HK1211488B - Method for treating cardiac infarction using hmgb1 fragment - Google Patents

Description

Method for treating myocardial infarction using HMGB1 fragment

Technical Field

The present invention relates to a novel pharmaceutical composition for treating myocardial infarction, which contains a fragment peptide of HMGB1, and use thereof.

Background

Acute myocardial infarction, in which myocardial necrosis is caused by occlusion of coronary arteries, is a disease with poor prognosis and is a major basic disease of heart disease, which is the cause of death at position 2 in japan. Although the current treatment methods, i.e., acute-phase catheterization and coronary bypass surgery, are helpful in reducing myocardial damage and mortality, they have problems such as myocardial damage (ischemia-reperfusion damage) caused by interrupted coronary blood flow recanalization, and further development of a treatment method has been desired.

In recent years, it has been reported in basic experiments using animal models that bone marrow mesenchymal stem cell transplantation suppresses the development of myocardial infarction through myocardial regeneration caused by direct differentiation of transplanted cells into myocardial constituent cells and suppression of left ventricular remodeling caused by the paracrine effect of produced cytokines, and clinical tests of autologous cell transplantation have been performed in various administration routes. However, there are many problems to be overcome in the future, such as a burden on the body during cell collection, cost/labor in cell culture, and time required before cell transplantation, as problems of autologous cell transplantation. On the other hand, the mechanism by which the damaged tissue releases the bone marrow pluripotent stem cell mobilizing factor into the blood and induces regeneration of the damaged tissue has been recently identified. Tissue regeneration using the mechanism for inducing regeneration of damaged tissue in a living body by administration of bone marrow stem cell-inducing factor is a new concept of regenerative medicine which has not been known so far, and compared to cell therapy using stem cells, it is considered that the bone marrow stem cell-inducing factor has advantages in that it can be stably supplied without requiring a human hand and can be administered at the initial stage of damage.

In previous studies, the present inventors identified HMGB1 protein as a novel bone marrow pluripotent stem cell mobilizing factor. It was confirmed that HMGB1 is a main component of non-histone nucleoproteins and is released from dendritic cells, phagocytes or necrotic cells aggregated in the damaged part to the outside of the cell, which is associated with various diseases.

Documents of the prior art

Patent document

Patent document 1: WO2008/053892

Patent document 2: WO2007/015546

Patent document 3: WO2009/133939

Patent document 4: WO2009/133940

Patent document 5: WO2009/133940

Patent document 6: WO2004/004763

Patent document 7: WO2002/074337

Non-patent document

Non-patent document 1: bustin et al, Mol Cell Biol, 19: 5237 5246, 1999

Non-patent document 2: hori et al, J.biol.chem., 270, 25752-25761, 1995

Non-patent document 3: wang et al, Science, 285: 248-

Non-patent document 4: muller et al, EMBO J, 20: 4337-4340, 2001

Non-patent document 5: wang et al, Science, 285: 248-

Non-patent document 6: germani et al, J Leukoc biol., 81 (1): 41-5, 2007

Non-patent document 7: palumbo et al, j.cell biol., 164: 441-449 and 2004

Non-patent document 8: merenmies et al, j.biol.chem., 266: 16722 16729, 1991

Non-patent document 9: wu Y et al, Stem cells, 25: 2648-2659 and 2007

Non-patent document 10: tamai et al, Proc Natl Acad Sci U A., 108 (16): 6609 year 6614 and 2011

Non-patent document 11: yang et al, J Leukoc biol., 81 (1): 59-66, 2007

Disclosure of Invention

Problems to be solved by the invention

In recent years, in diseases such as cerebral infarction, a mechanism has been confirmed in which damaged tissue releases bone marrow pluripotent stem cell mobilizing factors into the blood, thereby inducing tissue regeneration of the damaged portion, and this mechanism contributes to prevention of further expansion of damage. Therefore, the present inventors have developed a novel therapeutic agent for myocardial infarction using a fragment peptide derived from HMGB1 protein identified as a novel bone marrow pluripotent stem cell mobilizing factor as an object of the present invention.

