptide of the present application to atopic dermatitis model mice inhibited desquamation and edema, inhibited an increase in dermal thickness, and inhibited infiltration of CD45 positive cells. This is believed to be the result of mesenchymal stem cells being mobilized into peripheral blood by the action of the artificial sequence peptide of the present application, and the cells exerting an anti-inflammatory effect.

[0117] Reference example 3 Efficacy of the artificial peptide 1r10 for cerebral infarction

[0118] (1) Materials and methods i) Drugs Peptide 1r10 (TFA salt) described in Reference Example 1 was used as the test substance.

[0119] ii) Creation of a cerebral infarction model rat Crl:CD (SD) rats (7 weeks old, male) were obtained from Charles River Japan, and after 6 days of acclimation, they were divided into a control group (n = 8) and a peptide-administered group (n = 8). The day after grouping, the rats were fixed in a supine position under 2% isoflurane inhalation anesthesia (anesthesia background was nitrous oxide:oxygen = 7:3). To regulate body temperature during surgery, a thermometer probe connected to a digital thermometer was inserted into the rectum to monitor the change in rectal temperature before and after surgery. As a result, no decrease in rectal temperature was observed in any of the rats, so heating was not performed. The right common carotid artery, right external carotid artery, and right internal carotid artery were exposed, and the right common carotid artery and right external carotid artery were ligated with sutures. A No. 4 nylon thread (obturator) that had been previously coated with silicone and cut to a length of 19 mm was inserted from the bifurcation of the right external carotid artery and right internal carotid artery to occlude the right middle cerebral artery (MCA). After right MCA occlusion, the neck skin was sutured and the animal was released from anesthesia. 30 minutes after right MCA occlusion, the flexion of the forelimb on the contralateral side to the occlusion side was confirmed. Under inhalation anesthesia with 2% isoflurane, the right common carotid artery, right external carotid artery, and right internal carotid artery were exposed again, and 90 minutes after right MCA occlusion, the plug was removed to resume blood flow in the right MCA. After blood flow resumed in the right MCA, the right internal carotid artery was ligated and the neck skin was sutured, and the animal was released from anesthesia. Before the right MCA occlusion surgery, benzylpenicillin potassium was administered intramuscularly at a dose of 20,000 units / kg.

[0120] iii) Administration of peptides The peptide was administered by injecting a solution of peptide 1r10, adjusted to a concentration of 1 mg / mL in saline, into the tail vein at a volume of 2 mL / kg (peptide dose of 2 mg / kg) at 105 minutes and 24 hours after right MCA occlusion. In the control group, saline was injected into the tail vein at a volume of 2 mL / kg at 105 minutes and 24 hours after right MCA occlusion.

[0121] iv) Evaluation of the effect of peptide administration Calculation of cerebral infarction volume ratio 48 hours after right MCA occlusion, animals were decapitated under anesthesia with sodium pentobarbital (50 mg / kg, intraperitoneal administration), and the whole brain was removed and sliced ​​2 mm thick. Brain sections were cut at positions that obtained coronal planes 4 mm anterior to Bregma, 2 mm anterior to Bregma, above Bregma, 2 mm posterior to Bregma, 4 mm posterior to Bregma, and 6 mm posterior to Bregma, based on the Paxinos and Watson brain atlas (Paxinos G. and Watson C., The rat brain in stereotaxic coordinates, second edition. Academic Press Inc.; 1986). Brain sections were immersed in 1 w / v% 2,3,5-triphenyltetrazolium chloride (TTC) solution at room temperature, stained, and photographed. The obtained photographs were analyzed, and the area of ​​the cerebral infarction and the cross-sectional area of ​​the brain were measured. The volume of the cerebral infarction from 4 mm anterior to 6 mm posterior to the Bregma and the total brain volume were calculated using the following formula. The cerebral infarction volume rate was calculated from the cerebral infarction volume and the total brain volume (cerebral infarction volume rate (%) = cerebral infarction volume / total brain volume × 100). TIFF0007672636000004.tif14170V: Cerebral infarct volume or total brain volume (mm 3 ) a: Cerebral infarct area on the section 4 mm anterior to the Bregma or cross-sectional area of ​​the brain (mm 2 ) b: Cerebral infarct area on the section 2 mm anterior to the Bregma or cross-sectional area of ​​the brain (mm 2) c: Area of ​​cerebral infarction on the Bregma section or cross-sectional area of ​​the brain (mm 2 ) d: Cerebral infarct area on the section 2 mm posterior to the Bregma or cross-sectional area of ​​the brain (mm 2 ) e: Area of ​​cerebral infarction on the section 4 mm posterior to the Bregma or cross-sectional area of ​​the brain (mm 2 ) f: Cerebral infarct area on the section 6 mm posterior to the Bregma or brain cross-sectional area (mm 2 )

[0122] (2) Results The cerebral infarction volume ratios of the control group and the peptide-administered group were 34.1±1.1% and 27.2±1.2% (mean±standard error), respectively (Figure 13; see "Control" for the control group and "1r10" for the peptide-administered group). The cerebral infarction volume ratios of the peptide-administered group were lower than those of the control group, and a statistically significant difference was observed (Mann-Whitney U test, p<0.01).

