WO2014065348A1 - Hmgb1断片を利用した脊髄の損傷に対する新規治療法 - Google Patents
Hmgb1断片を利用した脊髄の損傷に対する新規治療法 Download PDFInfo
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- WO2014065348A1 WO2014065348A1 PCT/JP2013/078759 JP2013078759W WO2014065348A1 WO 2014065348 A1 WO2014065348 A1 WO 2014065348A1 JP 2013078759 W JP2013078759 W JP 2013078759W WO 2014065348 A1 WO2014065348 A1 WO 2014065348A1
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- the present invention relates to a novel pharmaceutical composition for treatment of spinal cord injury comprising the HMGB1 fragment and use thereof.
- Bone marrow mesenchymal stem cells are pluripotent in vivo stem cells and are known to differentiate into osteoblasts, adipocytes, cartilage, and the like. In recent years, it has begun to be reported that healing of tissue damage can be promoted by administering own bone marrow mesenchymal stem cells to patients with tissue damage such as cerebral infarction. However, since bone marrow mesenchymal stem cells are rare cells in the bone marrow, the amount that can be collected from a patient is limited. Therefore, it is difficult to secure an amount of bone marrow mesenchymal stem cells necessary for treatment of a wide range of tissue damage.
- bone marrow mesenchymal stem cells are once cultured and proliferated to ensure the number of cells necessary for treatment.
- it is extremely difficult to culture while maintaining the undifferentiated state of bone marrow mesenchymal stem cells.
- contamination of viruses and bacteria, and canceration of cells there are also many accompanying problems to be solved, such as contamination of viruses and bacteria, and canceration of cells.
- the cost for culturing cells with guaranteed safety and quality is extremely large.
- bone marrow mesenchymal stem cells have a healing promoting effect on spinal cord injury. This is because pluripotent bone marrow mesenchymal stem cells differentiate into neurons, and bone marrow mesenchymal stem cells accumulated in damaged tissues supply components that have tissue damage improving effects such as growth factors It is expected that.
- spinal cord injury is a disease that causes a loss of quality of life for patients, such as difficulty in walking due to paralysis of the lower body, spontaneous healing cannot be expected, and no effective treatment has been established.
- regenerative medicine such as cell therapy is currently expected to be applied to the treatment of spinal cord injury, but is still under development. Even if it is put into practical use, problems such as expensive treatment costs have not been solved.
- HMGB1 High-mobility-group-box-1
- HMGB1 is a protein having the activity of stimulating the migration of mesenchymal stem cells in the bone marrow and mobilizing it into the blood.
- HMGB1 is known as a major component of non-histone nucleoprotein, and has two DNA binding domains, BoxA and BoxB, in its molecule.
- BoxA and BoxB The function of HMGB1 in the nucleus is known to relax the nucleosome structure and construct an optimal structure for transcription reaction.
- HMGB1 is secreted extracellularly and exhibits various activities, even though it does not have a secretion signal. The most researched is its function as a mediator of inflammation.
- TLR4 is known as a receptor candidate.
- the most famous receptor of HMGB1 is RAGE, and it has been reported that the binding between this receptor and HMGB1 affects cell migration activity and inflammatory signaling.
- a novel therapeutic pharmaceutical composition for spinal cord injury containing the HMGB1 fragment and use thereof are disclosed.
- the present inventors obtained a peptide consisting of amino acids 1 to 44 of the HMGB1 protein (SEQ ID NO: 5), an HMGB1 fragment consisting of amino acids 11 to 44 (SEQ ID NO: 4), Prepared by peptide synthesis. Each prepared HMGB1 fragment was administered to a disease model mouse capable of evaluating the therapeutic effect of spinal cord injury, and the therapeutic effect of the fragment on spinal cord injury was confirmed.
- a pharmaceutical composition used for treating spinal cord injury comprising an HMGB1 fragment peptide.
- 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.
- 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.
- a method for treating spinal cord injury comprising the step of administering an HMGB1 fragment peptide.
- 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.
- 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.
- HMGB1 fragment peptide used to treat spinal cord injury 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.
- HMGB1 fragment peptide of [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.
- HMGB1 fragment peptide of [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.
- 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.
- 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.
- the HMGB1 fragment (11-44) administration group significantly improved the neurological symptoms.
- (* P ⁇ 0.05, ** p ⁇ 0.01 VS. PBS) It is a figure of HE staining of the spinal cord.
- the size of the spinal cord injury site was reduced, and a therapeutic effect was also observed in the pathological tissue.
- the present invention provides a pharmaceutical composition used for treating spinal cord injury, comprising an HMGB1 fragment peptide having cell migration stimulating activity.
- the pharmaceutical composition used to treat spinal cord injury of the present invention is also referred to herein as a medicament, agent or pharmaceutical composition.