Means for solving the problems

The present inventors have first elucidated that aggregation of PDGFR α -positive cells can be promoted at and near the infarct site by preparing a fragment peptide (1-44) of HMGB1 protein having cell migration activity in a myocardial infarction model and systemically administering the fragment peptide. It is also clear that the fragment peptides (1 to 44) have an effect of improving long-term cardiac function in an animal model of myocardial infarction in the group to which the fragment peptides were administered. These findings suggest that the fragment peptides (1 to 44) induce tissue regeneration using a mechanism for inducing regeneration of damaged tissue in vivo and inhibit the development of acute myocardial infarction. Therefore, the fragment peptide of the present invention shows the possibility of making a therapeutic agent useful for acute myocardial infarction in humans with poor prognosis.

The invention discloses a novel pharmaceutical composition for treating myocardial infarction containing a fragment peptide of HMGB1 and application thereof.

Specifically, the present inventors produced peptides (SEQ ID NO: 3) composed of amino acids 1 to 44 of the HMGB1 protein by peptide synthesis. Each of the prepared HMGB1 fragments was administered to a mouse model capable of evaluating the therapeutic effect on myocardial infarction, and the therapeutic effect on myocardial infarction of the fragment was confirmed.

The present application provides the following inventions based on this knowledge.

[1] A pharmaceutical composition for treating myocardial infarction, which contains HMGB1 fragment peptide.

[2] The pharmaceutical composition according to [1], wherein the HMGB1 fragment peptide is a peptide comprising the amino acid sequence of SEQ ID NO. 3.

[3] The pharmaceutical composition according to [1], wherein the HMGB1 fragment peptide is a peptide consisting of the amino acid sequence of SEQ ID NO. 3.

[4] A method for treating myocardial infarction, which comprises a step of administering an HMGB1 fragment peptide.

[5] The method according to [4], wherein the HMGB1 fragment peptide is a peptide comprising the amino acid sequence of SEQ ID NO. 3.

[6] The method according to [4], wherein the HMGB1 fragment peptide is a peptide consisting of the amino acid sequence of SEQ ID NO. 3.

[7] An HMGB1 fragment peptide for use in the treatment of myocardial infarction.

[8] The HMGB1 fragment peptide according to [7], wherein the HMGB1 fragment peptide is a peptide comprising the amino acid sequence of SEQ ID NO. 3.

[9] The HMGB1 fragment peptide according to [7], wherein the HMGB1 fragment peptide is a peptide consisting of the amino acid sequence of SEQ ID NO. 3.

[10] Use of a fragment peptide of HMGB1 in the manufacture of a medicament for the treatment of myocardial infarction.

[11] The use according to [10], wherein the HMGB1 fragment peptide is a peptide comprising the amino acid sequence of SEQ ID NO. 3.

[12] The use according to [10], wherein the HMGB1 fragment peptide is a peptide consisting of the amino acid sequence of SEQ ID NO. 3.

Drawings

Fig. 1A is a diagram showing the numbers of PDGFR α -positive bone marrow mesenchymal stem cells in the infarction portion (inflanct), the BORDER portion (BORDER), and the non-infarction portion (REMOTE) 7 days after myocardial infarction. Fragment peptide (1-44) administration group (1-44; N ═ 4) compared to the negative control group (control group; N ═ 4), the PDGFR α -positive bone marrow mesenchymal stem cells were significantly mobilized in all regions of infarction, border, and non-infarct (1-WAY ANOVA, relative to 1-44:. P < 0.05).

FIG. 1B is a fluorescent immunostaining image at the border of mice administered with the fragment peptide (1-44). The green fluorescence-positive cells are GFP-positive bone marrow derived cells, and the red fluorescence-positive cells are labeled with PDGFR α -expressing cells. GFP positive and PDGFR α positive cells are shown with arrows.

