Spinal cord injury treatment

Abstract

[Problem] To provide a therapeutic agent for spinal cord injury, namely, chronic incomplete spinal cord injury or chronic complete spinal cord injury, for which treatment has been conventionally difficult. [Solution] The present invention was accomplished upon confirming that this agent has a spinal injury therapeutic effect, a motor function improvement effect, and a urinary function improvement effect for chronic incomplete spinal cord injury or chronic complete spinal cord injury, the agent comprising: (a) a hepatocyte growth factor; a carrier which carries said (a) and can release said (a) in a sustained manner; and iPS cell-derived neural stem and / or progenitor cells.

Description

Technical Field

[0001] The present invention relates to a therapeutic agent for spinal cord injury, and particularly to a therapeutic agent for chronic incomplete spinal cord injury or chronic complete spinal cord injury. Further, the present invention relates to an agent for improving motor dysfunction, an agent for improving urinary dysfunction, an agent for increasing lower limb muscle mass, an agent for suppressing syringomyelia of the spinal cord, an anti-inflammatory agent for the injured site, an angiogenesis promoter, an agent for suppressing scar formation at the injured site, and an agent for promoting axonal extension at the injured site in chronic incomplete spinal cord injury or chronic complete spinal cord injury. In addition, each agent of the present invention may be collectively referred to as "the agent of the present invention". This application also claims the priority of PCT / JP2022 / 10976, which is incorporated herein by reference.

Background Art

[0002] (Spinal Cord Injury) Spinal cord injury is a pathological condition in which paralysis of the sensory, motor, and autonomic nervous systems below the injured part occurs due to injury to the spinal cord parenchyma caused by trauma or the like. The number of patients in Japan has reached over 150,000 in total, and about 5,000 new patients occur every year. However, it is considered that the central nervous system of mammals does not regenerate once damaged, and currently, no effective treatment method has been established yet. The pathological condition of spinal cord injury varies depending on the time after injury. Particularly in the chronic phase, nerve fibers in the injured part of the spinal cord are shed, and nerve regeneration is inhibited by syringomyelia and glial scars formed around it, so the difficulty of treatment is extremely high. Furthermore, in a completely transected spinal cord called a complete injury, functional recovery by nerve regeneration has been considered difficult because there are no nerve fibers crossing at the injured part. Although research on acute or subacute incomplete spinal cord injury is actively conducted worldwide, no effective treatment methods for chronic incomplete spinal cord injury or complete spinal cord injury, which account for the majority of spinal cord injury patients, have been reported, and the development of treatment methods for chronic incomplete spinal cord injury and complete spinal cord injury is an urgent task.

[0003] (Hepatocyte Growth Factor) Hepatocyte Growth Factor (HGF) protein was discovered as a physiologically active protein with proliferation-promoting activity against mature hepatocytes. Subsequent research has shown that HGF protein acts on c-Met receptors not only in hepatocytes but also in many epithelial cells and vascular endothelial cells, and is involved in the repair and regeneration of tissue and organ damage. HGF protein can be mass-produced as a recombinant protein using biotechnology, and recombinant HGF protein is expected to be applied as a therapeutic agent not only for hepatitis and cirrhosis but also for kidney disease and wounds. On the other hand, numerous recent studies involving gene expression and functional analysis, including knockout / knock-in mouse techniques, have revealed that HGF proteins also promote neuronal survival and neurite extension, making them important neurotrophic factors. HGF protein exhibits neurotrophic activity towards nerve cells such as hippocampal neurons, dopaminergic neurons, cerebellar granule cells, sensory neurons, and motor neurons. In particular, HGF protein shows a potent survival-promoting effect on motor neurons, and its activity is comparable to that of glial cell line-derived neurotrophic factor (GDNF), which is known to have the most potent survival-promoting effect on motor neurons. Based on this neurotrophic activity, HGF protein has been reported to be usable as a therapeutic agent for various neurological diseases, including amyotrophic lateral sclerosis (ALS) and spinal cord injury (see Patent Document 1).

[0004] (Prior non-patent literature) Non-patent document 1 discloses the effectiveness of cell transplantation in acute spinal cord injury, but also states that "improvement in motor function cannot be obtained with cell transplantation alone in chronic spinal cord injury." Non-patent document 2 discloses that "administering hepatocyte growth factor and iPS cell-derived NSCs after spinal cord injury is expected to be effective in promoting regeneration after spinal cord injury." However, it does not disclose or suggest the composition of the agent of the present invention. Non-patent document 3 discloses "a therapeutic method involving the application of a combination of gelatin-furfurylamine (FA) hydrogel and CBD-HGF or HGF to animals with spinal cord compression injury." However, document 3 discloses verification in an acute spinal cord injury model and mentions that the recovery was not satisfactory. Furthermore, document 3 does not disclose or suggest the composition of the agent of the present invention. Non-patent document 4 states that "further therapeutic effects can be expected by combining iPS-derived neural stem cell transplantation and HGF." However, it neither discloses nor suggests any specific configuration or method.

[0005] Patent Document 2 discloses a "cylindrical HGF protein-containing sustained-release formulation obtained by mixing an aqueous HGF protein solution with a phosphate buffer solution of atelocollagen, then freeze-drying and compression-molding it." However, it does not disclose or suggest the composition of the agent of the present invention. Patent Document 3 discloses a "neural injury treatment agent containing differentiated cell-derived pluripotent stem cells." However, it does not disclose or suggest the composition of the agent of the present invention. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2018-44000 [Patent Document 2] Japanese Patent No. 5419045 [Patent Document 3] Japanese Patent Publication No. 2009-215191 [Non-patent literature]

[0007] [Non-Patent Document 1] Molecular Brain volume 6, Article number: 3 (2013) [Non-Patent Document 2] Int. J. Mol. Sci. 2019, 20(15),3838 [Non-Patent Document 3] Sci. Rep. 2018, 8: 917. [Non-Patent Document 4] Medical Forum, 2020, Vol. 60, No. 9, pp. 4-6 [Overview of the project] [Problems that the invention aims to solve]

[0008] The objective is to provide a therapeutic agent for spinal cord injuries that have been difficult to treat with conventional methods, particularly chronic incomplete spinal cord injuries or chronic complete spinal cord injuries. [Means for solving the problem]

[0009] The present invention was completed by confirming that agents containing the following 1) to 3) have therapeutic effects on spinal cord injury in chronic incomplete spinal cord injury or chronic complete spinal cord injury, effects on improving motor function impairment, and effects on improving urinary function impairment. 1) HGF protein 2) A carrier capable of supporting and sustained-release of the HGF protein. 3) iPS cell-derived neural stem and / or progenitor cells