- injury of spinal cord means extrinsic or intrinsic damage in the spinal cord.
- Exogenous damage in the spinal cord includes, but is not limited to, traumatic damage in the spinal cord, for example. Further, in the present specification, exogenous damage in the spinal cord is also simply referred to as “spinal cord injury”.
- the cell migration stimulating activity means an activity for stimulating cell migration.
- cell migration stimulating activity is also expressed as cell migration-inducing activity or cell-inducing activity.
- the administration / addition site of the pharmaceutical composition of the present invention is not limited. That is, the composition can exert its effect even if it is administered to any site such as a damaged site of the spinal cord that needs to be regenerated, a site different from the damaged site, or blood.
- the composition can exert its effect even if it is administered to any site such as a damaged site of the spinal cord that needs to be regenerated, a site different from the damaged site, or blood.
- cells are mobilized at or near the administration / addition site to induce or promote regeneration of damage.
- cells are mobilized to the injury, and regeneration of the injury is induced or promoted.
- peripheral circulation is also referred to as “blood circulation” and “peripheral circulation blood flow”.
- administering means administration to a site other than the site that requires regeneration (a site different from the site that requires regeneration). Therefore, “a tissue different from the tissue that needs to be regenerated” refers to a site that is different from the tissue that needs to be regenerated, a site that is different from the site that needs to be regenerated, a site away from the tissue that needs to be regenerated, It can also be expressed as a site remote from the site, a site distal to the site that needs regeneration, a tissue distal to the tissue that needs to be regenerated, a distal portion, and a distal tissue.
- the composition of the present invention is effectively used for regenerating a tissue in which it is difficult to administer a drug directly from outside the body.
- tissue different from the tissue that needs to be regenerated include blood tissue, muscle tissue, subcutaneous tissue, intradermal tissue, and abdominal cavity.
- cells that are stimulated to migrate or cells that are mobilized from bone marrow to peripheral blood include, but are not limited to, undifferentiated cells and cells in various stages of differentiation.
- cells that are stimulated to migrate or cells that are mobilized from bone marrow to peripheral blood include, but are not limited to, stem cells and non-stem cells.
- Stem cells include circulating stem cells or non-circulating stem cells. Examples of non-circulating stem cells include tissue stem cells that are resident in tissues. Examples of circulating stem cells include blood circulating stem cells.
- hematopoietic stem cells refers to blood cells such as neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, as well as red blood cells, platelets, mast cells, dendritic cells, etc. It is known that the stem cells can be differentiated into these cells, and the markers are CD34 positive, CD133 positive in humans, CD34 negative in mice, c-Kit positive, Sca-1 positive, and Lineage marker-negative.
- hematopoietic stem cells are characterized by difficulty in culturing alone when cultured in a culture dish, and requiring co-culture with stromal cells.
- bone marrow cell means a cell existing in the bone marrow
- bone marrow-derived cell means “bone marrow cell” mobilized from the bone marrow to the outside of the bone marrow.
- bone marrow cells include cells that contain a population of tissue precursor cells present in the bone marrow.
- the “bone marrow-derived cell” may be a cell containing mesoangioblast or a cell excluding mesoangioblast.
- Tissue progenitor cells are defined as undifferentiated cells that have the ability to unidirectionally differentiate into specific tissue cells other than the blood system, and differentiate into mesenchymal tissue, epithelial tissue, neural tissue, parenchymal organ, and vascular endothelium as described above. Including undifferentiated cells having the ability.
- bone marrow mesenchymal stem cell is a cell that is present in the bone marrow and directly from the bone marrow or other It is indirectly collected from tissues (blood, skin, fat, other tissues) and can be cultured and proliferated as adherent cells on culture dishes (plastic or glass).
- Mesenchymal tissues such as bone, cartilage, and fat (Mesenchymal stem cells), or cells that have the characteristic of differentiating into skeletal muscle, myocardium, or even neural tissue or epithelial tissue (pluripotent stem cells), and can be obtained by collecting bone marrow cells It is.
- “Bone marrow cells” and “bone marrow derived cells” are hematopoietic stem cells and differentiated cells such as leukocytes, erythrocytes, platelets, osteoblasts and fibroblasts, or bone marrow mesenchymal stem cells or bone marrow stroma so far Stem cells represented by cells called pluripotent stem cells or bone marrow pluripotent stem cells.
- “bone marrow stem cell” means a stem cell existing in the bone marrow
- “bone marrow-derived stem cell” means “bone marrow stem cell” mobilized from the bone marrow to the outside of the bone marrow.
- cells that are stimulated to migrate or cells that are mobilized from bone marrow to peripheral blood include, but are not limited to, “bone marrow-derived stem cells”.