FIG. 1C is a graph showing the mRNA levels of inflammatory cytokines TNF α in the INFARCT (INFARCT), BORDER (Border) and non-INFARCT (REMOTE) 7 or 56 days after the generation of myocardial infarction. Compared with a negative control group (control group; N ═ 4), inflammatory cytokines (TNF alpha, IL-1 beta) in the non-infarcted area were significantly low in the group to which the fragment peptide (1-44) was administered (1-WAY ANOVA, 1-44 relative to the control group: # P < 0.05).

FIG. 1D is a graph showing the mRNA levels of the inflammatory cytokine IL-1. beta. in the INFARCT (INFARCT), BORDER (Border) and non-INFARCT (REMOTE) 7 or 56 days after the generation of myocardial infarction. Compared with a negative control group (control group; N ═ 4), inflammatory cytokines (TNF alpha, IL-1 beta) in the non-infarcted area were significantly low in the group to which the fragment peptide (1-44) was administered (1-WAY ANOVA, 1-44 relative to the control group: # P < 0.05).

FIG. 1E is a graph showing the left ventricular Ejection Fraction (EF), the left ventricular end-Diastolic Diameter (DD), and the left ventricular end-systolic Diameter (DS) of the negative control group and the group to which the fragment peptide (1-44) was administered 56 days after myocardial infarction.

Detailed Description

The invention provides a pharmaceutical composition for treating myocardial infarction, which contains HMGB1 fragment peptide with cell migration stimulating activity. The pharmaceutical composition for treating myocardial infarction of the present invention is also expressed as a drug, medicament or pharmaceutical composition in the present specification.

In the present invention, the cell migration stimulating activity means an activity of stimulating cell migration. In the present specification, the cell migration stimulating activity is also expressed as a cell migration inducing activity or a cell inducing activity.

The pharmaceutical composition of the present invention is not limited to the site of administration or addition. That is, the composition can exert its effect when administered to any site such as a myocardial infarction site requiring regeneration, a site different from the infarction site, or blood. For example, by administering, adding the composition, cells are mobilized to the site at or near the site of administration, addition, inducing or promoting regeneration of the lesion. For another example, by administering or adding the composition to or near an infarct site, cells are mobilized to the infarct site, and regeneration of the infarct site is induced or promoted. For example, the bone marrow cell is mobilized from the bone marrow through the distal circulation to a site requiring regeneration by administering and adding the composition to a tissue other than the tissue requiring regeneration, thereby inducing or promoting regeneration. Here, the "peripheral circulation" is also referred to as "blood circulation" or "peripheral circulation blood flow".

Administration to a tissue different from the tissue requiring regeneration means administration to a site other than the site requiring regeneration (a site different from the site requiring regeneration). Thus, "tissue different from tissue requiring regeneration" may also be expressed as a site different from tissue requiring regeneration, a site different from a site requiring regeneration, a site distant from tissue requiring regeneration, a site distant from a site requiring regeneration, a site distal to a site requiring regeneration, a tissue distal to a tissue requiring regeneration, a distal end portion, a distal end tissue. That is, the composition of the present invention can be effectively used for regenerating tissues that are difficult to directly administer an agent from the outside of the body. Examples of the tissue different from the tissue to be regenerated include hemorrhaging tissue, muscle tissue, subcutaneous tissue, intradermal tissue, and abdominal cavity.

In the present invention, the cells that are stimulated to migrate or are bone marrow-mobilized into the peripheral blood include undifferentiated cells and cells at various stages of differentiation, but are not limited thereto. In the present invention, the cells that are stimulated to migrate or cells that are bone marrow-mobilized into the peripheral blood include, but are not limited to, stem cells, non-stem cells, and the like. The stem cells include circulating stem cells or non-circulating stem cells. Examples of the non-circulating stem cells include tissue stem cells resident in a tissue. Examples of the circulating stem cells include circulating stem cells in blood.