[0010] The present invention includes the following: 1. Therapeutic agents for chronic spinal cord injury or chronic complete spinal cord injury, including the following: 1) (a) Hepatocyte growth factor protein or a substance having equivalent c-Met phosphorylation activity to hepatocyte growth factor protein, or (b) A gene encoding hepatocyte growth factor protein or a gene encoding a substance having equivalent c-Met phosphorylation activity to hepatocyte growth factor protein. 2) A carrier capable of supporting and slowly releasing (a) or (b); and 3) pluripotent stem cells, Here, the carrier is placed at the site of spinal cord injury, and the pluripotent stem cells are administered to the carrier after placement or to the site of spinal cord injury. A therapeutic agent. 2. The therapeutic agent according to paragraph 1, wherein the pluripotent stem cells are iPS cell-derived neural stem and / or progenitor cells. 3. The pluripotent stem cells are the therapeutic agent according to item 1 or 2 above, which is administered to the carrier after installation. 4. The substance having the phosphorylation effect on c-Met is a hepatocyte growth factor protein, and the pluripotent stem cells are iPS cell-derived neural stem and / or progenitor cells. The therapeutic agent according to item 1 above. 5. The treatment is characterized by promoting the regeneration of the injured spinal cord and / or improving the accompanying symptoms associated with spinal cord injury. The therapeutic agent according to any one of items 1 to 4 above. 6. The promotion of the regeneration of the injured spinal cord is characterized by promoting axonal extension, increasing nerve fibers, neurogenesis of endogenous stem cells, angiogenesis, suppressing scar formation, suppressing inflammation, angiogenesis, and / or suppressing syringomyelia. The therapeutic agent according to item 5 above. 7. The accompanying symptoms associated with the injured spinal cord are characterized by motor dysfunction and / or urinary dysfunction. The therapeutic agent according to item 5 above. 8. The motor dysfunction is lower limb motor dysfunction. The therapeutic agent according to item 7 above. 9. The motor dysfunction is caused by a decrease in muscle mass. The therapeutic agent according to item 7 above. 10. An agent for improving urinary dysfunction in chronic incomplete spinal cord injury or chronic complete spinal cord injury, comprising: 1) A hepatocyte growth factor protein, a carrier capable of carrying and sustainedly releasing the hepatocyte growth factor protein; and 2) iPS cell-derived neural stem and / or progenitor cells, wherein the carrier carrying the hepatocyte growth factor protein is installed at the spinal cord injury site, and further, the iPS cell-derived neural stem and / or progenitor cells are administered to the carrier after installation or the spinal cord injury site. An agent for improving urinary dysfunction. 11. The iPS cell-derived neural stem and / or progenitor cells are administered to the carrier after installation. The therapeutic agent according to item 10 above. 12. A therapeutic agent for chronic incomplete or chronic complete spinal cord injury, comprising a hepatocyte growth factor protein and a carrier capable of carrying and sustainedly releasing the hepatocyte growth factor protein, Here, the carrier carrying the hepatocyte growth factor protein is placed at the spinal cord injury site, and iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or the spinal cord injury site after placement. A treatment agent for chronic spinal cord injury or chronic complete spinal cord injury. 13. A spinal cord injury treatment agent comprising iPS cell-derived nerve trunk and / or progenitor cells, Here, the iPS cell-derived nerve trunk and / or progenitor cells are administered to the spinal cord injury site or to a carrier loaded with hepatocyte growth factor protein placed at the spinal cord injury site. A drug used to treat spinal cord injuries. 14. Agents for improving motor function impairment in chronic incomplete spinal cord injury or chronic complete spinal cord injury, including the following: 1) Hepatocyte growth factor protein, a carrier capable of supporting and sustainably releasing the hepatocyte growth factor protein; and 2) iPS cell-derived neural stem and / or progenitor cells, And, The carrier carrying the hepatocyte growth factor protein is placed at the site of spinal cord injury, and the iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or the site of spinal cord injury after placement. A drug for improving motor function disorders. 15. The motor function is lower limb motor function, as described in paragraph 14 above, an agent for improving motor function disorders. 16. Lower limb muscle mass increasing agents in chronic incomplete spinal cord injury or chronic complete spinal cord injury, including the following: 1) Hepatocyte growth factor protein, a carrier capable of supporting and sustainably releasing the hepatocyte growth factor protein; and 2) iPS cell-derived neural stem and / or progenitor cells, And, The carrier carrying the hepatocyte growth factor protein is placed at the site of spinal cord injury, and the iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or the site of spinal cord injury after placement. A drug that increases lower limb muscle mass. 17. Spinal cord cavitation inhibitors in chronic incomplete spinal cord injury or chronic complete spinal cord injury, including the following: 1) Hepatocyte growth factor protein, a carrier capable of supporting and sustainably releasing the hepatocyte growth factor protein; and 2) iPS cell-derived neural stem and / or progenitor cells, And, The carrier carrying the hepatocyte growth factor protein is placed at the site of spinal cord injury, and the iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or the site of spinal cord injury after placement. A drug that inhibits the formation of a cavity in the spinal cord. 18. Anti-inflammatory agents for the site of injury in chronic incomplete spinal cord injury or chronic complete spinal cord injury, including the following: 1) Hepatocyte growth factor protein, a carrier capable of supporting and sustainably releasing the hepatocyte growth factor protein; and 2) iPS cell-derived neural stem and / or progenitor cells, And, The carrier carrying the hepatocyte growth factor protein is placed at the site of spinal cord injury, and the iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or the site of spinal cord injury after placement. Anti-inflammatory drug. 19. Angiogenic agents for the site of injury in chronic incomplete spinal cord injury or chronic complete spinal cord injury, including the following: 1) Hepatocyte growth factor protein, a carrier capable of supporting and sustainably releasing the hepatocyte growth factor protein; and 2) iPS cell-derived neural stem and / or progenitor cells, And, The carrier carrying the hepatocyte growth factor protein is placed at the site of spinal cord injury, and the iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or the site of spinal cord injury after placement. Angiogenesis promoter. 20. Scar formation inhibitors for injury sites in chronic incomplete spinal cord injury or chronic complete spinal cord injury, including the following: 1) Hepatocyte growth factor protein, a carrier capable of supporting and sustainably releasing the hepatocyte growth factor protein; and 2) iPS cell-derived neural stem and / or progenitor cells, And, The carrier carrying the hepatocyte growth factor protein is placed at the site of spinal cord injury, and the iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or the site of spinal cord injury after placement. Scar formation inhibitor. 21. Axonal extension promoters for injury sites in chronic incomplete spinal cord injury or chronic complete spinal cord injury, including the following: 1) Hepatocyte growth factor protein, a carrier capable of supporting and sustainably releasing the hepatocyte growth factor protein; and 2) iPS cell-derived neural stem and / or progenitor cells, And, The carrier carrying the hepatocyte growth factor protein is placed at the site of spinal cord injury, and the iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or the site of spinal cord injury after placement. Axon extension promoting agent. 22. A treatment method for chronic spinal cord injury or chronic complete spinal cord injury, comprising the following steps: 1) A step of placing a carrier capable of sustained release on the spinal cord injury site of a subject with a spinal cord injury, which carries (a) hepatocyte growth factor protein or a substance having c-Met phosphorylation activity equivalent to that of hepatocyte growth factor protein, or (b) a gene encoding hepatocyte growth factor protein or a gene encoding a substance having c-Met phosphorylation activity equivalent to that of hepatocyte growth factor protein, and 2) A step of administering pluripotent stem cells to the carrier after placement or to the spinal cord injury site. 23. A treatment kit for chronic spinal cord injury or chronic complete spinal cord injury, including the following: 1) (a) Hepatocyte growth factor protein or a substance having equivalent c-Met phosphorylation activity to hepatocyte growth factor protein, or (b) A gene encoding hepatocyte growth factor protein or a gene encoding a substance having equivalent c-Met phosphorylation activity to hepatocyte growth factor protein. 2) A carrier capable of supporting and slowly releasing (a) or (b); and 3) pluripotent stem cells, And, The carrier carrying (a) or (b) is placed at the site of spinal cord injury in a spinal cord injury patient, and the pluripotent stem cells are further administered to the carrier or the site of spinal cord injury after placement. A treatment kit for chronic spinal cord injury or chronic complete spinal cord injury. [Effects of the Invention]

[0011] The agent of the present invention has one or more of the following effects. 1) Inhibitory effect on syringomyelia formation at the site of spinal cord injury 2) Effect of improving transplant cell survival rate 3) Effect of increasing nerve fibers derived from transplanted cells 4) Effect of increasing host-derived nerve fibers at the spinal cord injury site 5) Increased neovascularization effect at the site of spinal cord injury 6) Inhibitory effect on fibrous scarring at spinal cord injury sites 7) Inhibitory effect on scar formation at the site of spinal cord injury 8) Improvement of motor function impairment due to spinal cord injury 9) Improvement of urinary dysfunction caused by spinal cord injury 10) Sustained-release effect of HGF protein at spinal cord injury sites 11) Anti-inflammatory effect at the site of spinal cord injury 12) Neuroprotective effects at the site of spinal cord injury 13) Effect of promoting axonal extension at the site of spinal cord injury (particularly, effect of increasing the number of regenerated axons and improving the density of regenerated axons) 14) Endogenous nerve trunk and progenitor cell proliferation effect at the spinal cord injury site 15) Differentiation induction effect of endogenous nerve trunk progenitor cells into neurons at the site of spinal cord injury [Brief explanation of the drawing]

[0012] [Figure 1]Evaluation method for HGF protein-containing scaffolds in the spinal cord microenvironment after chronic complete spinal cord injury. Three groups were investigated during the chronic phase, 42 days after complete spinal cord transection: an HGF protein-containing scaffold group, a scaffold-only group, and a control group (no treatment). Spinal cord samples were collected on days 7 and 14 and evaluated by immunohistochemistry and Western blot. [Figure 2] Confirmation of the sustained-release effect of HGF protein by scaffolds (in vivo). [Figure 3] Evaluation of the angiogenesis-promoting effect of HGF protein-containing scaffolds at the center of injury (the left figure shows "Neovascularization evaluation at the center of injury, 7 days after placement," and the right figure shows "Neovascularization evaluation (7 and 14 days after placement), HGF-containing scaffold group"). [Figure 4] Evaluation of the anti-inflammatory effect of HGF protein-containing scaffolds (Arginase 1). [Figure 5] Evaluation of anti-inflammatory effects of HGF protein-containing scaffolds (Arginase1-positive cells / iba1-positive cells). [Figure 6] Evaluation of the anti-inflammatory effect (TNF-α suppression) of HGF protein-containing scaffolds. [Figure 7] Evaluation of neuroprotective effects (BDNF) using HGF protein-containing scaffolds. [Figure 8] Evaluation of scar formation inhibition (TGF-β inhibition) by HGF protein-containing scaffolds. [Figure 9] Evaluation of axon regeneration and axon regrowth effects using HGF protein-containing scaffolds (number of axons (left figure) and axon density (right figure)). [Figure 10] A method for evaluating the activation of endogenous neural stem cells and progenitor cells by HGF protein-containing scaffolds. [Figure 11] Evaluation of activation of endogenous neural stem progenitor cells by HGF protein-containing scaffolds (immunostaining images of SOX2, Musashi1, and Edu). [Figure 12]Evaluation of activation of endogenous neural stem progenitor cells by HGF protein-containing scaffolds (immunostaining images for GFAP, Musashi1, and Edu). [Figure 13] Quantitative evaluation of activation of endogenous neural stem progenitor cells by HGF protein-containing scaffolds. [Figure 14] Immunostaining images showing the differentiation effect of HGF protein-containing scaffolds on endogenous neural stem progenitor cells into neurons. [Figure 15] A method for evaluating a therapeutic agent (the agent of the present invention) used in combination with administration of iPS-derived neural stem progenitor cells. [Figure 16] Spinal cord findings at the site of injury 42 days after administration of iPS-derived neural trunk progenitor cells. [Figure 17] Evaluation of the survival and engraftment rate of administered iPS-derived neural stem progenitor cells. [Figure 18] Evaluation of transplanted cell-derived nerve fibers in the area surrounding the injury (mid-sagittal section). [Figure 19] Evaluation of host-derived nerve fibers in the area surrounding the injury (mid-sagittal section). [Figure 20] Evaluation of serotonergic neurons in the area surrounding the injury (mid-sagittal section). [Figure 21] Evaluation of neovascularization in the area surrounding the injury (mid-sagittal section). [Figure 22] Evaluation of fibrous scarring and cavitation (mid-sagittal section). [Figure 23] Evaluation of glial scars (mid-sagittal section). [Figure 24] Lower limb motor function assessment (BBB score, Digigait stride length, and calf muscle weight). [Figure 25] Motor function assessment (MEP). [Figure 26] Urinary function assessment (Bladder wall). [Figure 27] Urinary function assessment (Lumbar enlargement). [Figure 28] Expression level of HGF protein in the area surrounding the injury. [Modes for carrying out the invention]

