JP2007238487A - Method for reconstructing neurological function by using hgf for olfactory mucosa graft to spinal cord injury - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicine for potentiating a regenerative effect in the regeneration treatment of reconstructing spinal cord by grafting olfactory nerve sheath cells or mucosal tissue containing the above cells. <P>SOLUTION: This medicine for potentiating effects in the regeneration treatment of reconstructing the spinal cord by grafting the olfactory nerve sheath cells or mucosal tissue containing the above cells is characterized by containing a HGF (hepatocyte growth factor) protein or a nucleic acid encoding the same as an active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an effect enhancer for regenerative treatment of spinal cord injury and a regenerative treatment method using the same, and more particularly, reconstructs the spinal cord by transplanting olfactory nerve sheath cells or mucosal tissue containing the cells into a spinal cord injury patient. The present invention relates to an effect enhancer for regenerative treatment, and a spinal cord regeneration treatment method using the effect enhancer.

  Spinal cord injury is a pathological condition that causes paralysis of the peripheral motor, sensory, and autonomic nervous system below the injured part due to trauma such as a spinal dislocation fracture caused by a traffic accident or a fall of a high altitude. is there. Currently, the number of spinal cord injuries is estimated to be approximately 100,000 in Japan and approximately 250,000 in the United States. The number of patients in Japan is 5,000 and more than 10,000 in the United States each year. With the advancement of medical treatment in recent years, it has become possible to survive even after being injured. However, the inconvenience of daily life and the mental burden are causing social problems, resulting in social problems. It is.

  For many years, it has been thought that the central nervous system (brain and spinal cord) of an adult mammal does not regenerate once damaged. The central nervous system is difficult to regenerate, but it has long been known that the peripheral nervous system regenerates easily, and the difference is that in the central nervous system, oligodendrocytes, myelin, or extracellular matrix suppresses axonal outgrowth. It is explained that the factor exists, whereas such a factor does not exist in the peripheral nervous system. In other words, the peripheral nervous system axon environment is permissive as a whole for newly growing axons, whereas the central nervous system is non-permissive, ie, axonal neoplasia. Is in an environment where If this explanation is correct, it will be necessary to tolerately alter the rejecting environment to promote regeneration of the mammalian central nervous system. One of the attempts includes transplantation of olfactory nerve sheath cells (Non-patent Document 1).

  Olfactory neurons are specific cells that continue to regenerate throughout life in adult mammalian cells. Lu et al. Transplanted rat olfactory mucosa cells from the olfactory mucosa of a rat into a completely cut rat spinal cord, and as a result, motor function was remarkably restored, and descending suppression of the spinal cord reflex was markedly restored. The knowledge that the axon grew beyond that was obtained, and the possibility of applying it to the treatment of human spinal cord injury was shown. (Non-patent document 2).

  Since the olfactory mucosa can be collected even in adults, the expectation has rapidly increased in that it can be autotransplanted, and clinical trials are being conducted. For example, in Portugal, Carlos Lima et al. Reported that autologous transplantation of the olfactory mucosa to spinal cord injury patients had a therapeutic effect (Non-patent Document 3). Although it is not autotransplantation, in China, led by Huang et al., Clinical trials of human fetal olfactory nerve sheath cell transplantation for patients with spinal cord injury have been conducted since 2002, and some patients reported improvements. (Non-Patent Document 4).

  As described above, the method of treating spinal cord injury by transplanting olfactory sheath cells or olfactory mucosa to the injured site has achieved a certain result, but its effect is still limited, and further functional recovery is possible. In order to gain, it is necessary to improve the method of treatment.

  On the other hand, hepatocyte growth factor (hereinafter abbreviated as HGF) was first identified as a potent mitogen for mature hepatocytes, and its gene cloning was performed in 1989 (Non-patent Documents 5 and 6). Although HGF was discovered as a hepatocyte growth factor, numerous recent studies in expression and functional analysis, including the knockout / knock-in mouse approach, also revealed that HGF is a novel neurotrophic factor ( Non-Patent Documents 7 and 8).