- “Bone marrow cells” and “bone marrow-derived cells” can be isolated by bone marrow collection (bone marrow cell collection) or peripheral blood collection.
- Hematopoietic stem cells are non-adherent cells, but some of the “bone marrow cells” and “bone marrow-derived cells” are mononuclear cell fractionated cells obtained by bone marrow collection (bone marrow cell collection) and peripheral blood collection. It is obtained as an adherent cell by culture.
- “Bone marrow cells” and “bone marrow-derived cells” include mesenchymal stem cells, and can be identified by osteoblasts (identified by the presence of calcium deposition when differentiation is induced), chondrocytes (positive for Alcian blue staining, safranin) -O staining positive, etc.), adipocytes (identified by Zudan III staining positive), mesenchymal cells such as fibroblasts, smooth muscle cells, stromal cells, tendon cells, nerve cells, It preferably has an ability to differentiate into epithelial cells (for example, epidermal keratinocytes, intestinal epithelial cells express cytokeratin family) and vascular endothelial cells.
- the cells after differentiation are not limited to the above cells, but also include the ability to differentiate into parenchymal organ cells such as liver, kidney, pancreas and the like.
- bone marrow derived bone marrow mesenchymal stem cells “bone marrow derived bone marrow stromal pluripotent cells” or “bone marrow derived bone marrow pluripotent stem cells” mobilized from the bone marrow to the bone marrow are collected from peripheral blood, It is a cell that can be obtained by collection from mesenchymal tissue such as fat, epithelial tissue such as skin, and nerve tissue such as brain.
- these cells can be obtained directly after collection or by administering cells once attached to a culture dish to a damaged part of a living body, for example, epithelial tissues such as keratinocytes constituting the skin, nervous tissues constituting the brain. It also has the feature of having differentiation ability.
- Bone marrow mesenchymal stem cells, bone marrow stromal pluripotent stem cells, bone marrow pluripotent stem cells, or these cells mobilized from the bone marrow to the outside of the bone marrow are identified by osteoblasts (calcification is recognized when differentiation is induced, etc.
- chondrocytes (identifiable by Alcian blue staining positive, safranin-O staining positive, etc.), adipocytes (identifiable by Zudan III staining positive, etc.), for example, fibroblasts, smooth muscle cells, skeletal muscle Cells, stromal cells, tendon cells and other mesenchymal cells, nerve cells, pigment cells, epidermal cells, hair follicle cells (expressing cytokeratin family, hair keratin family, etc.), epithelial cells (eg epidermal keratinocytes, Intestinal epithelial cells express cytokeratin family etc.), endothelial cells, and preferably have the ability to differentiate into parenchymal organ cells such as liver, kidney, pancreas, etc.
- the cell is not limited to the above cell.
- human bone marrow cells and human bone marrow derived cells include cells that can be obtained as adherent cells by obtaining bone marrow collection (bone marrow cell collection), peripheral blood collection, and fat collection, and culturing directly or after separating the mononuclear cell fraction. Yes, but you are not limited to this.
- markers for human bone marrow cells and human bone marrow derived cells PDGFR ⁇ positive, Lin negative, CD45 negative, CD44 positive, CD90 positive, CD29 positive, Flk-1 negative, CD105 positive, CD73 positive, CD90 positive, CD71 positive, Stro- Examples of all or part of 1-positive, CD106-positive, CD166-positive, and CD31-negative can be exemplified, but are not limited thereto.
- mouse bone marrow cells and mouse bone marrow-derived cells can be obtained as adherent cells by obtaining bone marrow collection (bone marrow cell collection), peripheral blood collection, or fat collection, and culturing directly or after separation of the mononuclear cell fraction.
- bone marrow collection bone marrow cell collection
- peripheral blood collection peripheral blood collection
- fat collection or fat collection
- markers for mouse bone marrow cells and mouse bone marrow derived cells CD44 positive, PDGFR ⁇ positive, PDGFR ⁇ positive, CD45 negative, Lin negative, Sca-1 positive, c-kit negative, CD90 positive, CD29 positive, Flk-1 negative are all Or a part can be illustrated, but it is not limited to these.
- examples of cells whose migration is stimulated include PDGFR ⁇ -positive cells, but are not limited thereto.
- the PDGFR ⁇ -positive cells that stimulate migration are not particularly limited, but are preferably bone marrow-derived PDGFR ⁇ -positive cells.
- markers other than PDGFR ⁇ include all or part of CD29 positive, CD44 positive, CD90 positive, CD271 positive, CD11b negative and Flk-1 negative, but are not limited thereto.