[0013] (Target of this invention) The subject of the present invention (the agent of the present invention) is a spinal cord injury treatment agent, in particular a chronic incomplete spinal cord injury treatment agent or a chronic complete spinal cord injury treatment agent, which is an agent for improving motor function and urinary function in spinal cord injury, an agent for increasing lower limb muscle mass in spinal cord injury, an agent for inhibiting spinal cord cavitation, an anti-inflammatory agent for the injury site, an angiogenesis promoter, an inhibitor of scar formation at the injury site, and an axonal extension promoter at the injury site. The present invention comprises the following: 1) (a) HGF protein or a substance having c-Met phosphorylation activity equivalent to HGF, or (b) a gene encoding HGF or a gene encoding a substance having c-Met phosphorylation activity equivalent to HGF. 2) A carrier that supports (a) or (b) and is capable of releasing it slowly. 3) Pluripotent stem cells

[0014] (Spinal cord injury) The agent of the present invention can be used to treat any of the acute, subacute, chronic incomplete spinal cord injury, and chronic complete spinal cord injury, but it is preferably used to treat chronic incomplete spinal cord injury or chronic complete spinal cord injury, which were difficult to treat with conventional treatment methods. Treatment includes improvement, alleviation, prevention of recurrence, and complete cure.

[0015] (Target for treatment) The therapeutic targets of the agent of the present invention are not particularly limited as long as they have a spinal cord. Examples include humans, monkeys, cattle, horses, pigs, sheep, dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, and the like.

[0016] (HGF protein and the gene encoding HGF) The HGF protein used in this invention is not limited to any particular species, and various animal-derived HGF proteins (natural HGF proteins or recombinant proteins produced by genetic engineering technology) can be suitably used. In this invention, for example, it is preferable to use an HGF protein derived from an animal to which the agent of the present invention is applied. For example, when the agent of the present invention is applied to humans, a human-derived HGF protein (hereinafter sometimes referred to as human HGF protein) is suitably used as the HGF protein used in this invention. More preferably, recombinant human HGF protein. Furthermore, the HGF protein used in the present invention may be a deletion type (dHGF) in which five amino acid residues are deleted.

[0017] Human HGF proteins are preferably proteins encoded by DNA consisting of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. More specifically, proteins consisting of the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 are preferred. Among these, human HGF proteins having the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 are preferred, and proteins consisting of the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 are more preferred. For example, an HGF protein consisting of the amino acid sequence represented by SEQ ID NO: 6 is a 5-amino acid deficiency HGF protein (dHGF) in which five amino acid residues from positions 131 to 135 of the amino acid sequence represented by SEQ ID NO: 5 are deleted. Both proteins having the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 are naturally occurring HGF proteins (natural HGF proteins) in the human body and possess mitogen activity, motoren activity, etc., as HGF.

[0018] The HGF proteins used in this invention include proteins that have at least about 80% sequence identity with HGF proteins derived from various animals (natural HGF proteins), preferably about 90% or more sequence identity, and more preferably about 95% or more sequence identity, and that also possess biological activity as HGF (mitogen activity and motogen activity). With respect to amino acid sequences, "sequence identity" means the degree of agreement between sequences when comparing the primary structures of proteins, and "% or more" means the degree of that agreement. The presence of the above-mentioned mitogen activity and mortogen activity of the HGF protein can be confirmed, for example, by following the method described in J. Biol. Chem. 273, 22913-22920, 1998. It is preferable to use a protein in which the mitogen activity and mortogen activity measured according to J. Biol. Chem. 273, 22913-22920, 1998 is usually about 50% or more, preferably about 70% or more, more preferably about 80% or more, and even more preferably about 90% or more compared to the natural HGF protein.

[0019] Examples of proteins having sequence identity with HGF protein include proteins that have biological activity as HGF, such as amino acid sequences in which one to several amino acid residues are substituted, deleted, and / or inserted from the amino acid sequence represented by Sequence ID No. 5 or 6, or amino acid sequences in which one to several amino acid residues are modified. "Several" usually means 1 to 8 (1, 2, 3, 4, 5, 6, 7, 8), and is usually 8, preferably 6, more preferably 5, even more preferably 3, and particularly preferably 2. The inserted or substituted amino acids are preferably natural amino acids, but may also be non-natural amino acids other than the 20 amino acids encoded by genes. Non-natural amino acids may be any compound as long as they have an amino group and a carboxyl group, for example, γ-aminobutyric acid.

[0020] Substitution of amino acid residues means replacing one amino acid residue in a polypeptide with another amino acid residue, preferably a conservative substitution. A "conservative substitution" means replacing one to several amino acid residues with other chemically similar amino acid residues in such a way that the activity of the polypeptide does not substantially change. Examples include substituting one hydrophobic amino acid residue with another hydrophobic amino acid residue, or substituting one polar amino acid residue with another polar amino acid residue having the same charge. Functionally similar amino acids that can be substituted in this way are known in the art for each amino acid. Amino acids with nonpolar (hydrophobic) side chains include glycine, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Among amino acids with polar side chains, neutral ones include serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Amino acids with a positive charge (basic) include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0021] The HGF protein contained in the agent of the present invention may be only one type, or it may be two or more of the HGF proteins described above.

[0022] The HGF protein used in the agent of the present invention can be prepared by various methods, as long as it is purified to a degree suitable for pharmaceutical use. Various methods are known for preparing HGF protein, and for example, it can be obtained by extraction and purification from organs such as the liver, spleen, lungs, bone marrow, brain, kidneys, and placenta of mammals such as rats, cattle, horses, and sheep, as well as from blood cells such as platelets and white blood cells, or from plasma, serum, etc. As a method for extracting and purifying HGF protein from the above-mentioned biological tissues, for example, rats can be given carbon tetrachloride intraperitoneally to induce a hepatitis state, and the livers of these rats can be excised, pulverized, and purified using conventional protein purification methods such as column chromatography or HPLC with S-Sepharose or heparin-Sepharose.

[0023] Alternatively, HGF protein can be obtained by culturing primary or established cell lines that produce HGF protein, and then separating and purifying the HGF protein from the culture (culture supernatant, cultured cells, etc.). Or, by using genetic engineering techniques, the gene encoding HGF protein (preferably DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2) can be incorporated into a suitable vector, which can then be inserted into a suitable host for transformation, and the desired recombinant HGF protein can be obtained from the culture of this transformant (see, for example, Biochem. Biophys. Res. Commun. 180: 1151-1158, 1991; J. Clin.Invest. 87: 1853-1857, 1991; Protein Expr. Purif. 70: 231-235, 2010, etc.). The host cells mentioned above are not particularly limited, and various host cells that have been conventionally used in genetic engineering techniques, such as Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant or animal cells, can be used. For example, when using animal cells as host cells, animal cells, such as Chinese hamster ovary (CHO) cells, mouse C127 cells, or monkey COS cells, can be transformed using an expression vector incorporating cDNA encoding the amino acid sequence of human HGF protein. The culture supernatant can then be separated and purified by column chromatography or other methods to obtain HGF protein.

[0024] The HGF protein obtained in this manner may have an amino acid sequence in which one or more amino acids [for example, one to several (where "several" has the same meaning as above, for example, 1 to 8, preferably 1 to 6, more preferably 1 to 5, even more preferably 1 to 3, particularly preferably 1 to 2; the same applies hereinafter)] are substituted, deleted, and / or inserted in the amino acid sequence of the natural HGF protein, as long as it has biological activity as HGF. Substitutions are preferably conservative substitutions. Similarly, the HGF protein may have substituted, deleted, or inserted sugar chains. Here, with respect to the amino acid sequence, "deletion, substitution, and / or insertion of one or more amino acids" means that a number of amino acids (usually one to several) that can occur naturally, or that are deleted, substituted, and / or inserted, by well-known technical methods such as genetic engineering or site-directed mutagenesis, or by methods that can occur naturally. HGF proteins in which sugar chains are substituted, deleted, or inserted refer to HGF proteins in which sugar chains attached to natural HGF proteins have been removed by enzyme treatment or other means, or natural HGF proteins in which the amino acid sequence of the sugar chain attachment site has been mutated so that sugar chains cannot be attached, or natural HGF proteins in which the amino acid sequence has been mutated so that sugar chains are attached to a site different from the natural sugar chain attachment site.

