HK1213201B - Novel method for treating spinal cord injury using hmgb1 fragment - Google Patents

Description

Novel method for treating spinal cord injury using HMGB1 fragment

Technical Field

The present invention relates to a novel spinal cord injury-targeting pharmaceutical composition comprising an HMGB1 fragment and use thereof.

Background

The bone marrow mesenchymal stem cell is a pluripotent stem cell in a living body, and is known to be capable of differentiating into an osteoblast, an adipocyte, a cartilage, and the like. Recently, it has been reported that the autologous bone marrow mesenchymal stem cells can promote the recovery of tissue damage by administering the autologous bone marrow mesenchymal stem cells to a patient having tissue damage such as cerebral infarction. However, since the bone marrow mesenchymal stem cell is a rare cell present in the bone marrow, the amount that can be collected from the patient is limited. Therefore, it is difficult to secure the bone marrow mesenchymal stem cell in an amount necessary for the treatment of a wide range of tissue injuries. Therefore, a method of temporarily culturing bone marrow mesenchymal stem cells and proliferating the bone marrow mesenchymal stem cells to secure the number of cells required for therapy has been developed. However, it is extremely difficult to culture the bone marrow mesenchymal stem cells in an undifferentiated state. In addition, there are many problems to be solved, such as contamination with viruses and bacteria, canceration of cells, and the like. In addition, the cost for culturing cells with guaranteed safety and quality is extremely considerable.

On the other hand, the bone marrow is known to have a recovery promoting effect on the spinal cord injury. This effect is presumed to be due to a component having a tissue damage improving effect, such as differentiation of the bone marrow mesenchymal stem cell having pluripotency into a nerve cell or collection of the bone marrow mesenchymal stem cell supplying growth factor in the damaged tissue.

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: WO2009/133943

Patent document 5: WO2009/133940

Patent document 6: WO2004/004763

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, 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 USA, 108 (16): 6609 year 6614 and 2011

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

Non-patent document 12: basso et al, J Neurotrauma, 23 (5): 635 + 659, 2006

Non-patent document 13: Rahimi-Movaghar et al, Int J Neurosci, 118 (10): 1359 and 1373, 2008

Non-patent document 14: quertainmont R et al, PloS one, 7 (6): e39500, 2012

Disclosure of Invention

Problems to be solved by the invention

The spinal cord injury is a disease in which the quality of life of a patient is greatly lost due to difficulty in walking caused by paralysis of the lower body, but natural recovery is not expected, and an effective treatment method has not yet been established. As described above, the application of regenerative medicine such as cell therapy to spinal cord injury therapy is expected at present, but the application is still under development. Even if the practical use is made, the problem of high treatment cost and the like has not been solved yet.

However, if the recovery of the spinal cord injury can be promoted by administering a drug having an activity of mobilizing the bone marrow stem cells to the injury site, it is expected that an inexpensive and safe treatment method can be provided to a patient having the spinal cord injury for which there is almost no effective treatment method at present.

Means for solving the problems

To date, the inventors have found that HMGB1 (High mobility group protein-1) is a protein having an activity of stimulating the migration and mobilization of mesenchymal stem cells in the bone marrow. Conventionally, HMGB1 has been known as a major component of non-histone nucleoproteins, and has 2 DNA binding regions of BoxA and BoxB in the molecule. It is also known that the function of intranuclear HMGB1 is to relax the nucleosome structure and to construct a structure that is most suitable for the transcription reaction. However, in recent years, it has been clarified that the nuclear protein HMGB1, although having no secretion signal, is secreted outside the cell to exhibit various activities. The most intensive study was the function as a mediator (mediator) of inflammation. For example, it was clarified that HMGB1 secreted from phagocytes under TNF α stimulation is a mediator of sepsis in a sepsis model (LPS administration model) in mice (Wang et al: Science 1999; 285: 248-251) and TLR4 has been known as a candidate receptor. In addition, the best-known receptor for HMGB1 is RAGE, and binding of this receptor to HMGB1 has been reported to affect cellular migratory activity and inflammatory signaling.

The present invention discloses a novel therapeutic pharmaceutical composition for spinal cord injury containing an HMGB1 fragment and use thereof.

Specifically, the present inventors produced peptides composed of amino acids 1 to 44 of the HMGB1 protein (sequence No. 5) and HMGB1 fragments composed of amino acids 11 to 44 (sequence No. 4) by peptide synthesis. The prepared HMGB1 fragment is administered to a mouse disease model capable of evaluating the therapeutic effect of the spinal cord injury, and the therapeutic effect of the fragment on the spinal cord injury is confirmed.