Patent Document 1 discloses an example in which the action effect of the HGF gene on the model rat was examined behaviorally and histologically using Parkinson's disease model rats. Shows that the midbrain substantia nigra dopamine neurons were protected from the neurotoxin 6-OHDA and the symptoms of Parkinson's disease model rats were suppressed. And in this patent document 1, based on such experimental results, the HGF gene is not only Parkinson's disease, but also Alzheimer's disease, spinocerebellar degeneration, multiple sclerosis, striatal nigra degeneration, spinal muscle Atrophy, Huntington's chorea, Shy-Drager syndrome, Charcot-Marie-Tooth disease, Friedreich ataxia, myasthenia gravis, Willis arterial obstruction, amyloidosis, Pick disease, SMON disease, dermatomyositis / polymyositis, Kreuzfeld-Jakob disease, Behcet's disease, systemic lupus erythematosus, sarcoidosis, periarteritis nodosa, posterior longitudinal ligament ossification, mixed spinal stenosis, mixed connective tissue disease, diabetic peripheral neuritis, ischemic cerebrovascular It can also be applied to the treatment of neurological disorders such as disorders (cerebral infarction, cerebral hemorrhage, etc.). It is.
WO2003 / 045439 Kawaguchi, “Experimental Medicine”, Vol. 20, no. 5 (Special Issue), 2002, pages 796-803 Lu et al. , Brain (2002), 125, 14-21 pages. Laurance Johnston et al. "OLFACTORY-TISSUE TRANSPLANTATION FOR SCI: PORTUGAL CLINICAL TRIALS", [online], 2003, [February 15, 2006 search], Internet <URL: http: // www. healingtherapies. info / OlfactoryTissue2. htm> Iwanami et al., “PT Journal”, Vol. 39, No. 6, 2005, pp. 539-546 Biochem. Biophys. Res. Commun. 122, 1450-1459, 1984. Nature, 342, 440-443, 1989 Nat. Neurosci. 2, 213-217, 1999 Clin. Chim. Acta. 327, 1-23, 2003

  An object of the present invention is to provide a drug that enhances the effect of regenerative treatment when regenerating the spinal cord by regenerating the spinal cord by transplanting olfactory nerve sheath cells or mucosal tissue containing the cells to the injury site of spinal cord injury. Is. Another object of the present invention is to provide a method for enhancing the effect of the regenerative treatment using the drug.

  As a result of various studies to solve the above-mentioned problems, the present inventors perform regenerative treatment for reconstructing the spinal cord by transplanting olfactory sheath cells or mucosal tissue containing the cells into the damaged site for spinal cord injury. At that time, it has been found that the regenerative treatment effect can be remarkably enhanced by administering HGF protein or a nucleic acid encoding the same (for example, DNA) to the patient, and the present invention has been completed through further studies. It was.

That is, the present invention
[1] An effect-enhancing agent in regenerative treatment for transplanting olfactory nerve sheath cells or mucosal tissue containing the cells to a spinal cord injury patient to reconstruct the spinal cord, comprising HGF protein or nucleic acid encoding HGF protein as an active ingredient An effect enhancer characterized by
[2] The effect enhancer according to the above [1], wherein the active ingredient is HGF protein,
[3] The HGF protein is a protein comprising the amino acid sequence represented by SEQ ID NO: 1 or 2, or a protein comprising an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or 2, and as HGF The effect enhancer according to the above [2], which is a protein that acts,
[4] The effect enhancer according to any one of the above [1] to [3] for local application to a spinal cord injury site,
[5] The effect enhancer according to any one of the above [1] to [4], which is a dosage form of a sustained release preparation,
[6] The effect enhancer according to the above [5], wherein the sustained-release preparation is a preparation in which a porous gelatin sponge is impregnated with HGF protein,
[7] The effect enhancer according to the above [5], wherein the sustained-release preparation is an injection that can be intermittently administered using a pump,
[8] The effect enhancer according to any one of [1] to [7], wherein the mucosal tissue containing olfactory sheath cells is the olfactory mucosa,
[9] The effect enhancer according to the above [8], wherein the olfactory mucosa is an autologous olfactory mucosa,
[10] It is characterized by administering HGF protein or a nucleic acid encoding an HGF protein to the patient during spinal cord injury patients in regenerative treatment in which olfactory sheath cells or a mucosal tissue containing the cells are transplanted to reconstruct the spinal cord. A spinal cord regeneration therapy, and [11] an HGF protein or an HGF protein for producing an effect enhancing agent in regenerative therapy for transplanting an olfactory sheath cell or a mucosal tissue containing the cell to a spinal cord injury patient to reconstruct the spinal cord To the use of nucleic acids.