- PDGFR ⁇ -positive cells include PDGFR ⁇ -positive bone marrow-derived cells, PDGFR ⁇ -positive bone marrow-derived mesenchymal stem cells, PDGFR ⁇ -positive tissue cells (eg, fibroblasts), PDGFR ⁇ -positive cells
- PDGFR ⁇ -positive tissue cells eg, fibroblasts
- PDGFR ⁇ -positive cells examples include bone marrow collection (bone marrow cell collection) and cells obtained as adherent cells by mononuclear cell fractionation in blood obtained by peripheral blood collection, but are not limited thereto. It is not something.
- HMGB1 protein in the present invention examples include a human-derived HMGB1 protein containing the amino acid sequence shown in SEQ ID NO: 1, and DNAs encoding the protein include DNA containing the base sequence shown in SEQ ID NO: 2. However, it is not limited to these.
- HMGB1 fragment peptide having cell migration stimulating activity means a peptide comprising a part of HMGB1 protein having cell migration stimulating activity.
- the fragment peptide consisting of a part of the HMGB1 protein of the present invention is not particularly limited as long as it has cell migration stimulating activity, but is preferably the smallest of the fragments having cell migration stimulating activity confirmed by experiments by the present inventors.
- examples of the peptide having HMGB1 fragment having cell migration stimulating activity include the following fragments, but are not limited thereto.
- the HMGB1 fragment having cell migration stimulating activity is a fragment peptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, Examples thereof include HMGB1 fragment peptides having migration stimulating activity.
- fragment peptides include a fragment peptide containing at least the fragment peptide (17-25) of SEQ ID NO: 3 and the upper limit of the fragment peptide (11-44) of SEQ ID NO: 4, or SEQ ID NO: 4
- Examples include, but are not limited to, a fragment peptide having an upper limit of the fragment peptide (1-44) of No. 5.
- the HMGB1 fragment peptide having cell migration stimulating activity is a fragment peptide consisting of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. Examples thereof include HMGB1 fragment peptides having cell migration stimulating activity.
- the administration method of the composition of the present invention includes oral administration or parenteral administration, and specific examples of the parenteral administration method include injection administration, nasal administration, pulmonary administration, and transdermal administration. However, it is not limited to these.
- parenteral administration method include injection administration, nasal administration, pulmonary administration, and transdermal administration.
- injection administration include, for example, intravenous, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like, and the composition of the present invention is systemically or locally (for example, subcutaneous, intradermal, skin surface, eyeball or eyelid conjunctiva).
- Examples of the method for administering the composition of the present invention include intravascular administration (intraarterial administration, intravenous administration, etc.), intravascular administration, intramuscular administration, subcutaneous administration, intradermal administration, and intraperitoneal administration. However, it is not limited to these.
- the administration site includes a tissue site that requires regeneration or the vicinity thereof, a site different from the tissue that requires regeneration, or a site that is distal and different from the tissue that requires regeneration.
- examples include blood (intraarterial, intravenous, etc.), muscle, subcutaneous, intradermal, and intraperitoneal, but are not limited thereto.
- the administration method can be appropriately selected depending on the age and symptoms of the patient.
- the dose can be selected in the range of 0.0000001 mg to 1000 mg per kg of body weight per administration.
- the dose can be selected within the range of 0.00001 to 100,000 mg / body per patient.
- the pharmaceutical composition of the present invention is not limited to these doses.
- the HMGB1 fragment peptide of the present invention can be obtained as a recombinant by incorporating DNA encoding the peptide into an appropriate expression system, or can be synthesized artificially.
- the pharmaceutical composition of the present invention can be formulated according to conventional methods (for example, Remington's Pharmaceutical, Science, Latest Edition, Mark Publishing Company, Easton, USA) together with pharmaceutically acceptable carriers and additives. It may be included.
- pharmaceutically acceptable carriers and additives for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, flavoring agents
- surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, flavoring agents can be used as appropriate.
- At least the synthetic peptides (11-34), (1-34), (11-44), (1-44), and (11-30) showed activity equal to or higher than that of the positive control (FIG. 1A). . Moreover, activity was recognized also in the synthetic peptide (11-25) (FIG. 1A).
- a synthetic peptide (11-25) consisting of the 11th to 25th amino acid sequences of HMGB1;
- a synthetic peptide (12-25) consisting of the 12th to 25th amino acid sequence,
- a synthetic peptide consisting of the amino acid sequence from the 13th to the 25th (13-25),
- a synthetic peptide consisting of the amino acid sequence from the 14th to the 25th (14-25),
- a synthetic peptide (15-25) consisting of the amino acid sequence from the 15th to the 25th
- a synthetic peptide (17-25) consisting of the 17th to 25th amino acid sequence.
- a 1-day-old mouse skin (one mouse) was immersed in PBS and incubated at 4 ° C. for 12 hours, and a supernatant (skin extract) produced with HEK293 and a full-length mouse HMGB1 (1-215 (HEK)) produced using HEK293 were used.