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

[1] A pharmaceutical composition for the treatment of spinal cord injury, comprising an HMGB1 fragment peptide.

[2] The pharmaceutical composition according to [1], wherein the HMGB1 fragment peptide is a peptide comprising an amino acid sequence selected from the group consisting of sequence No. 3, sequence No. 4, and sequence No. 5.

[3] The pharmaceutical composition according to [1], wherein the HMGB1 fragment peptide is a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

[4] A method for treating spinal cord injury, comprising a step of administering an HMGB1 fragment peptide.

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

[6] The method according to [4], wherein the HMGB1 fragment peptide is a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

[7] An HMGB1 fragment peptide for the treatment of spinal cord injury.

[8] The HMGB1 fragment peptide according to [7], wherein the HMGB1 fragment peptide is a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

[9] The HMGB1 fragment peptide according to [7], wherein the HMGB1 fragment peptide is a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

[10] Use of an HMGB1 fragment peptide in the manufacture of a medicament for the treatment of spinal cord injury.

[11] The use according to [10], wherein the HMGB1 fragment peptide is a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

[12] The use according to [10], wherein the HMGB1 fragment peptide is a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

Drawings

Fig. 1A is a photograph showing the migration stimulating activity of PDGFR α -positive marrow derived mesenchymal stem cells of various synthetic peptides. All fragment peptides comprising the smallest fragment, i.e., fragment peptide of HMGB1 (17-25), showed cell migration stimulatory activity.

Fig. 1B is a photograph showing the migration stimulating activity of the PDGFR α -positive marrow derived mesenchymal stem cell possessed by the HMGB1 fragment peptides having different lengths. Each peptide was prepared by peptide synthesis. The accompanying figures were obtained by quantifying the cell migration stimulating activity of each fragment peptide. By this experiment, the HMGB1 fragment peptide (17-25) was shown to be the smallest fragment with migratory stimulatory activity.

In fig. 2A, significant improvement effect of neurological symptoms was seen in the HMGB1 fragment (11-44) administered group compared to the negative control group (PBS administered). (relative to PBS:. p <0.05,. p <0.01)

FIG. 2B is a spinal cord HE stain. In the group administered with the HMGB1 fragments (11-44), the size of the spinal cord injury site was reduced compared to the negative control group (administered with PBS), and a therapeutic effect was observed in pathological tissues.

Fig. 3 is a graph comparing the effects of improving neurological symptoms of the group administered full-length HMGB1 and HMGB1 fragment (11-44) in the production of a spinal cord injury model of animal diseases with time.

Fig. 4 is a graph comparing the effect of improving the neurological symptoms of the HMGB1 fragment (11-44 and 1-44) administration group in the spinal cord injury-producing animal disease model. (#: 1-44, relative to PBS: # p < 0.05;. 11-44, relative to PBS: # p <0.05)

Detailed Description

The present invention provides a pharmaceutical composition for the treatment of spinal cord injury, which contains a peptide of fragment HMGB1 having a cell migration stimulating activity. The pharmaceutical composition for treating spinal cord injury of the present invention is also referred to as a drug, a medicament, or a pharmaceutical composition in the present specification.

The spinal cord injury refers to an external or internal cause injury of the spinal cord. The spinal cord external injury includes, but is not limited to, a traumatic injury such as the spinal cord. In the specification, the spinal cord injury is also referred to as the spinal cord injury.

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 damaged part of the spinal cord, a part different from the damaged part, or blood, which requires regeneration. 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 applying or adding the composition to or near the site of injury, cells are mobilized to the injury, inducing or promoting regeneration of the injury site. For example, the bone marrow cell is mobilized from the bone marrow through the distal circulation to the tissue to be regenerated by administering and adding the composition to the tissue other than the tissue to be regenerated, 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, a "hematopoietic stem cell" is a stem cell that can differentiate into a leukocyte such as a neutrophil, an eosinophil, a basophil, a lymphocyte, a monocyte, or a macrophage, and a blood cell system cell such as a erythrocyte, a platelet, a mast cell, or a dendritic cell; as the markers, CD 34-positive and CD 133-positive markers are known for human, and CD 34-negative, c-Kit-positive, Sca-1-positive and Lineagarker-negative markers are known for mouse. 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 excluding a hemangioblast cell.

The 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 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 bone marrow cells.

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, nerve cells, epithelial cells (for example, epidermal keratinocytes, and intestinal epithelial cell keratin family expression 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-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 which can be obtained by collecting blood from the distal blood and collecting the same 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 to the lesion of the living body or the cells temporarily attached to the culture dish can be differentiated into epithelial tissues such as keratinocytes constituting the skin and nervous tissues constituting the brain.