[0025] In this invention, while the HGF protein used is preferably derived from humans when applied to humans, it may also be an HGF protein derived from mammals other than humans (for example, monkeys, cattle, horses, pigs, sheep, dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, etc.). Examples of such HGF proteins include, but are not limited to, those registered in the NCBI database, such as mouse-derived HGF proteins (e.g., Accession No. AAB31855, NP_034557, BAA01065, BAA01064, etc.), rat-derived HGF proteins (e.g., Accession No. NP_58713, etc.), bovine-derived HGF proteins (e.g., Accession No. NP_001026921, XP874086, BAD02475, etc.), cat-derived HGF proteins (e.g., Accession No. NP_001009830, BAC10545, BAB21499, etc.), dog-derived HGF proteins (e.g., Accession No. NP_001002964, BAC57560, etc.), or chimpanzee-derived HGF proteins (e.g., Accession No. XP519174, etc.). The HGF protein used in the present invention may have a carboxyl group (-COOH), a carboxylate [-COOM (where M represents a metal)], an amide (-CONH2), or an ester (-COOR) at its C-terminus. Here, as R in the ester, for example, a C1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, or n-butyl; a C3-8 cycloalkyl group such as cyclopentyl or cyclohexyl; a C6-12 aryl group such as phenyl or α-naphthyl; a C7-14 aralkyl group such as phenyl-C1-2 alkyl groups such as benzyl or phenethyl, or an α-naphthyl-C1-2 alkyl group such as α-naphthylmethyl; and a C2-6 alkanoylmethyl group such as acetyloxymethyl or pivaloyloxymethyl. If the HGF protein used in the present invention has a carboxyl group or carboxylate in addition to the C-terminus, HGF proteins in which the carboxyl group or carboxylate is amidated or esterified are also included in the HGF protein of the present invention. In this case, the ester can be, for example, the C-terminal ester mentioned above. Furthermore, the HGF proteins used in the present invention also include, in the above-mentioned proteins, those in which the amino group of the N-terminal methionine residue is protected by a protecting group (for example, a formyl group, a C1-6 acyl group such as a C2-6 alkanoyl group such as acetyl), those in which the glutamyl group produced by cleavage of the N-terminal side in vivo is pyroglutamine-oxidized, those in which the reactive groups on the side chains of amino acids within the molecule (for example, -OH, -SH, amino group, imidazolyl group, indolyl group, guanidino group, etc.) are protected by an appropriate protecting group (for example, a formyl group, a C1-6 acyl group such as a C2-6 alkanoyl group such as acetyl), or complex proteins such as so-called glycoproteins to which sugar chains are attached.

[0026] In the present invention, "gene encoding HGF protein" refers to a gene capable of expressing the aforementioned HGF protein. Examples of preferred human-derived HGF protein encoding DNA (hereinafter sometimes referred to as HGF protein-encoding DNA) include those described in Nature, 342, 440 (1989); Japanese Patent No. 2777678; Biochem. Biophys. Res. Commun., 1989, Vol. 163, pp. 967-973; Proc. Natl. Acad. Sci. USA, 1991, Vol. 88 (No. 16), pp. 7001-7005, etc., and registered in GenBank / EMBL / DDBJ as Accession No. M69718, M73240, AC004960, AY246560, M29145, or M73240, etc. Furthermore, while the DNA encoding the HGF protein used in this invention is preferably human-derived when applied to humans, it may also be DNA encoding the HGF protein derived from mammals other than humans (for example, monkeys, cattle, horses, pigs, sheep, dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, etc.). Examples of DNA encoding HGF proteins include, but are not limited to, those registered in the NCBI database, such as DNA encoding mouse-derived HGF proteins (e.g., Accession No. S71816, NM_010427, D10213, D10212, etc.), DNA encoding rat-derived HGF proteins (e.g., Accession No. NM_017017, etc.), DNA encoding bovine-derived HGF proteins (e.g., Accession No. NM_001031751, AB110822, etc.), DNA encoding cat-derived HGF proteins (e.g., Accession No. NM_001009830, AB080187, AB046610, etc.), DNA encoding dog-derived HGF proteins (e.g., Accession No. NM_001002964, AB090353, etc.), or DNA encoding chimpanzee-derived HGF proteins (e.g., Accession No. XM_519174, etc.). Furthermore, a specific example of DNA encoding HGF protein is, for example, DNA having the base sequence represented by SEQ ID NO: 1 or 2. Here, the base sequence represented by SEQ ID NO: 1 corresponds to the base sequence located at nucleotides 73 to 2259 of the base sequence of Accession No. M60718, and the DNA consisting of this base sequence also corresponds to the DNA encoding HGF protein consisting of the amino acid sequence represented by SEQ ID NO: 3. In addition, in DNA recombination technology, when HGF protein expressed and produced in cells (SEQ ID NO: 3) is secreted outside the cell, the signal sequence is cleaved to become HGF protein consisting of the amino acid sequence shown by SEQ ID NO: 5. Therefore, the DNA consisting of the base sequence shown by SEQ ID NO: 1 also corresponds to the DNA that encodes (produces) HGF protein consisting of the amino acid sequence shown by SEQ ID NO: 5. The base sequence represented by SEQ ID NO: 2 corresponds to the base sequence located at nucleotides 66 to 2237 of the base sequence of Accession No. M73240, and the DNA consisting of this base sequence also corresponds to the DNA encoding HGF protein consisting of the amino acid sequence shown by SEQ ID NO: 4. This HGF protein (SEQ ID NO: 4) also undergoes DNA recombination technology, and when secreted outside the cell, its signal sequence is cleaved, resulting in an HGF protein consisting of the amino acid sequence shown in SEQ ID NO: 6. Therefore, the DNA consisting of the base sequence shown in SEQ ID NO: 2 also corresponds to the DNA that codes for (produces) the HGF protein consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0027] (A substance having c-Met phosphorylation activity equivalent to that of HGF protein, and the gene encoding said substance) HGF proteins, which have the phosphorylating effect of c-Met, are known to induce phosphorylation of c-Met receptors, thereby exhibiting anti-apoptosis, angiogenesis, morphogenesis, cell division promotion, and axon elongation effects (see: Proc Jpn Acad Ser B Phys Biol Sci. 2010;86(6):588-610). In other words, a substance having the same c-Met phosphorylation activity as HGF protein can be used as an active ingredient in the agent of the present invention, just like HGF protein. Examples of substances having c-Met phosphorylation activity equivalent to that of HGF protein include, but are not limited to, the following. For example, small molecule compounds such as ANG-3777 (Angion: a Hepatocyte Growth Factor (HGF) Mimetic) and ATH-1017 (Athira Pharma); cyclic peptide dimers such as aML5, aMD4, and aMD5; HGF substitute peptides (product code: PG-001; c-Met agonist; PeptiGrowth Co., Ltd.); anti-Met antibody AGMB-101 (AGOMAB: AGMB-101 is a full MET agonist); HGF molecular fragment modifiers such as NK1 dimer or K1K1; DNA aptamers, RNA aptamers; Internalin (Listeria monocytogenes protein); and peptides having substantially the same activity as HGF protein are preferred. A peptide having substantially the same activity as the HGF protein (hereinafter sometimes abbreviated as HGF partial peptide) may be any of the HGF protein partial peptides described above that have substantially the same activity as the HGF protein. In the present invention, the number of amino acids in the HGF partial peptide is preferably about 20 or more, preferably about 50 or more, and more preferably about 100 or more amino acids from the constituent amino acid sequence of the HGF protein described above. Specifically, for example, a peptide represented by the sequence from the N-terminal hairpin loop to the first kringle domain of the human HGF amino acid sequence is preferred. In the HGF partial peptide of the present invention, the C-terminus may be any of a carboxyl group (-COOH), a carboxylate [-COOM], an amide (-CONH2), or an ester (-COOR). Furthermore, the HGF partial peptide also includes complex peptides such as those in which the amino group of the N-terminal methionine residue is protected by a protecting group, those in which the N-terminal side is cleaved in vivo and the resulting Gln is pyroglutamine-oxidized, those in which substituents on the side chains of amino acids within the molecule are protected by appropriate protecting groups, or so-called glycopeptides to which sugar chains are attached. Examples of genes encoding substances that phosphorylate c-Met used in the present invention include genes encoding the above-mentioned peptides that phosphorylate c-Met.

[0028] (sustained-release carrier) The sustained-release carrier of the present invention is not particularly limited as long as it can sustainably release HGF protein or a substance having c-Met phosphorylation activity equivalent to that of HGF protein, a gene encoding hepatocyte growth factor, or a gene encoding a substance having c-Met phosphorylation activity equivalent to that of HGF protein (hereinafter, this may be abbreviated as "the active ingredient of the present invention" in this specification) at the spinal cord injury site. Examples of the shape of the sustained-release carrier include, but are not limited to, a membrane (sheet-like, film-like), sponge-like, gel-like, spherical, particulate, or fibrous form. The sustained-release carrier can be manufactured according to known methods. The biodegradable polymer used in this sustained-release carrier can be appropriately selected from known biodegradable polymers. Examples include polysaccharides such as starch, dextran, hyaluronan (hyaluronic acid) or its salts or chitosan; proteins such as atelocollagen, collagen or gelatin; polyamino acids such as polyglutamic acid, polylysine, polyleucine, polyalanine or polymethionine; polyesters such as polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, polycaprolactone, poly-β-hydroxybutyric acid, polymalic acid, polyacid anhydride or fumaric acid-polyethylene glycol-vinylpyrrolidone copolymer; polyalkylcyanoacrylic acids such as polyorthoester or polymethyl-α-cyanoacrylic acid; and polycarbonates such as polyethylene carbonate or polypropylene carbonate. Preferred sustained-release carriers are lactic acid-glycolic acid copolymers or collagen.

[0029] When using a lactic acid-glycolic acid copolymer, the composition ratio (lactic acid / glycolic acid) (mol%) varies depending on the sustained release period. For example, if the sustained release period is about 2 weeks to 3 months, preferably about 2 weeks to 1 month, the ratio is about 100 / 0 to 50 / 50. The weight-average molecular weight of the lactic acid-glycolic acid copolymer is generally about 5,000 to 20,000. The lactic acid-glycolic acid copolymer can be produced according to known manufacturing methods, for example, the manufacturing method described in Japanese Patent Publication No. 61-28521. There are no particular limitations on the mixing ratio of biodegradable polymer and HGF protein, but for example, the amount of HGF protein relative to the biodegradable polymer is about 0.01 to 30 w / w%. As the lactic acid-glycolic acid copolymer, for example, Neuro-SpinalScaffold (highly porous bioresorbable polymer composed of poly(lactic-co-glycolic acid)-b-poly-(l-lysine); INVIVO) can be preferably used. In this specification, unless otherwise specified, % refers to mass%.