  Regeneration is performed by administering HGF protein or a nucleic acid (eg, DNA) to the patient during regenerative treatment in which the olfactory sheath cell or a mucosal tissue containing the cell is transplanted into a spinal cord injury patient to reconstruct the spinal cord. The effect of treatment can be significantly enhanced.

  The effect-enhancing agent of the present invention is an effect-enhancing agent in regenerative treatment for reconstructing the spinal cord by transplanting olfactory sheath cells or mucosal tissue containing the cells into a spinal cord injury patient, and an HGF protein or a nucleic acid encoding the HGF protein (For example, DNA) as an active ingredient.

  As used herein, “olfactory nerve sheath cell” refers to a cell that surrounds the axon of an olfactory nerve cell. The position of olfactory nerve sheath cells in humans is shown in FIG.

  Specific examples of the “mucosal tissue containing the cells” in the present invention include “olfactory mucosa”.

Referred to in the present invention "olfactory mucosa" is in part surrounded by the upper nasal turbinate top of left and right nasal隔上portion of the nasal cavity as a main olfactory, for example in humans those having an area of about the right and left each about 2 cm ² is there. The olfactory mucosa is composed of the olfactory epithelium and the olfactory mucosa lamina, and olfactory nerve sheath cells are mainly present in the olfactory mucosa lamina.

In carrying out the regenerative treatment according to the present invention, first, “olfactory nerve sheath cells or mucosal tissues containing the cells” are prepared.
Preparation of mucosal tissue containing olfactory nerve sheath cells is performed by removing mucosal tissue containing "olfactory mucosa" present in the nose of mammals, preferably removing the olfactory mucosa, more preferably the olfactory mucosa lamina propria Is done. The site shown in FIG. 2 is preferable as the excision site in humans, but if the olfactory mucosa is the main component, respiratory mucosa (not including olfactory nerve sheath cells) may be mixed. The excised olfactory mucosa can be made into a form that can be easily transplanted to a spinal cord injury site by chopping as necessary.

  In addition, olfactory nerve sheath cells derived from the olfactory mucosa can be cultured and isolated for use, and the culture and isolation method therefor can be carried out by a method known per se. For example, the mucosal tissue containing the extracted olfactory nerve sheath cells is cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum for 3 to 4 days, and then cultured in DMEM serum-free medium for 2 to 3 weeks. The olfactory nerve sheath cells may be isolated by using trypsin-EDTA after the latter medium is added twice a week and cultured. Further, the isolated olfactory nerve sheath cells can be suspended in an appropriate medium (for example, DMEM, serum-added DMEM, etc.) and used.

  Olfactory nerve sheath cells and the source of mucosal tissue containing the cells include adult humans (autologous or allogeneic transplantation), human fetuses (allografts), human cadaver (allografts), and other mammals such as pigs (xenotransplantation) In consideration of donor problems, problems of immune rejection that can occur after transplantation, ethical problems, and the like, human adults who undergo transplantation (autologous transplantation) are preferable.

Transplantation of olfactory nerve sheath cells and mucosal tissues containing the cells is performed at the site of spinal cord injury. The transplantation can be performed by various methods, for example, as follows.
Under general anesthesia, the patient is made a midline incision and the muscles are removed under the periosteum at an appropriate level. Carefully handle muscles, bones, and epidural space during dural opening. A laminectomy is performed over the entire lesion. The entire vertebral arch is excised to avoid spinal deformity due to surgical procedures and to allow structural regeneration after olfactory nerve sheath cell transplantation. Inject the cultured suspension of olfactory sheath cells into the site adjacent to the cranial and caudal side of the spinal cord lesion, or fill the void in the excision site with mucosal tissue containing olfactory sheath cells Transplant by.

  The timing of transplantation may be performed immediately after spinal cord injury or 1 to several weeks later depending on the condition of the patient. Furthermore, chronic phase transplantation is also applicable to cases where months to years have passed since injury, and in some cases, it may be performed on patients who are unable to recover due to rehabilitation or the like.