The bone marrow-derived mesenchymal stem cell, the bone marrow-derived pluripotent stem cell, or the cells that are bone marrow-derived are preferably capable of differentiating 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 follicle cells (which express keratin family or hair keratin family), endothelial cells, liver cells, etc., mesenchymal cells such as nerve cells, pigment cells, epidermal cells, hair follicle cells (which express keratin family, hair keratin family), endothelial cells, liver cells, etc., and the like, 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 (fig. 1).

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

In the present invention, as the HMGB1 fragment having cell migration stimulating activity, a fragment peptide comprising an amino acid sequence selected from the amino acid sequences of seq id nos. 3, 4 and 5, 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 fragment peptide including at least the fragment peptide (17-25) of SEQ ID NO. 3 and having the fragment peptide (11-44) of SEQ ID NO. 4 as an upper limit, a fragment peptide including at least the fragment peptide (11-44) of SEQ ID NO. 4 and having the fragment peptide (1-44) of SEQ ID NO. 5 as an upper limit, and a fragment peptide including at least the fragment peptide (17-25) of SEQ ID NO. 3 and having the fragment peptide (1-44) of SEQ ID NO. 5 as an upper limit.

In the present invention, an HMGB1 fragment peptide having cell migration stimulating activity includes a fragment peptide consisting of an amino acid sequence selected from the amino acid sequences of seq id nos. 3, 4 and 5, that is, an HMGB1 fragment peptide having cell migration stimulating activity.

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 systemically or locally (for example, subcutaneously, intradermally, on the skin surface, eyeball or palpebral conjunctiva, nasal mucosa, oral and digestive tract mucosa, vaginal/intrauterine 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 may be selected in the range of 0.0000001mg to 1000mg per 1kg body weight in, for example, 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 to prepare a recombinant (recombiant), or can be artificially synthesized. The Pharmaceutical composition of the present invention can be prepared into preparations according to conventional methods (e.g., 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

Confirmation of migration Activity of synthetic peptide

(method)

MBL (institute of medical and biological sciences, Ltd.) was requested to synthesize the following peptides by a solid phase method. Although peptides were synthesized based on the sequence of mouse HMGB1, mouse and human HMGB1 all agreed with each other from amino acid sequences 1 to 169, and they had 100% homology.

A synthetic peptide (1-10) consisting of the 1 st to 10 th amino acid sequences of HMGB1,

A synthetic peptide (1-34) consisting of the 1 st to 34 th amino acid sequences,

A synthetic peptide (11-20) consisting of an 11 th to 20 th amino acid sequence,

A synthetic peptide (11-25) consisting of an 11 th to 25 th amino acid sequence,

A synthetic peptide (11-30) consisting of an 11 th to 30 th amino acid sequence,

A synthetic peptide (11-34) consisting of an 11 th to 34 th amino acid sequence,

Synthetic peptides (11-44) consisting of 11-44 th amino acid sequences,

And, as a positive control, the full length HMGB1(1-215(HEK)) produced by HEK293 was adjusted to 100. mu.g/ml, placed in the lower layer of the chemotactic chamber, and the migration activity of the mouse bone marrow mesenchymal stem cell line (MSC-1 cells, produced by Yujing, Osaka university, etc. (Tamai et al, Proc Natl Acad Sci U A., 108 (16): 6609-6614, 2011) was studied.

(results)

At least the synthetic peptides (11-34), (1-34), (11-44), (1-44) and (11-30) showed activities equal to or higher than those of the positive control (FIG. 1A). In addition, synthetic peptides (11-25) were also shown to be active (FIG. 1A).

(method)

To further shrink the active center portion, short peptides were synthesized as follows.

A synthetic peptide (11-25) consisting of the 11 th to 25 th amino acid sequences of HMGB1,

A synthetic peptide (12-25) consisting of a 12 th to 25 th amino acid sequence,

A synthetic peptide (13-25) consisting of a 13 th to 25 th amino acid sequence,

A synthetic peptide (14-25) consisting of 14 th to 25 th amino acid sequences,

A synthetic peptide (15-25) consisting of a 15 th to 25 th amino acid sequence,

A synthetic peptide (16-25) consisting of a 16 th to 25 th amino acid sequence,

A synthetic peptide (17-25) consisting of the 17 th to 25 th amino acid sequences.