[0123] In this reference example, administration of the artificial sequence peptide of the present application to a cerebral infarction model rat resulted in the reduction of cerebral infarction lesions. This is believed to be the result of mesenchymal stem cells being mobilized into peripheral blood by the action of the artificial sequence peptide of the present application, and the cells exerting inflammation suppression effects, trophic effects (secretion of nutrient factors), tissue regeneration effects, etc. [Industrial Applicability]

[0124] The peptides of the present application can be used as compositions for mobilizing mesenchymal stem cells into peripheral blood, as well as therapeutic agents for inflammatory diseases, autoimmune diseases, fibrotic diseases, and diseases involving tissue damage / ischemia / necrosis.

Claims

1. A composition for use in mobilizing mesenchymal stem cells to peripheral blood, comprising a peptide in the range of 11 to 29 amino acids in length, wherein the peptide is selected from the following: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having an activity of mobilizing mesenchymal stem cells into peripheral blood; (b) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 in which one to two amino acids have been substituted, deleted, inserted or added, the peptide comprising the amino acid sequence of SEQ ID NO: 17 or 19 and having the activity of mobilizing mesenchymal stem cells into peripheral blood; and (c) A peptide consisting of a portion of an amino acid sequence having 93% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having the activity of mobilizing mesenchymal stem cells into peripheral blood.

2. A composition for use in treating a disease or pathological condition in a subject by mobilizing mesenchymal stem cells to peripheral blood, comprising a peptide in the range of 11-29 amino acids in length, wherein the peptide is selected from the following: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having an activity of mobilizing mesenchymal stem cells into peripheral blood; (b) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 in which one to two amino acids have been substituted, deleted, inserted or added, the peptide comprising the amino acid sequence of SEQ ID NO: 17 or 19 and having the activity of mobilizing mesenchymal stem cells into peripheral blood; and (c) A peptide consisting of a portion of an amino acid sequence having 93% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having the activity of mobilizing mesenchymal stem cells into peripheral blood.

3. The composition of claim 2 , wherein the treatment of a disease or pathological condition is selected from anti-inflammatory treatments, immunomodulatory treatments, treatments inducing tissue regeneration, and treatments inhibiting tissue fibrosis.

4. The composition of claim 2, wherein the disease or pathological condition is selected from inflammatory diseases, autoimmune diseases, diseases involving tissue damage, ischemia or necrosis, and fibrotic diseases.

5. The composition of claim 2, wherein the disease or pathological condition is inflammatory bowel disease.

6. The composition of claim 2, wherein the disease or pathological condition is ulcerative colitis.

7. The composition of claim 2, wherein the disease or pathological condition is atopic dermatitis.

8. The composition according to claim 2 , wherein the disease or pathological condition is cerebral infarction.

9. A composition for use in treating a disease selected from inflammatory bowel disease, atopic dermatitis and cerebral infarction, comprising a peptide in the range of 11 to 29 amino acids in length, wherein the peptide is selected from the following: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having an activity of mobilizing mesenchymal stem cells into peripheral blood; (b) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 in which one to two amino acids have been substituted, deleted, inserted or added, the peptide comprising the amino acid sequence of SEQ ID NO: 17 or 19 and having the activity of mobilizing mesenchymal stem cells into peripheral blood; and (c) A peptide consisting of a portion of an amino acid sequence having 93% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having the activity of mobilizing mesenchymal stem cells into peripheral blood.

10. The composition of any one of claims 1 to 9, wherein the peptide is selected from the following: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having an activity of mobilizing mesenchymal stem cells into peripheral blood; (b) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 in which one amino acid has been substituted, deleted, inserted or added, the peptide comprising the amino acid sequence of SEQ ID NO: 17 or 19 and having the activity of mobilizing mesenchymal stem cells into peripheral blood; and (c) A peptide consisting of a portion of an amino acid sequence having 96% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having the activity of mobilizing mesenchymal stem cells into peripheral blood.

11. A composition described in any one of claims 1 to 9, wherein the peptide is a peptide consisting of a portion of the amino acid sequence of SEQ ID NO: 1, contains the amino acid sequence of SEQ ID NO: 17 or 19, and has the activity of mobilizing mesenchymal stem cells into peripheral blood.