  In carrying out the regenerative treatment, “HGF protein or nucleic acid encoding HGF protein”, which is an active ingredient of the effect enhancer of the present invention, is prepared.

  The HGF protein used in the present invention is a known substance, and any protein prepared by various methods can be used as long as it is purified to the extent that it can be used as a medicine. As a method for producing the HGF protein, for example, primary cultured cells or established cells that produce the HGF protein can be cultured, separated from the culture supernatant, etc., and purified to obtain the HGF protein. Alternatively, a nucleic acid encoding the HGF protein is incorporated into an appropriate vector by genetic engineering techniques, and this is inserted into an appropriate host cell for transformation, and the desired recombinant HGF protein is obtained from the culture supernatant of the transformant. (See, for example, JP-A No. 5-111382, Biochem. Biophys. Res. Commun. 1989, 163, p. 967). The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, yeast or animal cells can be used. As long as the HGF protein thus obtained has substantially the same action as the natural HGF protein, one or a plurality of [for example, several (for example, 1 to 8); )]] May be substituted, deleted or added, and similarly, sugar chains may be substituted, deleted or added. Examples of such an HGF protein include the 5-amino acid deficient HGF protein described below. Here, with respect to the amino acid sequence, “one or a plurality of amino acids are deleted, substituted or added” is generated by a well-known technical method such as genetic engineering method, site-directed mutagenesis method, or the like. It means that as many numbers (1 to several) as possible are deleted, substituted or added. An HGF protein in which a sugar chain is substituted, deleted or added is, for example, an HGF protein in which a sugar chain added to a natural HGF protein is treated with an enzyme or the like so that the sugar chain is deleted, or a sugar chain is not added. The amino acid sequence of the glycosylation site is mutated, or the amino acid sequence is mutated so that the sugar chain is added to a site different from the natural glycosylation site. Specifically, for example, the Accession No. registered in the NCBI database. For the human HGF of NP_001010932, substitution of the glycosylation site at position 289 Asn to Gln, position 397 Asn to Gln, position 471 Thr to Gly, position 561 Asn to Gln, position 648 Asn to Gln HGF [Fukuta K et al. , Biochemical Journal, 388, 555-562 (2005)].

  Examples of the HGF protein include the amino acid sequence represented by SEQ ID NO: 1 or 2. The HGF protein represented by SEQ ID NO: 2 is a 5-amino acid deficient HGF protein in which the 5th amino acid residues from 161 to 165th of the amino acid sequence represented by SEQ ID NO: 1 have been deleted. Both of the proteins having the amino acid sequence represented by SEQ ID NO: 1 or 2 are human-derived natural HGF proteins, and have mitogenic activity, motogenic activity, etc. as HGF.

  A protein comprising an amino acid sequence that is substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or 2 is also included in the HGF protein of the present invention. Such a protein includes the amino acid sequence represented by SEQ ID NO: 1 or 2 From a protein comprising an amino acid sequence having at least about 80% or more, preferably about 90% or more, more preferably about 95% or more homology, such as the amino acid sequence represented by SEQ ID NO: 1 or 2, An amino acid sequence in which amino acid residues are inserted or deleted, an amino acid sequence in which one to several amino acid residues are substituted with another amino acid residue, or an amino acid sequence in which one to several amino acid residues are modified It is preferable that it is a protein that acts as HGF. The amino acid to be inserted or substituted may be an unnatural amino acid other than the 20 amino acids encoded by the gene. The non-natural amino acid may be any compound as long as it has an amino group and a carboxyl group, and examples thereof include γ-aminobutyric acid.

  These proteins may be used alone or as a mixed protein thereof. Examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or 2 include Accession No. registered in the NCBI database. Examples include, but are not limited to, human-derived HGF such as BAA14348 or AAC71655.

  As the HGF protein used in the present invention, those derived from humans described above are preferably used when applied to humans, but mammals other than humans (for example, monkeys, cows, horses, pigs, sheep, dogs, cats) HGF protein derived from rat, mouse, chimpanzee, etc. Examples of such HGF include mouse-derived HGF (eg, Accession No. AAB31855, NP — 034557, BAA01065, BAA01064, etc.) and rat-derived HGF [eg, Accession No. NP — 58713 (protein consisting of the amino acid sequence represented by SEQ ID NO: 5), etc., bovine-derived HGF (eg, Accession No. NP — 0010269921, XP874740, BAD02475, etc.), cat-derived HGF (eg, Accession No. NP — 001009830, BAC10545, BAB21499, etc.), Examples include, but are not limited to, dog-derived HGF (eg, Accession No. NP — 001002964, BAC57560, etc.) or chimpanzee-derived HGF (eg, Accession No. XP519174, etc.).