As positive controls, a centrifuged supernatant (skin extract) obtained by immersing 1-day-old mouse skin (amount of 1 mouse) in PBS and incubating at 4 ℃ for 12 hours, and mouse full-length HMGB1(1-215(HEK)) produced by HEK293 were used. The marrow mesenchymal stem cell line (MSC-1) was inserted into the upper layer of the chemotactic chamber, and these proteins/synthetic peptides were inserted into the lower layer of the chemotactic chamber at a concentration of 5. mu.M or 10. mu.M. Migration analysis was performed in the same manner as described above.

(results)

Activity was seen for all synthetic peptides (fig. 1B). The HMGB1 fragment peptide (17-25) was shown to be the smallest fragment with migratory stimulatory activity.

Example 2

(method)

The subject used a female mouse C57BL6/J, 7-week-old. Inhalation anesthesia was performed with isoflurane, and the midline dorsal skin was incised to expose and resect the vertebral arch of the 9 th thoracic vertebra. The spinal cord was grasped from the dura mater for 3 seconds by a miniature needle holder so that the epidural spinal cord injury was produced. The spinal cord is manufactured and then the skin is sutured. The spinal cord injury was confirmed by evaluating paralysis of both hind limbs on the next day of the operation, and mice that did not show paralysis were excluded from the test. A test drug was prepared by diluting 100. mu.g of a fragment of HMGB1 (amino acid sequence: 11 th to 44 th amino acids, synthetic peptide, manufactured by MBL) to 200. mu.L of Dulbecco's PBS (D-PBS). Negative control used D-PBS 200. mu.L. The following day of surgery initial administration was performed from the tail vein, followed by a total of 5 administrations daily. Regarding evaluation of neurological symptoms, BMS (base Mouse Scale) scores were performed on days 1, 3, 7, 10, 14, 17, and 21 after surgery.

(results)

In the BMS score, significant improvement of neurological symptoms was observed from the 3 rd day after surgery in the group administered with HMGB1 fragment compared to the PBS-administered group. In particular, the symptoms were significantly improved on days 3, 7, 14 and 17 after the operation (fig. 2A). In addition, the spinal cord HE stained image of the injury site showed a wide range of injuries in the negative control group (D1), and the therapeutic effect was also shown in pathological tissues by reduction of the injury portion (D2) in the HMGB1 fragment (11-44 peptide) application group (fig. 2B).

(examination)

By administering the HMGB1 fragment, the spinal cord injury was visibly improved in neurological symptoms in mice. The HMGB1 fragment used in the test is expected to show the bone marrow mesenchymal stem cell activating activity and to have a therapeutic effect on the bone marrow damage caused by the activated bone marrow mesenchymal stem cell. The function of damaging the tissue of the bone marrow mesenchymal stem cell is expected to protect damaged tissue caused by growth factors, cytokines, and the like secreted by the bone marrow mesenchymal stem cell, in addition to tissue regeneration caused by multipotential differentiation into the nerve. In this experiment, it is assumed that the effect of the latter is short-term effect up to 1 week after the operation, and the latter effect exerts its influence again thereafter.

Example 3

(method)

The subject used a female mouse C57BL6/J, 7-week-old. Inhalation anesthesia was performed with isoflurane, and the midline dorsal skin was incised to expose and resect the vertebral arch of the 9 th thoracic vertebra. The spinal cord was grasped from the dura mater for 3 seconds by a miniature needle holder so that the epidural spinal cord injury was produced. The spinal cord is manufactured and then the skin is sutured. The spinal cord injury was confirmed by evaluating paralysis of both hind limbs on the next day of the operation, and mice that did not show paralysis were excluded from the test. A fragment of HMGB1 was prepared by diluting 100. mu.g (amino acid sequence: 11 th to 44 th amino acids, synthetic peptide, manufactured by MBL) into 200. mu.L of Dulbecco's PBS (D-PBS). The full-length protein of HMGB1 was prepared as reported by diluting HMGB1(100 μ g) expressed, produced and purified from HEK293 into 200 μ L of D-PBS. Negative control used D-PBS 200. mu.L. The first administration was performed from the tail vein the following day of surgery, followed by a total of 5 administrations daily. For evaluation of neurological symptoms, BMS scores were performed on days 1, 3, 7, and 14 after surgery.

(results)

The improvement effect of neurological symptoms was evaluated by BMS scores. The most effective treatment effect can be seen in the HMGB1 fragment (11 th-44 th amino acids) administration groups at any time of 7 th and 14 th days. Although the treatment effect was observed in the HMGB1 (full length) administration group compared to the negative control group, the treatment effect as in the HMGB1 fragment (11 th to 44 th amino acids) was not observed (fig. 3).