[0030] When using collagen, atelocollagen is preferred. Suitable atelocollagen materials include those with a sponge-like structure, such as collagen sponge sheets and artificial dermis (Pernac; Gunze Corporation). Atelocollagen can contain, for example, gelatin (preferably negatively charged gelatin). An example of a negatively charged gelatin-containing collagen sponge sheet is Pernac G Plus (Gunze Corporation), which contains 10% negatively charged gelatin in Pernac. By using Pernac G Plus, positively charged substances, such as HGF protein, can easily bind to the contained negatively charged gelatin, and the HGF protein can be released gradually along with the decomposition of the gelatin.

[0031] As for the loading method, the active ingredient of the present invention can be loaded onto a sustained-release carrier by impregnating it with a solution containing the active ingredient of the present invention, or by coating, spraying, or injecting the solution onto the sustained-release carrier. Preferably, the amount of active ingredient loaded onto the carrier includes at least the dosage amount of the active ingredient of the present invention. Furthermore, genes encoding HGF protein or substances having c-Met phosphorylation activity equivalent to that of HGF protein are supported on a sustained-release carrier in the form of a recombinant expression vector into which the DNA containing the gene is incorporated. Examples of such expression vectors include naked plasmids, detoxified retroviruses, adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus type I), vaccinia viruses, poxviruses, polioviruses, cymbisviruses, Sendai viruses, SV40, or DNA viruses or RNA viruses such as immunodeficiency virus (HIV). Among these, naked plasmids, herpes simplex virus type I (HSV-1) vectors, Sendai virus envelope (HVJ-E) vectors, adenovirus vectors, or adeno-associated virus (AAV) vectors are preferred.

[0032] (pluripotent stem cells) The pluripotent stem cells of the present invention are not particularly limited as long as they can exert the effects of the agent of the present invention. Examples of pluripotent stem cells include differentiated cell-derived pluripotent stem cells obtained by forcibly expressing the Oct3 / 4 gene, the Sox2 gene, and the Klf4 gene in differentiated cells (see: Japanese Patent Publication No. 2009-215191), pluripotent stem cells (see: Japanese Patent No. 5603282), embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells). Neural stem cells are cells that possess the ability to self-renew and differentiate into neural progenitor cells. Neural progenitor cells are cells that, although undifferentiated, have differentiated one step from neural stem cells, and they self-replicate and eventually differentiate into nerve cells. It is preferable that neural stem cells or neural progenitor cells are obtained by differentiation induction from pluripotent stem cells. The method for differentiating pluripotent stem cells into neural stem cells or neural progenitor cells is not particularly limited and may be any commonly used method. For example, neurospheres can be obtained by suspension culture of pluripotent stem cells in a medium containing at least one of bFGF and EGF. Alternatively, the obtained neurospheres may be dissociated into single cells and then suspension cultured again in a medium containing bFGF to form new neurospheres, and this process may be repeated multiple times. For example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells are available (see: WO2021 / 045217). iPS cell-derived neural stem and / or progenitor cells can preferably be exemplified by neural stem cells and / or neural progenitor cells derived from human umbilical cord blood-derived human iPS cells (YZWJs513 hiPSc).

[0033] (Method for installing sustained-release carriers) The method for installing the sustained-release carrier is not particularly limited. Although HGF protein is used as an example in this explanation, sustained-release carriers containing other active ingredients can be installed in the same manner. It is preferable to place a sustained-release carrier loaded with HGF protein at the spinal cord injury site (particularly the cavity where the spinal cord has been severed). The dosage of HGF protein is appropriately selected according to the severity of the disease or age, but is usually 1 μg to 100 mg, preferably 10 μg to 50 mg, of HGF protein per dose. The administration method can also be a single dose or multiple doses with intervals between doses. Furthermore, the timing of administration can be appropriately selected according to the severity of the disease or the patient's age; for example, it can be administered immediately after injury. However, as described in the following examples, the agent of the present invention is also effective in treating chronic incomplete spinal cord injury or chronic complete spinal cord injury. Therefore, it is not limited to the acute phase (approximately 2 weeks after injury) and the subacute phase (2-3 weeks after injury), but can be administered in the chronic phase after the subacute phase.

[0034] (Method of administering pluripotent stem cells) Methods of administering pluripotent stem cells include, but are not limited to, transplantation and injection. For example, methods of administration include administering them to a pre-installed sustained-release carrier as described above (particularly, administering pluripotent stem cells to multiple locations on the carrier), and administering pluripotent stem cells to the spinal cord injury site (particularly the cavity where the spinal cord was severed) before or after the placement of the sustained-release carrier. They can also be administered by intravenous injection to reach the spinal cord injury site. The timing of administration of pluripotent stem cells may be before the placement of the sustained-release carrier, but it is preferable to administer them after the placement of the carrier. Examples include the period or duration during which HGF is being sustainably released from the carrier, or immediately after the placement of the carrier. Typically, this occurs 1 to 40 days after the installation of the sustained-release carrier (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 days later), with 1 to 30 days being preferred. Furthermore, if HGF is administered multiple times, the timing of pluripotent stem cell administration can be added. There is no particular limit to the number of times pluripotent stem cells are administered; it is at least once, and usually between 1 and 5 times. If HGF is administered multiple times, the number of times pluripotent stem cells are administered can also be increased as appropriate. The number of pluripotent stem cells administered is not particularly limited, but is at least 100,000 cells / per (usually 100,000 to 50 million cells / per, 100,000 to 10 million cells / per, 100,000 to 5 million cells / per, 200,000 to 3 million cells / per, or 300,000 to 2.5 million cells / per).

[0035] (Spinal cord injury treatment agent) The spinal cord injury treatment agent of the present invention comprises the following: 1) (a) HGF protein or a substance having equivalent c-Met phosphorylation activity to HGF protein, or (b) a gene encoding HGF or a gene encoding a substance having equivalent c-Met phosphorylation activity to HGF protein. 2) A carrier that supports (a) or (b) and is capable of releasing it slowly. 3) Pluripotent stem cells Furthermore, another embodiment of the spinal cord injury treatment agent of the present invention includes the following: 1) HGF protein and a carrier capable of supporting and sustainably releasing the HGF protein The carrier carrying the HGF protein is placed at the spinal cord injury site, and then iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or the spinal cord injury site. Furthermore, another embodiment of the spinal cord injury treatment agent of the present invention includes the following: 1) iPS cell-derived neural stem and / or progenitor cells Furthermore, iPS cell-derived neural trunk and / or progenitor cells are administered to the spinal cord injury site where a carrier loaded with HGF protein is placed.

[0036] (Treatment methods for spinal cord injury) The present invention provides a method for treating spinal cord injury, which includes the following steps. 1) A process of placing a carrier capable of sustained release on the spinal cord injury site of a person with a spinal cord injury, which carries (a) HGF protein or a substance having c-Met phosphorylation activity equivalent to that of HGF protein, or (b) a gene encoding HGF protein or a gene encoding a substance having c-Met phosphorylation activity equivalent to that of HGF protein. 2) The process of administering pluripotent stem cells to the carrier after placement in 1) or to the spinal cord injury site.

[0037] (Treatment kit for spinal cord injury) The spinal cord injury treatment kit of the present invention includes the following: 1) (a) HGF protein or a substance having equivalent c-Met phosphorylation activity to HGF protein, or (b) a gene encoding HGF protein or a gene encoding a substance having equivalent c-Met phosphorylation activity to HGF protein. 2) A carrier that supports (a) or (b) and is capable of releasing it slowly. 3) Pluripotent stem cells The carrier supporting (a) or (b) is placed at the site of spinal cord injury in a spinal cord injury patient, and the pluripotent stem cells are administered to the carrier or the site of spinal cord injury after placement.

[0038] Preferred embodiments of the spinal cord injury treatment agent, spinal cord injury treatment method, and spinal cord injury treatment kit of the present invention include a substance having c-Met phosphorylating activity being HGF protein, pluripotent stem cells being iPS cell-derived neural trunk and / or progenitor cells, and spinal cord injury being chronic incomplete spinal cord injury or chronic complete spinal cord injury. Furthermore, in a preferred embodiment of the spinal cord injury therapeutic agent, spinal cord injury treatment method, and spinal cord injury treatment kit of the present invention, a carrier carrying and capable of sustained-release of HGF protein is placed at the injury site, and iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or injury site after placement.

[0039] As an example of treatment using the spinal cord injury treatment agent, method for treating spinal cord injury, and treatment kit for spinal cord injury of the present invention, the invention promotes the regeneration of the injured spinal cord and / or improves accompanying symptoms associated with spinal cord injury. Examples of promoting the regeneration of damaged spinal cord include promoting axonal extension, increasing nerve fibers, neurogenesis of endogenous stem cells, angiogenesis, suppression of scar formation, suppression of inflammation, and / or suppression of syringomyelia. Examples of accompanying symptoms associated with spinal cord injury include motor dysfunction and / or urinary dysfunction. Examples of motor function impairment include lower limb motor dysfunction and muscle loss.

[0040] (An agent for improving urinary dysfunction in chronic incomplete spinal cord injury or chronic complete spinal cord injury) The present invention provides an agent for improving urinary dysfunction in chronic incomplete spinal cord injury or chronic complete spinal cord injury, and includes the following: 1) HGF protein, and a carrier capable of supporting and sustainably releasing the HGF protein. 2) iPS cell-derived neural stem and / or progenitor cells A carrier loaded with HGF protein is placed at the spinal cord injury site, and iPS cell-derived nerve trunk and / or progenitor cells are then administered to the carrier or the spinal cord injury site after placement.