12. The composition of any one of claims 1 to 9, wherein the peptide is selected from the following: (1) a peptide consisting of the amino acid sequence from the 1st to the 27th amino acids of SEQ ID NO: 1 (SEQ ID NO: 2); (2) a peptide consisting of the amino acid sequence from the 1st to the 26th amino acids of SEQ ID NO: 1 (SEQ ID NO: 3); (3) a peptide consisting of the 1st to 25th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 4); (4) a peptide consisting of the 1st to 24th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 5); (5) A peptide consisting of the 1st to 23rd amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 6); (6) A peptide consisting of the 1st to 19th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 7); (7) A peptide consisting of the 1st to 18th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 8); (8) A peptide consisting of the 1st to 16th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 9); (9) A peptide consisting of the 6th to 30th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 11); (10) A peptide consisting of the amino acid sequence from the 11th to the 30th amino acids of SEQ ID NO: 1 (SEQ ID NO: 12); (11) A peptide consisting of the amino acid sequence from the 12th to the 30th amino acids of SEQ ID NO: 1 (SEQ ID NO: 13); (12) A peptide consisting of the amino acid sequence from the 13th to the 30th amino acids of SEQ ID NO: 1 (SEQ ID NO: 14); (13) A peptide consisting of the amino acid sequence from the 16th to the 30th amino acids of SEQ ID NO: 1 (SEQ ID NO: 15); (14) A peptide consisting of the amino acid sequence from the 17th to the 30th amino acids of SEQ ID NO: 1 (SEQ ID NO: 16); (15) A peptide consisting of the amino acid sequence from the 18th to the 30th positions of SEQ ID NO: 1 (SEQ ID NO: 17); and (16) A peptide consisting of the amino acid sequence from the 6th to the 16th of SEQ ID NO: 1 (SEQ ID NO: 19).

13. A peptide ranging from 11 to 29 amino acids in length, the peptide being selected from the following: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having an activity of mobilizing mesenchymal stem cells into peripheral blood; (b) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 in which one to two amino acids have been substituted, deleted, inserted or added, the peptide comprising the amino acid sequence of SEQ ID NO: 17 or 19 and having the activity of mobilizing mesenchymal stem cells into peripheral blood; and (c) A peptide consisting of a portion of an amino acid sequence having 93% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having the activity of mobilizing mesenchymal stem cells into peripheral blood.

14. The peptide of claim 13, selected from the following: (a) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having an activity of mobilizing mesenchymal stem cells into peripheral blood; (b) a peptide consisting of a part of the amino acid sequence of SEQ ID NO: 1 in which one amino acid has been substituted, deleted, inserted or added, the peptide comprising the amino acid sequence of SEQ ID NO: 17 or 19 and having the activity of mobilizing mesenchymal stem cells into peripheral blood; and (c) A peptide consisting of a portion of an amino acid sequence having 96% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of SEQ ID NO: 17 or 19, and having the activity of mobilizing mesenchymal stem cells into peripheral blood.

15. The peptide described in claim 13, which is a peptide consisting of a portion of the amino acid sequence of SEQ ID NO: 1, contains the amino acid sequence of SEQ ID NO: 17 or 19, and has the activity of mobilizing mesenchymal stem cells into peripheral blood.

16. The peptide of claim 13, selected from the following: (1) a peptide consisting of the amino acid sequence from the 1st to the 27th amino acids of SEQ ID NO: 1 (SEQ ID NO: 2); (2) a peptide consisting of the amino acid sequence from the 1st to the 26th amino acids of SEQ ID NO: 1 (SEQ ID NO: 3); (3) a peptide consisting of the 1st to 25th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 4); (4) a peptide consisting of the 1st to 24th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 5); (5) A peptide consisting of the 1st to 23rd amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 6); (6) A peptide consisting of the 1st to 19th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 7); (7) A peptide consisting of the 1st to 18th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 8); (8) A peptide consisting of the 1st to 16th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 9); (9) A peptide consisting of the 6th to 30th amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 11); (10) A peptide consisting of the amino acid sequence from the 11th to the 30th amino acids of SEQ ID NO: 1 (SEQ ID NO: 12); (11) A peptide consisting of the amino acid sequence from the 12th to the 30th amino acids of SEQ ID NO: 1 (SEQ ID NO: 13); (12) A peptide consisting of the amino acid sequence from the 13th to the 30th amino acids of SEQ ID NO: 1 (SEQ ID NO: 14); (13) A peptide consisting of the amino acid sequence from the 16th to the 30th amino acids of SEQ ID NO: 1 (SEQ ID NO: 15); (14) A peptide consisting of the amino acid sequence from the 17th to the 30th amino acids of SEQ ID NO: 1 (SEQ ID NO: 16); (15) A peptide consisting of the amino acid sequence from the 18th to the 30th positions of SEQ ID NO: 1 (SEQ ID NO: 17); and (16) A peptide consisting of the amino acid sequence from the 6th to the 16th of SEQ ID NO: 1 (SEQ ID NO: 19).

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