- the established bone marrow mesenchymal stem cell line (MSC-1) was placed in the upper layer of the chemotaxis chamber, and these proteins and synthetic peptides were inserted into the lower layer of the chemotaxis chamber at a concentration of 5 ⁇ M or 10 ⁇ M.
- the migration assay was performed in the same manner as described above.
- the test drug was prepared by diluting 100 ⁇ g of HMGB1 fragment (amino acid sequence: amino acids 11 to 44, synthetic peptide, manufactured by MBL) into 200 ⁇ L of Dulbecco's PBS (D-PBS). For negative control, 200 ⁇ L of D-PBS was used. The first administration was performed from the tail vein on the day after the operation, and then 5 times daily. Neurological symptoms were evaluated by BMS (Basso Mouse Scale) score on days 1, 3, 7, 10, 14, 17, and 21 after surgery.
- BMS Baseso Mouse Scale
- the administration of the HMGB1 fragment showed a clear improvement in the neurological symptoms in mice that had injured the spinal cord.
- the HMGB1 fragment used in this study showed mobilization activity of bone marrow mesenchymal stem cells, and was expected to have a therapeutic effect on spinal cord injury by mobilized bone marrow mesenchymal stem cells.
- a protective effect on damaged tissues by growth factors, cytokines, etc. secreted by bone marrow mesenchymal stem cells can be expected.
- the latter effect is expected to be affected in the short term until the first week after surgery, and the former effect is expected to be affected after that.
- HMGB1 A fragment of HMGB1 was prepared by diluting 100 ⁇ g (amino acid sequence: amino acids 11-44, synthetic peptide, manufactured by MBL) into 200 ⁇ L of Dulbecco's PBS (D-PBS).
- D-PBS Dulbecco's PBS
- the full length protein of HMGB1 was prepared by diluting HMGB1 (100 ⁇ g) expressed in HEK293, purified and purified in 200 ⁇ L of D-PBS as previously reported.
- As a negative control 200 ⁇ L of D-PBS was used.
- the first administration was performed from the tail vein on the day after the operation, and thereafter, a total of 5 administrations were performed every day. Neurological symptoms were evaluated by BMS score on days 1, 3, 7, and 14 after surgery.
- the HMGB1 full-length administration group showed an intermediate therapeutic effect between the HMGB1 fragment (11th to 44th amino acids) administration group and the negative control group.
- the HMGB1 fragment (11th to 44th amino acids) administration group showed an extremely good improvement compared to the other groups.
- This experiment revealed that the therapeutic effect was more effective than spinal cord injury than the full-length protein.
- Peptides that can be chemically synthesized, such as this fragment are considered to be extremely useful in practical use because they can produce a large amount of inexpensive and uniform products in pharmaceutical production.
- HMGB1 amino acid sequence: amino acids 11 to 44 and amino acids 1 to 44, synthetic peptide, manufactured by MBL
- the first administration was performed from the tail vein on the day after the operation, and thereafter, a total of 5 administrations were performed every day. Neurological symptoms were evaluated on the 1st, 3rd, 7th, 10th, 14th, 17th, 21st and 28th days after surgery.
- HMGB1 fragment 11-44 and 1-44 were effective in treating spinal cord injury.
- the inventors revealed that one of the core domains of HMGB1 bone marrow mesenchymal stem cell mobilization activity is a peptide consisting of the 17th to 25th amino acids.
- Both the 11-44 fragment and the 1-44 fragment contain 17-25, and the core peptide showing the efficacy of these peptides is expected to be the 17-25 sequence.
- HMGB1 full-length protein does not mediate RAGE when mobilizing bone marrow mesenchymal stem cells (2011 PNAS Tamai, etc.), and 11-44 fragments and 1-44 fragments are also reported in RAGE etc. Since there is no report as a receptor ligand, it is expected that these fragments target a previously unknown receptor.
- the present invention provides a novel use of the HMGB1 fragment peptide that maintains the mobilization activity of PDGFR ⁇ -positive cells for treating spinal cord injury.
- the HMGB1 fragment peptide of the present invention has a molecular weight of about 20 percent or less with respect to the HMGB1 protein having a total length of 215 amino acids. Since such a fragment peptide can be produced by chemical synthesis using a peptide synthesizer, an improvement in purification purity, stable production, and cost reduction are expected in the situation where the peptide is produced as a pharmaceutical product.