(examination)

The spinal cord injury was produced, and the therapeutic effect was observed at an early stage in the group that administered the full length of the HMGB1, at a level between the group that administered the HMGB1 fragment (11 th to 44 th amino acids) and the negative control group, but the spinal cord injury was produced at 14 th day after production, which was extremely better than that of the group that administered the HMGB1 fragment (11 th to 44 th amino acids). It is clear from this experiment that the bone marrow damage-specific protein is effective in terms of therapeutic effects. Since a peptide chemically synthesized as in this fragment can be produced in large quantities as a cheap and uniform product in pharmaceutical production, it is considered to be extremely useful in practical use.

Example 4

(method)

The subject used a female mouse C57BL6/J, 7-week-old. Inhalation anesthesia was performed with isoflurane, and the midline dorsal skin was incised to expose and resect the vertebral arch of the 9 th thoracic vertebra. The spinal cord was grasped from the dura mater for 3 seconds by a miniature needle holder so that the epidural spinal cord injury was produced. The spinal cord is manufactured and then the skin is sutured. The spinal cord injury was confirmed by evaluating paralysis of both hind limbs on the next day of the operation, and mice that did not show paralysis were excluded from the test. The fragment of HMGB1 was prepared by diluting 100. mu.g (amino acid sequence: 11 th to 44 th amino acids and 1 st to 44 th amino acids, synthetic peptide, manufactured by MBL) into 200. mu.L of D-PBS. Negative control used D-PBS 200. mu.L. The following day of surgery initial administration was performed from the tail vein, followed by a total of 5 administrations daily. For evaluation of neurological symptoms, BMS scores were performed on days 1, 3, 7, 10, 14, 17, 21, 28 after surgery.

(results)

The improvement effect of neurological symptoms was evaluated by BMS scores. At any time of days 17, 21 and 28, the treatment effect was observed in the group to which the HMGB1 fragment (amino acids 11 to 44 and amino acids 1 to 44) was administered, compared with the negative control group. The therapeutic effects of the fragment of HMGB1 consisting of the 11 th to 44 th amino acids and the fragment of HMGB1 consisting of the 1 st to 44 th amino acids were substantially the same (FIG. 4).

(examination)

The HMGB1 fragments 11-44 and 1-44 are all effective in the spinal cord injury treatment effect. As shown in fig. 1, the inventors have found that one of the core regions of the bone marrow mesenchymal stem cell mobilizing activity of HMGB1 is a peptide consisting of 17 th to 25 th amino acids. The fragments 11 to 44 and 1 to 44 each contain 17 to 25, and the core peptide of these peptides, which is assumed to exhibit a medicinal effect, has a sequence of 17 to 25. It is assumed that the full-length protein of HMGB1 mobilizes bone marrow mesenchymal stem cells, which is reported not via RAGE (PNAS Yujing, 2011) but also that fragments 11 to 44 and fragments 1 to 44 are not reported as ligands for known receptors such as RAGE.

Industrial applicability

The present invention provides a novel use of an HMGB1 fragment peptide that maintains the mobilization activity of PDGFR α -positive cells for the treatment of spinal cord injury. The molecular weight of the HMGB1 fragment peptide is less than about 20% relative to the HMGB1 protein consisting of 215 amino acids in the whole length. Such a fragment peptide can be produced by chemical synthesis using a peptide synthesizer, and therefore, when a peptide is produced as a drug, improvement in purification purity, stable production, and cost reduction can be expected.

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 the treatment of spinal cord injury can be developed.

Regeneration of a spinal cord injury in need of regeneration can be induced or promoted by administering the HMGB1 fragment of the present invention directly to or near the site of the injury. In addition, in a method such as intravenous administration, by administering the HMGB1 fragment of the present invention to a site different from a site where regeneration is required, regeneration of spinal cord injury can also be induced or promoted. As described above, according to the present invention, since the treatment of spinal cord injury can be performed by intravenous administration which is widely performed in general medical treatment, the therapeutic agent can be safely and easily administered at an arbitrary frequency and 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 is considered to be highly safe because it does not include a step of taking out cells to the outside of the body or a step of adding manual work. This is also one of the excellent aspects of the present invention as compared with conventional treatment methods.

Claims (2)

1. Use of an HMGB1 fragment peptide for the manufacture of a medicament for the treatment of spinal cord injury, wherein the HMGB1 fragment peptide is a fragment peptide comprising at least the fragment peptide of SEQ ID NO. 3 with the fragment peptide of SEQ ID NO. 5 as the upper limit.
2. Use of an HMGB1 fragment peptide for the manufacture of a medicament for the treatment of spinal cord injury, wherein the HMGB1 fragment peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5.