The bone marrow derived cell or hematopoietic stem cell may be mentioned as the stimulated or bone marrow derived cell or cell that is bone marrow secreted into the peripheral blood, but the invention is not limited thereto. In the present specification, the term "hematopoietic stem cell" refers to a stem cell that can differentiate into neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages and other leukocytes, and into erythrocytes, platelets, mast cells, dendritic cells and other blood cell lines, and is known to be positive for human CD34 and positive for CD133, and negative for mouse CD34, c-Kit, Sca-1 and Lineagemarker. Furthermore, hematopoietic stem cells are characterized in that they are difficult to culture alone and require co-culture with stromal cells when cultured in a culture dish.

The "bone marrow cell" as used herein refers to a cell present in the bone marrow, and the "bone marrow derived cell" refers to a bone marrow cell that drives the bone marrow. The "bone marrow cell" includes a cell including a tissue precursor cell group present in the bone marrow. The "bone marrow-derived cell" may be a cell including a hemangioblast cell (mesendoblast) or a cell other than a hemangioblast cell.

Tissue precursor cells are defined as undifferentiated cells having a unidirectional differentiation ability to differentiate into specific tissue cells other than blood systems, and include undifferentiated cells having an ability to differentiate into the above-mentioned mesenchymal tissue, epithelial tissue, neural tissue, solid organ, and vascular endothelium.

The "bone marrow cell" and the "bone marrow derived cell" are stem cells represented by hematopoietic stem cells, leukocytes, erythrocytes, platelets, osteoblasts, fibroblasts and other differentiated cells derived from hematopoietic stem cells, or cells known as bone marrow mesenchymal stem cells or bone marrow pluripotent stem cells. The "bone marrow stem cell" in the specification refers to a stem cell present in the bone marrow, and the "bone marrow stem cell" refers to a bone marrow stem cell that is located outside the bone marrow. In the present invention, the bone marrow derived stem cell is not limited to the bone marrow derived stem cell as the cell that is stimulated to migrate or that is driven from the bone marrow to the peripheral blood. The bone marrow cell and the bone marrow derived cell may be isolated by bone marrow collection or peripheral blood collection. The hematopoietic stem cell is a non-adherent cell, and some of the "bone marrow cell" and the "bone marrow derived cell" are obtained as adherent cells by culturing mononuclear cell fraction cells in blood collected from the bone marrow cell and the peripheral blood.

The term "bone marrow cell" or "bone marrow derived cell" includes mesenchymal stem cells, and preferably has the ability to differentiate into osteoblasts (which can be confirmed by confirmation of calcium deposition upon differentiation induction), chondrocytes (which can be confirmed by staining with alcyon blue, staining with safranin-O, or the like), adipocytes (which can be confirmed by staining with sudan III), mesenchymal cells such as fibroblasts, smooth muscle cells, stromal cells, tendon cells, and the like, and nerve cells, epithelial cells (for example, epidermal keratinocytes, intestinal epithelial cell-expressing keratin family cells), and vascular endothelial cells. The differentiated cells are not limited to the above-mentioned cells, but include the ability to differentiate into cells of solid organs such as liver, kidney, pancreas, and the like.

The "bone marrow mesenchymal stem cell", "bone marrow pluripotent cell", or "bone marrow pluripotent stem cell" in the present specification is a cell present in the bone marrow and has the following characteristics: the bone marrow is a cell which is directly collected from the bone marrow or indirectly collected from other tissues (blood, skin, fat, and other tissues), can be cultured and proliferated as adherent cells attached to a culture dish (made of plastic or glass), has the ability to differentiate into mesenchymal tissues (mesenchymal stem cells) such as bone, cartilage, and fat, skeletal muscle, cardiac muscle, neural tissue, and epithelial tissue (pluripotent stem cells), and can be collected from the bone marrow cells.