- HMGB1 full-length HMGB1 is known to have a binding activity to LPS (Lipopolysaccharide), a kind of endotoxin, but if a small amount of LPS is mixed with a pharmaceutical, it causes fever and often serious side effects. Therefore, the incorporation of LPS into pharmaceuticals is strictly regulated. Since HMGB1 has an affinity for LPS, it is difficult to completely remove LPS mixed in a pharmaceutical product. However, since the affinity with LPS is reduced by peptideization, it is expected that the incorporation of LPS into pharmaceuticals can be reduced. Therefore, by using the HMGB1 fragment containing the PDGFR ⁇ -positive cell mobilization part specified in the present invention, it is possible to develop a safer pharmaceutical composition for treating spinal cord injury.
- LPS Lipopolysaccharide
- HMGB1 fragment of the present invention By directly administering the HMGB1 fragment of the present invention at the site of spinal cord injury that requires regeneration or in the vicinity thereof, regeneration of the injury can be induced or promoted. Furthermore, the regeneration of spinal cord injury can also be induced or promoted by administering the HMGB1 fragment of the present invention to a site different from the site requiring regeneration by a method such as intravenous administration.
- a method such as intravenous administration.
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Abstract
Description
[1]
HMGB1断片ペプチドを含有する、脊髄の損傷を治療するために用いられる医薬組成物。
[2]
前記HMGB1断片ペプチドが、配列番号:3、配列番号:4および配列番号:5からなる群から選択されるアミノ酸配列を含むペプチドである、[1]の医薬組成物。
[3]
前記HMGB1断片ペプチドが、配列番号:3、配列番号:4および配列番号:5からなる群から選択されるアミノ酸配列からなるペプチドである、[1]の医薬組成物。
[4]
HMGB1断片ペプチドを投与する工程を含む、脊髄の損傷の治療方法。
[5]
前記HMGB1断片ペプチドが、配列番号:3、配列番号:4および配列番号:5からなる群から選択されるアミノ酸配列を含むペプチドである、[4]の方法。
[6]
前記HMGB1断片ペプチドが、配列番号:3、配列番号:4および配列番号:5からなる群から選択されるアミノ酸配列からなるペプチドである、[4]の方法。
[7]
脊髄の損傷を治療するために使用するHMGB1断片ペプチド。
[8]
前記HMGB1断片ペプチドが、配列番号:3、配列番号:4および配列番号:5からなる群から選択されるアミノ酸配列を含むペプチドである、[7]のHMGB1断片ペプチド。
[9]
前記HMGB1断片ペプチドが、配列番号:3、配列番号:4および配列番号:5からなる群から選択されるアミノ酸配列からなるペプチドである、[7]のHMGB1断片ペプチド。
[10]
HMGB1断片ペプチドを含有する、脊髄の損傷を治療するために用いられる医薬の製造のための使用。
[11]
前記HMGB1断片ペプチドが、配列番号:3、配列番号:4および配列番号:5からなる群から選択されるアミノ酸配列を含むペプチドである、[10]の使用。
[12]
前記HMGB1断片ペプチドが、配列番号:3、配列番号:4および配列番号:5からなる群から選択されるアミノ酸配列からなるペプチドである、[10]の使用。
(方法)
以下のペプチドを、MBL(株式会社医学生物学研究所)に依頼し固相法で合成した。なお、マウスHMGB1の配列に基づき、ペプチドを合成したが、マウス、およびヒトのHMGB1は1番目から169番目までのアミノ酸配列はすべて一致し、100%相同性を保っている。
HMGB1の1番目から10番目のアミノ酸配列からなる合成ペプチド(1-10)、
1番目から34番目のアミノ酸配列からなる合成ペプチド(1-34)、