[0041] (An agent for improving motor function impairment in chronic incomplete spinal cord injury or chronic complete spinal cord injury) The present invention provides an agent for improving motor function impairment in chronic incomplete spinal cord injury or chronic complete spinal cord injury, and includes the following: 1) HGF protein, and a carrier capable of supporting and sustainably releasing the HGF protein. 2) iPS cell-derived neural stem and / or progenitor cells A carrier loaded with HGF protein is placed at the spinal cord injury site, and iPS cell-derived nerve trunk and / or progenitor cells are then administered to the carrier or the spinal cord injury site after placement. An example of motor function is lower limb motor function.

[0042] (Lower limb muscle mass increasing agent in chronic incomplete spinal cord injury or chronic complete spinal cord injury) The lower limb muscle mass increasing agent for chronic incomplete spinal cord injury or chronic complete spinal cord injury of the present invention comprises the following: 1) HGF protein, and a carrier capable of supporting and sustainably releasing the HGF protein. 2) iPS cell-derived neural stem and / or progenitor cells A carrier loaded with HGF protein is placed at the spinal cord injury site, and iPS cell-derived nerve trunk and / or progenitor cells are then administered to the carrier or the spinal cord injury site after placement.

[0043] (An agent that inhibits spinal cord cavitation in chronic incomplete spinal cord injury or chronic complete spinal cord injury) The spinal cord cavity formation inhibitor for chronic incomplete spinal cord injury or chronic complete spinal cord injury of the present invention comprises the following: 1) HGF protein, and a carrier capable of supporting and sustainably releasing the HGF protein. 2) iPS cell-derived neural stem and / or progenitor cells A carrier loaded with HGF protein is placed at the spinal cord injury site, and iPS cell-derived nerve trunk and / or progenitor cells are then administered to the carrier or the spinal cord injury site after placement.

[0044] (Anti-inflammatory agents for the injury site in chronic incomplete spinal cord injury or chronic complete spinal cord injury) The anti-inflammatory agent for the injury site in chronic incomplete spinal cord injury or chronic complete spinal cord injury according to the present invention comprises the following: 1) HGF protein, and a carrier capable of supporting and sustainably releasing the HGF protein. 2) iPS cell-derived neural stem and / or progenitor cells A carrier loaded with HGF protein is placed at the spinal cord injury site, and iPS cell-derived nerve trunk and / or progenitor cells are then administered to the carrier or the spinal cord injury site after placement.

[0045] (Angiogenesis promoters for the injury site in chronic incomplete spinal cord injury or chronic complete spinal cord injury) The present invention provides an angiogenesis promoter for the site of injury in chronic incomplete spinal cord injury or chronic complete spinal cord injury, comprising the following: 1) HGF protein, and a carrier capable of supporting and sustainably releasing the HGF protein. 2) iPS cell-derived neural stem and / or progenitor cells A carrier loaded with HGF protein is placed at the spinal cord injury site, and iPS cell-derived nerve trunk and / or progenitor cells are then administered to the carrier or the spinal cord injury site after placement.

[0046] (An agent that inhibits scar formation at the site of injury in chronic incomplete spinal cord injury or chronic complete spinal cord injury) The present invention provides a scar formation inhibitor for the injury site in chronic incomplete spinal cord injury or chronic complete spinal cord injury, comprising the following: 1) HGF protein, and a carrier capable of supporting and sustainably releasing the HGF protein. 2) iPS cell-derived neural stem and / or progenitor cells A carrier loaded with HGF protein is placed at the spinal cord injury site, and iPS cell-derived nerve trunk and / or progenitor cells are then administered to the carrier or the spinal cord injury site after placement.

[0047] (Axonal extension promoter for the injured area in chronic incomplete spinal cord injury or chronic complete spinal cord injury) The axonal extension promoting agent for the injury site in chronic incomplete spinal cord injury or chronic complete spinal cord injury of the present invention comprises the following: 1) HGF protein, and a carrier capable of supporting and sustainably releasing the HGF protein. 2) iPS cell-derived neural stem and / or progenitor cells A carrier loaded with HGF protein is placed at the spinal cord injury site, and iPS cell-derived nerve trunk and / or progenitor cells are then administered to the carrier or the spinal cord injury site after placement.

[0048] The present invention will be described in detail below with specific examples, but the present invention is not limited to these examples. All animal experiments were conducted in accordance with the guidelines of the Keio University Animal Experimentation Committee (Keio University Approval Number 7207). [Examples]

[0049] In this example, we confirmed the effect of hepatocyte growth factor (HGF) protein on damaged spinal cords during spinal cord regeneration, as well as the effect of a scaffold that provides a support structure and sustained drug release through electrical charge.

[0050] (Materials and Methods) The recombinant human HGF protein, consisting of the amino acid sequence represented by Sequence ID No. 6, was produced using CHO cells in accordance with the method described in Biochem. Biophys. Res. Commun. 180: 1151-1158, 1991. A complete spinal cord injury model was created by transection of the spinal cord at the 10th thoracic vertebra in female Sprague-Dawley rats (body weight 160-200g). 42 days after injury (corresponding to the chronic phase), an HGF protein-containing sustained-release collagen gelatin scaffold (HGF+scaffold group) was surgically placed in the cavity created at the transection site. The control group consisted of three groups: one with a collagen gelatin scaffold containing phosphate-buffered saline (scaffold group) placed in the injury site, and another with no treatment (control group) (see Figure 1, reference: J Surg Res., 2018 (221)173-182). Recombinant human HGF protein was used at a concentration of 10 mg / 10 μL. For the collagen gelatin scaffold, we used Pernac G Plus (a product of Gunze Corporation) in sheet form, measuring 5 mm x 5 mm x 3 mm. The size of the scaffold installed was adjusted as needed depending on the size of the cavity. The method for loading HGF protein onto a collagen gelatin scaffold involved impregnating the collagen gelatin scaffold with HGF protein. Spinal cord samples were collected from the area surrounding the implantation site on the 7th and / or 14th day after implantation. Immunostaining and Western blot were then used to measure the intensity of phosphorylated c-Met, evaluate neovascularization in the central area of ​​injury (RECA-1 postive area), evaluate anti-inflammatory effects (Arginase 1, Arginase 1-positive cells / iba1-positive cells, and TNF-a), evaluate neuroprotective effects (BDNF), evaluate scar formation inhibition (TGF-b), and evaluate axonal regeneration and axonal regrowth (co-staining of Tuj-1 and pGAP43). The references for each evaluation method were: "Regulation of axon growth by the JIP1–AKT axis, 2014", "Combined Action of GDNF and HGF Up-Regulates Axonal Growth by Increasing ERK1 / 2 Phosphorylation, 2018", "PI3K-GSK3 signaling regulates mammalian axon regeneration by inducing the expression of Smad1, 2013", and "The discovery of Hepatocyte Growth Factor (HGF) and its significance for cell biology, life sciences and clinical medicine, 2010".

[0051] (Confirmation of the sustained-release effect of HGF protein using a scaffold) As shown in the graph in Figure 2, phosphorylated c-Met was significantly detected in the HGF protein-containing scaffold group at a point 1 mm caudal to the center of the injury. Since the detection of phosphorylated c-Met indicates the sustained activation effect of HGF protein, it was confirmed that the HGF protein-containing scaffold released HGF protein for at least 7 days after installation. As a result, the HGF protein-containing scaffold has a sustained-release effect on HGF protein. In addition, scaffolds containing substances that have c-Met phosphorylation activity equivalent to that of HGF protein can continuously phosphorylate c-Met in the same way as scaffolds containing HGF protein.

[0052] (Evaluation of the angiogenesis-promoting effect of HGF protein-containing scaffolds at the center of injury) As shown in the graph on the left of Figure 3, the HGF protein-containing scaffold group showed significantly higher neovascularization compared to the control group. Furthermore, the HGF protein-containing scaffold group showed more neovascularization on day 14 compared to day 7 (Figure 3 right). As a result, HGF protein-containing scaffolds have an angiogenesis-promoting effect at the center of injury.

[0053] (Evaluation of anti-inflammatory effects of HGF protein-containing scaffolds) The anti-inflammatory effects of HGF protein-containing scaffolds were evaluated by measuring Arginase1, a marker for anti-inflammatory microglia and macrophages exhibiting neuroprotective effects; the percentage of Arginase1-positive cells among Iba1 (Ionized calcium binding adapter protein 1)-positive microglia and macrophages; and the measurement of TNF-α, an inflammatory cytokine. As is clear from the graph in Figure 4, we confirmed that Arginase1 levels were elevated in the HGF protein-containing scaffold group compared to the control group. As is clear from the graph in Figure 5, the HGF protein-containing scaffold group showed a significantly higher proportion of Arginase1-positive cells among iba1-positive cells compared to the control group. As is clear from the graph in Figure 6, the HGF protein-containing scaffold group showed lower levels of the inflammatory cytokine TNF-α compared to the control group. This indicates that the HGF protein-containing scaffold has an anti-inflammatory effect by sustained release of HGF protein. Furthermore, it was confirmed that the HGF protein-containing scaffold can also improve transplant cell viability through its TNF-α inhibitory effect.

[0054] (Evaluation of neuroprotective effects of HGF protein-containing scaffolds) As shown in the graph in Figure 7, we confirmed that the HGF protein-containing scaffold group showed elevated levels of BDNF, a neurotrophic factor, compared to the control group. As a result, the HGF protein-containing scaffold exhibits a neuroprotective effect by slowly releasing HGF protein.