The bone marrow-derived bone marrow-derived bone marrow stem cell and the bone marrow-derived bone marrow pluripotent cell or the bone marrow-derived bone marrow pluripotent cell are cells that can be obtained by collecting blood from a tip and collecting blood from a mesenchymal tissue such as fat, an epithelial tissue such as skin, a neural tissue such as brain, and the like.

In addition, these cells have the following characteristics: the cells collected and applied directly or after being temporarily attached to a culture dish can be applied to a damaged part of a living body, and can differentiate into epithelial tissue such as keratinocytes constituting the skin and nervous tissue constituting the brain.

The bone marrow-derived mesenchymal stem cell, the bone marrow-derived pluripotent stem cell, or the cells that are electrically driven from the bone marrow preferably have the ability to differentiate into osteoblasts (which can be identified by confirmation of calcium deposition upon differentiation induction), chondrocytes (which can be identified by staining with alcian blue or staining with safranin-O), adipocytes (which can be identified by staining with sudan III), mesenchymal cells such as fibroblasts, smooth muscle cells, epidermal cells, hair follicles (which express keratin family or hair keratin family), endothelial cells, and liver, and also preferably have the ability to differentiate into endothelial cells such as fibroblasts, epidermal cells, hair follicle (which express keratin family or hair keratin family), epithelial cells such as epidermal keratinocytes, and epidermal cells, The ability of cells of solid organs such as kidney and pancreas to differentiate, but the differentiated cells are not limited to the above cells.

The human bone marrow cell and the human bone marrow derived cell include, but are not limited to, cells obtained by bone marrow collection, peripheral blood collection, fat collection, and obtained as adherent cells by direct culture or culture after isolation of a mononuclear cell fraction. The human bone marrow cell and the marker of the human bone marrow derived cell include: PDGFR alpha 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 all or part of, but not limited to.

The mouse bone marrow cell and the mouse bone marrow derived cell may be obtained by bone marrow collection, peripheral blood collection, fat collection, or may be obtained as adherent cells by direct culture or culture after isolation of a monocyte fraction, but the present invention is not limited thereto. The markers of the mouse bone marrow cell and the mouse bone marrow derived cell include: CD44 positive, PDGFR alpha positive, PDGFR beta positive, CD45 negative, Lin negative, Sca-1 positive, c-kit negative, CD90 positive, CD29 positive, Flk-1 negative, all or a part of these, but not limited to.

In the present invention, PDGFR α -positive cells are exemplified as the cells that are stimulated to migrate, but the present invention is not limited thereto. The stimulated and migrated PDGFR α -positive cell is not particularly limited, and is preferably a bone marrow derived PDGFR α -positive cell. In addition, as markers other than PDGFR α, there can be exemplified: all or a part of CD29 positive, CD44 positive, CD90 positive, CD271 positive, CD11b negative, and Flk-1 negative, but not limited thereto. Examples of PDGFR α -positive cells include: the bone marrow derived cell derived from PDGFR α, the bone marrow derived bone marrow stem cell derived from PDGFR α, a tissue cell (for example, a fibroblast cell may be exemplified) residing in a PDGFR α -positive tissue, the bone marrow derived cell derived from PDGFR α can be obtained by culturing a mononuclear cell fraction cell in blood obtained by bone marrow collection (bone marrow cell collection) or peripheral blood collection, and the like, but the present invention is not limited thereto.

Examples of the HMGB1 protein of the present invention include: the protein comprising the amino acid sequence shown in sequence No. 1 of HMGB1 protein derived from human and the DNA comprising the base sequence shown in sequence No. 2 of DNA encoding the protein are not limited to these.

The "HMGB 1 fragment peptide having cell migration stimulating activity" in the present invention refers to a peptide consisting of a part of HMGB1 protein having cell migration stimulating activity. The fragment peptide composed of a part of HMGB1 protein of the present invention is not particularly limited as long as it has cell migration stimulating activity, but is preferably the smallest peptide fragment among fragments having cell migration stimulating activity, which have been experimentally confirmed by the present inventors, that is, an HMGB1 fragment peptide containing at least the 17 th to 25 th amino acid sequences (sequence number 3) of HMGB1 protein.