11番目から20番目のアミノ酸配列からなる合成ペプチド(11-20)、
11番目から25番目のアミノ酸配列からなる合成ペプチド(11-25)、
11番目から30番目のアミノ酸配列からなる合成ペプチド(11-30)、
11番目から34番目のアミノ酸配列からなる合成ペプチド(11-34)、
11番目から44番目のアミノ酸配列からなる合成ペプチド(11-44)、および
陽性コントロールとしてHEK293で生産したマウス全長HMGB1(1-215(HEK))を、100μg/mlになるように調整し、ケモタキシスチャンバーの下層に入れ、マウス骨髄間葉系幹細胞株(MSC-1細胞、大阪大学玉井ら作製(Tamaiら、Proc Natl Acad Sci U S A.、108(16):6609-6614、2011年)に対するマイグレーション活性を検討した。
少なくとも、合成ペプチド(11-34)、(1-34)、(11-44)、(1-44)、(11-30)には、陽性コントロールと同等以上の活性を認めた(図1A)。また、合成ペプチド(11-25)にも活性を認めた(図1A)。
さらに活性中心部分を絞り込むために短いペプチドを以下のように合成した。
HMGB1の11番目から25番目のアミノ酸配列からなる合成ペプチド(11-25)、
12番目から25番目のアミノ酸配列からなる合成ペプチド(12-25)、
13番目から25番目のアミノ酸配列からなる合成ペプチド(13-25)、
14番目から25番目のアミノ酸配列からなる合成ペプチド(14-25)、
15番目から25番目のアミノ酸配列からなる合成ペプチド(15-25)、
16番目から25番目のアミノ酸配列からなる合成ペプチド(16-25)、
17番目から25番目のアミノ酸配列からなる合成ペプチド(17-25)。
全ての合成ペプチドで活性を認めた(図1B)。HMGB1断片ペプチド(17-25)が、遊走刺激活性を有する最小の断片であることが示された。
被験動物はマウスC57BL6/J, 7週齢雌マウスを使用した。イソフルランによる吸入麻酔を施行し、背部正中線の皮膚を切開し第9胸椎の椎弓を露出し切除した。同部位の硬膜を露出し、マイクロ持針器を用い硬膜上から脊髄を3秒間把持し、外傷性の脊髄の損傷を作製した。脊髄の損傷を作製した後に皮膚の縫合を行った。脊髄の損傷の確認は手術の翌日に両後肢の麻痺を評価することによって行い、麻痺の見られないマウスは試験から除外した。試験薬はHMGB1の断片(アミノ酸配列:11-44番目のアミノ酸、合成ペプチド、MBLにて製造)100μgをダルベッコーのPBS(D-PBS)200μLに希釈して調製した。陰性コントロールにはD-PBS200μLを使用した。手術翌日に初回の投与を尾静脈から行いその後毎日計5回投与を行った。神経症状の評価はBMS(Basso Mouse Scale)スコアを術後1,3,7,10,14,17,21日目に行なった。
BMSスコアで、PBS投与群に比較してHMGB1断片を投与した群にて手術後3日目から有意に神経症状の改善が観察された。特に術後3日目、7日目、14日、17日目の症状改善が顕著であった。(図2A) また、損傷部位の脊髄のHE染色像では陰性コントロール群では広範囲に損傷が認められる(D1)のに対し、HMGB1断片(11-44ペプチド)投与群では損傷部分(D2)が縮小しており病理組織上においても治療効果が認められた。(図2B)
HMGB1断片の投与によって脊髄に損傷を負わせたマウスの神経症状の明らかな改善が認められた。本試験で使用したHMGB1断片には骨髄間葉系幹細胞の動員活性が認められており、動員された骨髄間葉系幹細胞による脊髄の損傷の治療効果が期待された。骨髄間葉系幹細胞の組織損傷に対する作用としては、多能性による神経への分化による組織再生の他、骨髄間葉系幹細胞が分泌する成長因子、サイトカインなどによる損傷組織の保護作用が期待できる。今回の試験においては術後1週目までの短期的には後者の作用が、またそれ以降においてはさらに前者の作用が影響していると予想される。
被験動物はマウスC57BL6/J, 7週齢雌マウスを使用した。イソフルランによる吸入麻酔を施行し、背部正中線の皮膚を切開し第9胸椎の椎弓を露出し切除した。同部位の硬膜を露出し、マイクロ持針器を用い硬膜上から脊髄を3秒間把持し、外傷性の脊髄の損傷を作製した。脊髄の損傷を作製した後に皮膚の縫合を行った。脊髄の損傷の確認は手術の翌日に両後肢の麻痺を評価することによって行い、麻痺の見られないマウスは試験から除外した。HMGB1の断片は(アミノ酸配列:11-44番目のアミノ酸、合成ペプチド、MBLにて製造)100μgをダルベッコーのPBS(D-PBS)200μLに希釈して調製した。HMGB1の全長タンパクは既報の通りHEK293に発現させ製造し精製したHMGB1(100μg)をD-PBS200μLに希釈して調製した。陰性コントロールにはD-PBS 200μLを使用した。手術翌日に初回の投与を尾静脈から行い、その後毎日計5回投与を行った。神経症状の評価はBMSスコアを術後1,3,7,14日目に行なった。
神経症状の改善効果はBMSスコアを用いて評価した。7,14日目いずれの時点においても、HMGB1断片(11-44番目のアミノ酸)投与群において最も治療効果が見られた。HMGB1(全長)投与群は、陰性コントロール群に比較し治療効果が認められたもののHMGB1断片(11-44番目のアミノ酸)ほどの治療効果は認められなかった。(図3)