[0055] (Evaluation of scar formation inhibition by HGF protein-containing scaffolds) As shown in the graph in Figure 8, we confirmed that the HGF protein-containing scaffold group showed a decrease in TGF-β, a factor that forms glial scars, compared to the control group. As a result, the HGF protein-containing scaffold has an inhibitory effect on scar formation (particularly on glial scar formation) by slowly releasing HGF protein.

[0056] (Evaluation of axon regeneration and axon regrowth effects of HGF protein-containing scaffolds) Figure 9 shows the number and density of regenerated axons in the cranial stump of the injured area in co-stained axial images of Tuj-1 and the regenerated axon marker pGAP43. As shown in the graph in Figure 9, the HGF protein-containing scaffold group showed a higher number of regenerated axons (Figure 9 left) and a higher regenerated axon density (Figure 9 right) compared to the control group. Furthermore, comparing the HGF protein-containing scaffold group at 7 and 14 days after placement, it was confirmed that the number of regenerated axons was even greater at 14 days (not shown). As a result, the HGF protein-containing scaffold has an axon extension promoting effect by slowly releasing HGF protein.

[0057] (Overview of Example 1) We confirmed that the HGF protein-containing scaffold has the following effects. 1) Sustained-release effect of HGF protein for at least 14 days 2) Promotes angiogenesis at the site of spinal cord injury (especially in the central part of the injury). 3) Anti-inflammatory effect at the site of spinal cord injury 4) Improving transplant cell survival rate 5) Neuroprotective effect at the site of spinal cord injury 6) Suppression of scar formation at the site of spinal cord injury 7) Promotes axonal extension at the site of spinal cord injury (particularly increases the number of regenerated axons and improves the density of regenerated axons). [Examples]

[0058] In this example, the activation of endogenous neural stem cells and progenitor cells by an HGF protein-containing scaffold was evaluated.

[0059] (Materials and Methods) Three groups were prepared in the same manner as in Example 1. Furthermore, EdU labeling was performed by intraperitoneal administration of EdU (5-ethynyl-2'-deoxyuridine) immediately after the placement of HGF protein-containing sustained-release collagen gelatin scaffolds on day 42. Tissue samples were collected on day 7 after placement, and the tissues were subjected to immunostaining and Westernblots (SOX2, Musashi1GFAP, DCX, Hoechst, and Nestin) (see Figure 10). The references for each evaluation method were "Chondroitinase and Growth Factors Enhance Activation and Oligodendrocyte Differentiation of Endogenous Neural Precursor Cells after Spinal Cord Injury, 2012", "Cetuximab modified collagen scaffold directs neurogenesis of injury-activated endogenous neural stem cells for acute spinal cord injury repair, 2017", and "Regeneration of the central nervous system using endogenous repair mechanisms, 2007".

[0060] (Evaluation of activation of endogenous neural stem progenitor cells by HGF protein-containing scaffolds) The results of SOX2, musashi1, and EdU immunohistochemical staining are shown in Figure 11. The HGF protein-containing scaffold group showed a higher number of SOX2, EdU co-positive cells and Musashi1-positive cells compared to the control group. Figure 12 shows the SOX2-EdU co-positive cells and musashi1-EdU co-positive cells in the HGF protein-containing scaffold group. It was confirmed that the HGF protein-containing scaffold group had a high number of SOX2-EdU co-positive cells and musashi1-EdU co-positive cells. Figure 13 shows the quantitative evaluation of activation of endogenous neural stem progenitor cells by HGF protein-containing scaffolds. The HGF protein-containing scaffold group showed a significantly higher number of SOX2-expressing cells, SOX2·EdU co-positive cells, and musashi1-positive cells in the central area of ​​the injury. As a result, the HGF protein-containing scaffold has the effect of promoting the proliferation of endogenous neural stem and progenitor cells by slowly releasing HGF protein.

[0061] (Evaluation of the differentiation effect of endogenous neural stem progenitor cells into neurons by HGF protein-containing scaffolds) Figure 14 shows the results of DCX, Nestin, and Hoechst immunohistochemical staining. The HGF protein-containing scaffold group showed a higher proportion of co-stained cells (co-positive cells) for DCX, an immature neuron marker, and Nestin, an undifferentiated neuron marker. As a result, the HGF protein-containing scaffold has the effect of inducing the differentiation of endogenous neural stem progenitor cells into neurons by sustained release of HGF protein.

[0062] (General overview of Example 2) We confirmed that the HGF protein-containing scaffold has the following effects. 1) Effect of promoting the proliferation of endogenous nerve trunk and progenitor cells at the site of spinal cord injury. 2) Differentiation induction effect of endogenous nerve trunk progenitor cells into neurons at the site of spinal cord injury [Examples]

[0063] Examples 1 and 2 confirmed the effects of the HGF protein-containing scaffold. However, while the HGF protein-containing collagen gelatin scaffold significantly improved the poor spinal cord microenvironment, which inhibited nerve regeneration after chronic complete injury, it did not improve motor function. The inventors of this invention believe that the reason why motor function did not improve was that a large cavity had formed at the site of injury, resulting in a shortage of nerve cells and thus insufficient functional improvement. Therefore, in this embodiment, in addition to placing the HGF protein-containing scaffold at the injured site, iPS cell-derived neural stem progenitor cells were also administered. In other words, the agent of the present invention was evaluated.

[0064] (Materials and Methods) A complete spinal cord injury model was created by transection of the spinal cord at the 10th thoracic vertebral level in nude rat females (rnu / rnu, body weight 130-150g). On day 42 after injury, an HGF protein-containing scaffold (HGF protein-containing sustained-release collagen gelatin scaffold) was surgically implanted. On day 49 after injury, three clinical-grade iPS cell-derived neural trunk progenitor cells (YZWJs513 hiPSC-NS / PCs) were transplanted at the HGF protein-containing scaffold implantation site (TP+Combined group). The control group received no treatment on day 42 after injury and was administered the drug on day 49 after injury (TP group). Regarding the administered cells, YZWJs513 hiPSc-derived NS / PCs were injected a total of 1 × 10¹⁶ times at the cranial, central, and caudal ends of the injury site (3 injection points). 6Cells were administered, and then every other week, motor function of the hindlimbs of rats was evaluated using the Basso, Beattie, and Bresnahan (BBB) ​​score, and independent urination function was also evaluated. The observation period continued until 91 days after injury, after which the spinal cord was collected and evaluated by immunohistochemistry (see Figure 15). For more details, one point was 3.3 × 10⁶. 5 It is a cell, and the sum of the three points is 1 × 10 6 There were 1 million cells. The HGF protein-containing scaffold used is the same as in Example 1.

[0065] (Evaluation of the effect of suppressing cavitation at the spinal cord injury site) Figure 16 shows the spinal cord findings on day 42. In the TP+Combined group (the agent of the present invention), unlike the TP-alone group, the severed spinal cord remained connected, and no cavity formation was observed. As a result, the agent of the present invention has the effect of connecting severed spinal cords and filling the cavities (cavitation inhibitory effect).

[0066] (Evaluation of the survival and engraftment rate of administered iPS-derived neural stem progenitor cells) The survival rate of iPS-derived neural stem progenitor cells was measured using HNA (Human Neutrophil Alloantigen)-positive cells as a marker. The results are shown in Figure 17. The TP+Combined group (the agent of the present invention) showed a significantly improved survival rate of iPS-derived neural stem progenitor cells compared to the TP-alone group. As a result, the agent of the present invention has the effect of improving the survival rate of transplanted cells (pluripotent stem cells).

[0067] (Confirmation of differentiation tendencies in transplanted cells) The differentiation tendency of iPS-derived neural stem progenitor cells in the TP+Combined group (the agent of the present invention) was compared with that of the TP-alone group. Measurements of the percentage of pan-ELAVL (Embryonic Lethal Abnormal Vision-Like) positive cells and the percentage of GFAP (glial fibrillary acidic protein) positive cells revealed that in both groups, approximately 90% differentiated into neurons and approximately 5% into astrocytes (not shown). Measurement of the percentage of antigen-presenting cells (APCs) showed that approximately 4% of cells in both groups had differentiated into oligodendrocytes (not shown). Measurements of the percentage of Ki-positive cells (%) and the percentage of Nestin-positive cells (%) showed no neoplastic growth in either group, and undifferentiated markers were also low (not shown). This confirmed that the agent of the present invention does not cause adverse events in transplanted cells (pluripotent stem cells), particularly cancerous transformation of transplanted cells.

[0068] (Evaluation of transplanted cell-derived nerve fibers in the area surrounding the injury) STM121 was measured as a marker for nerve fibers derived from administered iPS-derived neural stem progenitor cells. The results are shown in Figure 18. The TP+Combined group (the agent of the present invention) showed an increase in transplanted cell-derived nerve fibers at the center of the injury (1 mm) compared to the TP alone group. Therefore, the agent of the present invention has the effect of increasing nerve fibers derived from transplanted cells.

[0069] (Evaluation of host-derived nerve fibers in the area surrounding the injury) Rat neurofilament H (NF-H) was measured as a marker for host-derived nerve fibers. The results are shown in Figure 19. The TP+Combined group (the agent of the present invention) showed an increase in host-derived nerve fibers at the center of the injury (1 mm) compared to the TP alone group. Therefore, the agent of the present invention has the effect of increasing host-derived nerve fibers.

[0070] (Evaluation of serotonergic neurons in the area surrounding the injury) We measured serotonergic neurons (5-HT), which play a crucial role in the recovery of lower limb motor function after spinal cord injury. The results are shown in Figure 20. In the TP-alone group, we observed dieback of 5-HT-positive nerve fibers in the cranial region. On the other hand, in the TP+Combined group (the agent of the present invention), we observed 5-HT-positive cells extending from the cranial region to the center of the injury. In addition, in the TP+Combined group, we observed many 5-HT-positive fibers at a point 4 mm caudal to the injury site. The TP+Combination group (the agent of the present invention) showed an increase in serotonergic neurons at the center of the injury (1 mm) compared to the TP monotherapy group. Therefore, the agent of the present invention has the effect of restoring lower limb motor function.