In the present invention, the following fragments are exemplified as the HMGB1 fragment peptide having cell migration stimulating activity, but the present invention is not limited thereto.

In the present invention, as the HMGB1 fragment peptide having cell migration stimulating activity, a peptide comprising the amino acid sequence of sequence No. 3, that is, an HMGB1 fragment peptide having cell migration stimulating activity, is exemplified. Examples of such a fragment peptide include, but are not limited to, a peptide including at least HMGB1 fragment peptide (1 to 44) and having as its upper limit any HMGB 1-derived fragment peptide excluding the full length of HMGB1 protein.

In the present invention, as the HMGB1 fragment peptide having cell migration stimulating activity, HMGB1 fragment peptide (1-44) can be exemplified.

The method of administering the composition of the present invention includes oral administration or non-oral administration, and specific examples of the non-oral administration include injection administration, nasal administration, pulmonary administration, and transdermal administration, but are not limited thereto. As examples of the injection administration, the composition of the present invention can be administered to the whole body or the part (for example, subcutaneous, intracutaneous, skin surface, eyeball or eyelid conjunctiva, nasal mucosa, oral and digestive tract mucosa, vagina/runner's mucosa or lesion site, etc.) by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

Examples of the method of administering the composition of the present invention include, but are not limited to, intravascular administration (intra-arterial administration, intravenous administration, etc.), intravascular administration, intramuscular administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

The site of application is not limited, and examples thereof include a site of a tissue to be regenerated or a site in the vicinity thereof, a site different from the tissue to be regenerated, or a site distal to and at a different position from the tissue to be regenerated. Examples thereof include, but are not limited to, blood (intra-arterial, intravenous), muscle, subcutaneous, intradermal, and intraperitoneal.

In addition, the method of administration may be appropriately selected according to the age and symptoms of the patient. In the case of administering the peptide of the present invention, the administration amount can be selected, for example, in the range of 0.0000001mg to 1000mg per 1kg body weight in 1 administration. Alternatively, the amount administered may be selected, for example, in the range of 0.00001 to 100000mg/body per patient. In the case of administering a cell secreting the peptide of the present invention or a vector for gene therapy into which a DNA encoding the peptide is inserted, the administration may be carried out such that the amount of the peptide is within the above range. However, the pharmaceutical composition of the present invention is not limited to these administration amounts.

The HMGB1 fragment peptide of the present invention can be obtained by incorporating DNA encoding the peptide into an appropriate expression system, preparing a recombinant (recombiant), or can be artificially synthesized. The Pharmaceutical composition of the present invention can be prepared by a conventional method (for example, Remington's Pharmaceutical Science, latest edition, Mark publishing Company, Easton, U.S. A) and can contain pharmaceutically acceptable carriers and additives. Examples thereof include: the surfactant, excipient, colorant, perfume, preservative, stabilizer, buffer, suspending agent, isotonic agent, binder, disintegrating agent, lubricant, fluidity promoter, flavoring agent, etc., but are not limited thereto, and other commonly used carriers may be appropriately used. Specifically, there may be mentioned: light silicic anhydride, lactose, microcrystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethylcellulose, corn starch, inorganic salts, and the like.

In addition, all prior art documents cited in the present specification are incorporated herein by reference.

The present invention is further illustrated by the following examples, but the present invention is not limited to the following examples.

Example 1

(purpose)

The therapeutic effect of the fragment peptides (1-44) of HMGB1 protein administered systemically was evaluated in general using a mouse model of acute myocardial infarction using a cardiac physiology/histopathology/molecular biology approach.