脊髄の損傷を作製した後、早期においてはHMGB1全長投与群では、HMGB1断片(11-44番目のアミノ酸)投与群と陰性コントロール群の中間程度の治療効果が認められたが、脊髄の損傷作製後14日目ではHMGB1断片(11-44番目のアミノ酸)投与群において他の群と比較して極めて良好な改善が認められた。今回の実験によって、治療効果においてはむしろ全長のタンパク以上に脊髄の損傷に対する有効性が明らかになった。本断片のように化学合成可能なペプチドは、医薬品製造において安価で均一な製品を大量に製造できることから実用化において極めて有用性が高いと考えられる。
被験動物はマウスC57BL6/J, 7週齢雌マウスを使用した。イソフルランによる吸入麻酔を施行し、背部正中線の皮膚を切開し第9胸椎の椎弓を露出し切除した。同部位の硬膜を露出し、マイクロ持針器を用い硬膜上から脊髄を3秒間把持し、外傷性の脊髄の損傷を作製した。脊髄の損傷を作製した後に皮膚の縫合を行った。脊髄の損傷の確認は手術の翌日に両後肢の麻痺を評価することによって行い、麻痺の見られないマウスは試験から除外した。HMGB1の断片は(アミノ酸配列:11-44番目のアミノ酸および1-44番目のアミノ酸、合成ペプチド、MBLにて製造)100μgをD-PBS 200μLに希釈して調製した。陰性コントロールにはD-PBS 200μLを使用した。手術翌日に初回の投与を尾静脈から行い、その後毎日計5回投与を行った。神経症状の評価はBMSスコアを術後1,3, 7,10,14,17,21,28日目に行なった。
神経症状の改善効果はBMSスコアを用いて評価した。17、21、28日いずれの時点においても、陰性コントロール群に比較するとHMGB1断片(11-44番目のアミノ酸および1-44番目のアミノ酸)投与群において治療効果が見られた。11-44番目のアミノ酸からなるHMGB1断片と1-44番目のアミノ酸からなるHMGB1断片の治療効果はほぼ同程度であった。(図4)
HMGB1断片11-44も1-44いずれも脊髄の損傷の治療効果において有効性が認められた。図1に示すように、発明者らはHMGB1の骨髄間葉系幹細胞動員活性のコアドメインの一つが17番目のアミノ酸から25番目のアミノ酸からなるペプチドであることを明らかにした。11-44の断片も1-44の断片も17-25を含んでおりこれらのペプチドの薬効を示すコアペプチドは17-25の配列であると予想される。HMGB1全長タンパクが骨髄間葉系幹細胞を動員する際にRAGEを介さないことが報告されており(2011年PNAS 玉井など)、また11-44の断片も1-44の断片もRAGE等の既報のレセプターのリガンドとしての報告がないことから、これらの断片はこれまで知られていないレセプターを標的としていることが予想される。
Claims (3)
- HMGB1断片ペプチドを含有する、脊髄の損傷を治療するために用いられる医薬組成物。
- 前記HMGB1断片ペプチドが、配列番号:3、配列番号:4および配列番号:5からなる群から選択されるアミノ酸配列を含むペプチドである、請求項1に記載の医薬組成物。
- 前記HMGB1断片ペプチドが、配列番号:3、配列番号:4および配列番号:5からなる群から選択されるアミノ酸配列からなるペプチドである、請求項1に記載の医薬組成物。
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Also Published As
Publication number | Publication date |
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PL2913059T3 (pl) | 2018-09-28 |
RU2015119523A (ru) | 2016-12-20 |
KR20150102957A (ko) | 2015-09-09 |
HK1212888A1 (zh) | 2016-06-24 |
BR112015008745B1 (pt) | 2022-08-02 |
JPWO2014065348A1 (ja) | 2016-09-08 |
TR201807769T4 (tr) | 2018-06-21 |
CA2889299A1 (en) | 2014-05-01 |
DK2913059T3 (en) | 2018-06-25 |
EP2913059A1 (en) | 2015-09-02 |
BR112015008745A2 (pt) | 2017-11-21 |
US9688733B2 (en) | 2017-06-27 |
CA2889299C (en) | 2023-02-14 |
MX2015005253A (es) | 2015-10-29 |
AU2013335685A1 (en) | 2015-05-14 |
SG11201503215XA (en) | 2015-06-29 |
MX365899B (es) | 2019-06-19 |
HK1213201A1 (zh) | 2016-06-30 |
AU2013335685B2 (en) | 2017-10-12 |
CN104955470B (zh) | 2017-06-16 |
ES2673861T3 (es) | 2018-06-26 |
US20150274792A1 (en) | 2015-10-01 |
KR102146822B1 (ko) | 2020-08-21 |
JP6253590B2 (ja) | 2017-12-27 |
CN104955470A (zh) | 2015-09-30 |
EP2913059B1 (en) | 2018-04-11 |
ZA201502829B (en) | 2020-11-25 |
EP2913059A4 (en) | 2016-03-30 |
RU2649069C2 (ru) | 2018-04-02 |
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