[0071] (Evaluation of neovascularization in the area surrounding the injury) CD31, an endothelial marker, was measured at the center of the injury. The results are shown in Figure 21. The TP+Combination group (the agent of the present invention) showed increased neovascularization at the center of the injury compared to the TP alone group. Therefore, the agent of the present invention has a neovascularization-increasing effect.

[0072] (Evaluation of fibrous scarring and cavitation) Fibrous scar tissue was stained using picrosirius staining, which stains collagen fibers red, and then quantified. The results are shown in Figure 22. The TP+Combination group (the agent of the present invention) showed a significant reduction in fibrous scars compared to the TP-alone group. As a result, the agent of the present invention has the effect of inhibiting fibrous scarring and cavitation.

[0073] (Evaluation of glial scars) Glial scarring was evaluated by assessing the GFAP (Glial fibrillary acidic protein) expression intensity around the injury in the GFAP-positive area. More specifically, Figure 23 shows the results after correcting the GFAP expression intensity at the center of the injury with the GFAP expression intensity at a distance from the injury site. The TP+Combination group (the agent of the present invention) showed a thinner GFAP-positive layer and significantly lower GFAP expression intensity compared to the TP-alone group. As a result, the agent of the present invention has a scar formation inhibitory effect.

[0074] (Equipment function assessment) Motor function was assessed (particularly lower limb motor function) using the BBB (Basso-Beattie-Bresnahan, Bassoetal.J.Neurotrauma vol.12, pp.1-21, 1995) score, stride length using the Digigait small animal gait analysis system, and lower leg muscle mass. The results are shown in Figure 24. Regarding the BBB score, the TP+Combination group (the agent of the present invention) showed an increase until day 91, but the TP monotherapy group did not show an increase. In the Digigait small animal gait analysis system, the TP+Combination group (agent of the present invention) showed a significant increase in lower limb stride length compared to the TP-alone group. Regarding the weight of the triceps surae muscle, the TP+Combination group (the agent of the present invention) showed a significant increase compared to the TP alone group. Figure 25 shows the results of electrophysiological testing using transcranial motor evoked potentials (MEP). Specifically, the spinal dura mater in the cranial lateral aspect of the injury was stimulated, and recordings were made in the quadriceps femoris muscle. Unlike the TP+Combination group (the agent of the present invention), waveforms could be detected in the TP+Combination group. These results show that the TP+Combination group (the agent of the present invention) was able to improve motor function, while the TP-alone group was not. As a result, the agent of the present invention has an effect of improving motor function disorders (particularly an effect of increasing lower limb muscle mass).

[0075] (Assessment of urinary function) It is known that the mucomuscular layer of the bladder wall thickens in spinal cord injuries. Therefore, urinary function was observed using bladder tissue. Regarding the HE stained image (top of Figure 26), it was confirmed that the muscle layer was thinned in the TP+Combination group (the agent of the present invention). In addition, regarding the total circumferential area of ​​the bladder wall (bottom of Figure 26), corrected for bladder walls of normal rats of the same age and without spinal cord injury, it was confirmed that the TP+Combination group (agent of the present invention) had a smaller area compared to the TP-alone group. We quantitatively evaluated GAD65, a GABAergic neuron marker in the lumbar spinal cord's dorsal horn region, which is involved in urinary function (Figure 27). We confirmed that the TP+Combination group (the agent of the present invention) showed higher levels compared to the TP-alone group. These results suggest that increased inhibitory input in the lumbar spinal cord's posterior horn region alleviated detrusor muscle spasm and improved urinary function. Therefore, the agent of the present invention has the effect of improving urinary dysfunction.

[0076] (HGF protein expression level in the area surrounding the injury) The expression level of HGF protein in the area surrounding the injury was measured 91 days after the injury. The measurement results are shown in Figure 28. The TP+Combination group (the agent of the present invention: "HGF+scaffold" in the figure) showed a higher level of HGF protein in the area surrounding the injury compared to the other groups. As a result, the agent of the present invention has a long-term sustained-release effect on HGF protein.

[0077] (General overview of the examples) Based on the results of Examples 1 to 3, the agent of the present invention was confirmed to have the following effects. 1) Inhibitory effect on syringomyelia formation at the site of spinal cord injury 2) Effect of improving transplant cell survival rate 3) Effect of increasing nerve fibers derived from transplanted cells 4) Effect of increasing host-derived nerve fibers at the spinal cord injury site 5) Increased neovascularization effect at the site of spinal cord injury 6) Inhibitory effect on fibrous scarring at spinal cord injury sites 7) Inhibitory effect on scar formation at the site of spinal cord injury 8) Improvement of motor function impairment due to spinal cord injury 9) Improvement of urinary dysfunction caused by spinal cord injury 10) Sustained-release effect of HGF protein at spinal cord injury sites 11) Anti-inflammatory effect at the site of spinal cord injury 12) Neuroprotective effects at the site of spinal cord injury 13) Effect of promoting axonal extension at the site of spinal cord injury (particularly, effect of increasing the number of regenerated axons and improving the density of regenerated axons) 14) Endogenous nerve trunk and progenitor cell proliferation effect at the spinal cord injury site 15) Differentiation of endogenous nerve trunk progenitor cells into neurons at the site of spinal cord injury In addition, substances that have a c-Met phosphorylation activity equivalent to that of HGF protein are thought to have similar effects to HGF protein. [Industrial applicability]

[0078] We can provide a therapeutic agent for spinal cord injury in chronic incomplete spinal cord injury or chronic complete spinal cord injury.

Claims

1. Therapeutic agents for chronic spinal cord injury or chronic complete spinal cord injury, including the following: 1) (a) hepatocyte growth factor protein, or (b) a gene encoding hepatocyte growth factor protein, 2) A carrier capable of supporting and slowly releasing (a) or (b); and 3) Neural trunk and / or progenitor cells, Here, the carrier is placed at the site of spinal cord injury, and the nerve trunk and / or progenitor cells are administered to the carrier or the site of spinal cord injury after placement. A therapeutic agent.

2. The therapeutic agent according to claim 1, wherein the nerve trunk and / or progenitor cells are iPS cell-derived nerve trunk and / or progenitor cells.

3. The therapeutic agent according to claim 1 or 2, wherein the nerve trunk and / or progenitor cells are administered to a carrier after placement.

4. The therapeutic agent according to claim 1, characterized in that the treatment promotes the regeneration of the injured spinal cord and / or improves accompanying symptoms associated with spinal cord injury.

5. The therapeutic agent according to claim 4, characterized in that the promotion of regeneration of the injured spinal cord is the promotion of axon extension, the increase of nerve fibers, neurogenesis of endogenous stem cells, angiogenesis, inhibition of scar formation, inhibition of inflammation, and / or inhibition of syringomyelia.

6. The therapeutic agent according to claim 4, characterized in that the accompanying symptoms associated with the spinal cord injury are motor dysfunction and / or urinary dysfunction.

7. The therapeutic agent according to claim 6, wherein the motor dysfunction is lower limb motor dysfunction.

8. The therapeutic agent according to claim 6, wherein the motor dysfunction is due to a decrease in muscle mass.

9. A therapeutic agent for chronic spinal cord injury or chronic complete spinal cord injury comprising hepatocyte growth factor protein and a carrier capable of supporting and sustained-release of the hepatocyte growth factor protein, Here, the carrier carrying the hepatocyte growth factor protein is placed at the spinal cord injury site, and iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or the spinal cord injury site after placement. A treatment agent for chronic spinal cord injury or chronic complete spinal cord injury.

10. A therapeutic agent for chronic spinal cord injury or chronic complete spinal cord injury, comprising iPS cell-derived neural trunk and / or progenitor cells, Here, the iPS cell-derived nerve trunk and / or progenitor cells are administered to the spinal cord injury site or to a carrier carrying hepatocyte growth factor protein placed at the spinal cord injury site. A drug used to treat spinal cord injuries.

11. The following are examples of agents for improving motor function impairment in chronic incomplete spinal cord injury or chronic complete spinal cord injury: 1) Hepatocyte growth factor protein, a carrier capable of supporting and sustainably releasing the hepatocyte growth factor protein; and 2) iPS cell-derived neural stem and / or progenitor cells, And, The carrier carrying the hepatocyte growth factor protein is placed at the site of spinal cord injury, and the iPS cell-derived nerve trunk and / or progenitor cells are administered to the carrier or the site of spinal cord injury after placement. A drug for improving motor function disorders.

12. The motor function is lower limb motor function, as described in claim 11.

13. A treatment kit for chronic spinal cord injury or chronic complete spinal cord injury, including the following: 1) (a) hepatocyte growth factor protein, or (b) a gene encoding hepatocyte growth factor protein, 2) A carrier capable of supporting and slowly releasing (a) or (b); and 3) Neural trunk and / or progenitor cells, And, The carrier carrying (a) or (b) is placed at the site of spinal cord injury in a spinal cord injury patient, and the nerve trunk and / or progenitor cells are further administered to the carrier or the site of spinal cord injury after placement. A treatment kit for chronic spinal cord injury (incomplete or complete).

14. The therapeutic agent according to claim 1, wherein the therapeutic agent has an effect of improving motor function.

15. The therapeutic agent according to claim 1, wherein the carrier is a lactic acid / glycolic acid copolymer or collagen.

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