(materials and methods)

The bone marrow chimeric mouse is produced by collecting a bone marrow cell from the thigh/lower leg bone of a 3-week-old mouse (C57BL6-CAG-GFP transgenic mouse), and administering a bone marrow cell suspension tail vein (300 μm L/mouse) to a radiation-irradiated (10GY) 6-week-old mouse (C57BL 6). The bone marrow was subjected to left intercostal thoracotomy in the supine position 60 days after the bone marrow transplantation, and the proximal part of the left anterior descending branch of the coronary artery was ligated to prepare a wide myocardial infarction model (coronary artery left anterior descending branch/circumcision occlusion model). In order to reduce the molecular weight of HMGB1 protein, a fragment peptide of HMGB1 protein was developed in consideration of maintaining the activity of mobilizing mesenchymal stem cells (1 to 44). After 6, 24, 48, 72, and 96 hours of infarction, 5 times of 5 μ g/300 μ l of the fragment peptide of HMGB1 protein (1-44) and 300 μ l of PBS (negative control group) were administered from the tail vein. Excess ketamine and xylazine were administered to euthanize the heart and the heart was harvested. Tissues were embedded in OTC compounds and frozen sections were prepared to a thickness of 5 μm. Immune tissue studies were performed using anti-mouse PDGFR α antibodies as follows. For the antibody 1, a PDGFR α rabbit anti-PDGF receptor α antibody (AB 61219, Abcam) was used. Alexa 555 goat anti-rabbit antibody (Molecular Probes) was used for 2 antibody applications, and 4, 6-diamino-2-phenylindole (DAPI, Molecular Probes) was used for nuclear staining. Confocal laser microscope (FV300, manufactured by Olympus) was used for the photography. The number of bone marrow derived mesenchymal stem cells that have accumulated in the tissue was measured for the number of cells positive for both GFP and PDGFR α present in the entire area of 5 randomly selected sections, and the average value was calculated. Further, RNA was extracted from myocardial tissue using RNeasy Kit (Qiagen), and a reverse transcription reaction was performed using Omniscript reverse transcription (Qiagen) to obtain cDNA. The amounts of mRNA for TNF α and IL-1 β were quantified as a ratio to the amount of mRNA for GAPDH by real-time PCR using Gene Amp (R) PCR System9700(Life Technologies Japan) and the above cDNA as a template. Evaluation of improvement of cardiac function was carried out by measuring LVDd (Dd: left ventricular end diastolic diameter), LVDs (Ds: left ventricular end systolic diameter), LVEF (EF: left ventricular ejection fraction) using Vivid 7echocardiography system and 12-MHZ transducer (General Electric).

By systemic administration of fragment peptides (1-44) of the HMGB1 proteinThe PDGFR α positive bone marrow-derived mesenchymal CELLS were significantly mobilized in the non-infarcted part, the boundary and the entire region of the infarcted part on the 7 th day of infarct creation (non-infarcted part: partial fragment of HMGBI protein (1-44 peptide), 52. + -. 11 #; negative control, 32. + -.9 CELLS/MM)²N-4 per group, relative to the negative control group: # P<0.05) (FIG. 1A) in addition, inflammatory cytokines (TNF α, IL-1 β) in the non-infarct portion of the fragment peptide (1-44) of HMGB1 protein were significantly low (TNF α: fragment peptide (1-44) of HMGB1 protein, 4. + -.2 #; negative control, 11. + -.3 GAPDH; N ═ 4 per group, relative to the negative control group: # P: # P<0.05), (IL-1 β: partial fragment of HMGBI protein (1-44 peptide), 2 + -1 #, negative control, 6 + -1 GAPDH, and N ═ 4 per group, relative to the negative control group, # P: #<0.05) (fig. 1C, D). As a result of the ultrasonic examination of the heart on day 56, the left ventricular systolic function impairment after myocardial infarction was significantly suppressed in the peptide fragment (1-44) of HMGB1 protein (EF: peptide fragment (1-44) of HMGB1 protein (N8), 26. + -. 4#, and 20. + -. 4% in the negative control group (N16), relative to the negative control group: # P<0.05). In addition, in the group of the fragment peptides (1-44) of HMGB1 protein, expansion of the left ventricle was significantly inhibited (DD: fragment peptide (1-44) of HMGB1 protein (N ═ 8), 5.8 ± 0.2#, negative control group (N ═ 16), 6.3 ± 0.2MM, relative to negative control group: # P<0.05) (fig. 1E).

In this study, it was demonstrated that the PDGFR α -positive bone marrow mesenchymal stem cells were mobilized to the site of infarction and its vicinity in the acute phase after infarction (day 7) by the 5-day administration of the peptide fragment of HMGB1 protein (1-44) (fig. 1A, B). In particular, it was shown that by administering the fragment peptides (1-44), mobilization of PDGFR α -positive mesenchymal stem cells to non-infarcted myocardial tissue was promoted. The bone marrow mesenchymal stem cells are known to have an inflammation inhibitory action and a tissue regeneration action, and in the present example, an action of inhibiting an inflammatory reaction is also confirmed (fig. 1C, D). It was also confirmed that administration of HMGB1 fragment peptide (1-44) significantly inhibited deterioration of left ventricular contraction function, left ventricular enlargement and left ventricular remodeling, and also confirmed a significant long-term improvement effect in myocardial function (fig. 1E).

Industrial applicability

The invention provides a new application of HMGB1 fragment peptide maintaining the mobilization activity of PDGFR alpha positive cells for treating myocardial infarction. The HMGB1 fragment peptide of the present invention contains a fragment peptide having a molecular weight of about 20% relative to an HMGB1 protein consisting of 215 amino acids in the full length. Since such a fragment peptide can be produced by chemical synthesis using a peptide synthesizer, improvement in purification purity, stable production, and cost reduction can be expected when the peptide is produced into a pharmaceutical product.

It is also known that full-length HMGB1 has an activity of binding to LPS (lipopolysaccharide), which is one of endotoxins, and causes fever and the like when LPS is mixed in a small amount in a drug, and is often associated with serious side effects, so that the mixing of LPS in a drug is strictly limited. Since HMGB1 has affinity for LPS, it is difficult to completely remove LPS mixed in a pharmaceutical product. However, since the affinity for LPS is lowered by the peptide formation, the contamination of LPS into the drug is expected to be reduced. Therefore, by using the HMGB1 fragment containing a PDGFR α -positive cell mobilization moiety identified in the present invention, a safer pharmaceutical composition for treating myocardial infarction can be developed.

By directly administering the HMGB1 fragment peptide of the present invention to a site of myocardial infarction requiring regeneration or a site in the vicinity thereof, regeneration of the infarction or tissue damage caused by the infarction can be induced or promoted. In addition, in a method such as intravenous administration, by administering the HMGB1 fragment peptide of the present invention to a site different from a site requiring regeneration, regeneration of myocardial infarction can be induced or promoted. As described above, according to the present invention, since myocardial infarction treatment can be performed by intravenous administration which is widely performed in general medical care, a therapeutic agent can be safely and easily administered at an arbitrary number of times and at an arbitrary concentration. This is one of the extremely superior aspects of the present invention compared to conventional treatment methods.

In addition, in the field of current regenerative medicine or cell therapy, rare bone marrow pluripotent stem cells derived from a patient are cultured outside a living body and proliferated for therapy, but the culturing process involves a risk of cell deterioration (canceration, contamination with bacteria, viruses, and the like), and therefore, sufficient safety management is required. In contrast, the therapeutic agent according to the present invention does not include a step of taking out cells from the body or a step of adding manual work, and therefore, is considered to be highly safe. This is also one of the excellent aspects of the present invention as compared with conventional treatment methods.

Claims (1)

1. Use of an HMGB1 fragment peptide in the manufacture of a medicament for the treatment of myocardial infarction, wherein the HMGB1 fragment peptide is a peptide consisting of the amino acid sequence of sequence No. 3.