In the HGF protein used in the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl C _6-12 aryl groups such as α-naphthyl, C _7- such as phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl _In addition to the ₁₄ aralkyl group, a pivaloyloxymethyl group and the like are used. When the HGF protein used in the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the HGF protein of the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used.

Further, in the HGF protein used in the present invention, the amino group of the N-terminal methionine residue is a protecting group (for example, C _1-6 such as a C _2-6 alkanoyl group such as formyl group, acetyl, etc.). An acyl group and the like, a glutamyl group produced by cleavage of the N-terminal side in vivo, pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, An amino group, an imidazole group, an indole group, a guanidino group, etc.) protected with an appropriate protecting group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as formyl group or acetyl), Alternatively, a complex protein such as a so-called glycoprotein to which a sugar chain is bound is also included.

  The HGF protein used in the present invention can be used in the form of a salt. Examples of such a salt include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. . Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

In addition, even if it is a partial peptide of the said protein, if it has an effect | action as HGF, it is contained in the category of the HGF protein of this invention.
A partial peptide of HGF protein can be produced according to a known peptide synthesis method or by cleaving HGF protein with an appropriate peptidase. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, when the partial peptide or amino acid capable of constituting the HGF protein is condensed with the remaining part and the product has a protecting group, the target peptide can be produced by removing the protecting group. Examples of known condensation methods and protecting group elimination include M.I. Bodanszky and M.M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966), Schroeder and Luebke, The Peptide, Academic Press, New 65, etc. After the reaction, a partial peptide of HGF protein can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted into a free form by a known method. Can do.

  The effect enhancer of the present invention can also contain a nucleic acid (eg, DNA) encoding an HGF protein as an active ingredient.

  Examples of the nucleic acid encoding HGF protein include DNA encoding HGF protein, for example, Nature, 342, 440 (1989); Japanese Patent No. 2777678; Biochem. Biophys, Res. Commun. 1989, 163, p. 967-973; Proc. Natl. Acad. Sci. U. S. A. 1991, Vol. 88 (No. 16), p. 7001-7005 etc., for example, Access No. in GeneBank / EMBL / DDBJ. Preferred examples include DNA encoding a human-derived HGF protein registered as M60718, M73240, AC004960, AY246560, M29145 or M73240.

  The DNA encoding the HGF protein of the present invention is DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA, and has substantially the same activity as the HGF protein. For example, DNA encoding a protein having mitogenic activity, motogenic activity, etc. is included.

  Specific examples of DNA encoding the HGF protein include, for example, DNA having the base sequence represented by SEQ ID NO: 3 or 4, or DNA comprising a base sequence complementary to the DNA having the base sequence represented by SEQ ID NO: 3 or 4 And DNA that encodes a protein that hybridizes under stringent conditions with a protein having substantially the same quality of activity as the HGF protein, such as mitogenic activity, motogenic activity, and the like.

  The DNA that hybridizes with the DNA having the base sequence represented by SEQ ID NO: 3 or 4 uses, for example, a colony hybridization method, a plaque hybridization method, or a Southern blot hybridization method using the above DNA as a probe. Means the DNA obtained by Specifically, hybridization is performed at about 65 ° C. in the presence of about 0.7 to 1.0 M sodium chloride using a filter on which colony or plaque-derived DNA is immobilized, and then about 0. By washing the filter under a condition of about 65 ° C. using an SSC solution having a concentration of 1 to 2 times (the composition of the SSC solution having a concentration of 1 consists of 150 mM sodium chloride and 15 mM sodium citrate). Mention may be made of DNA that can be identified.

  Specifically, the DNA that hybridizes with the DNA having the base sequence represented by SEQ ID NO: 3 or 4 above is about 80% or more, preferably about 90% or more, with the base sequence represented by SEQ ID NO: 3 or 4. More preferred is DNA having a base sequence having a homology of about 95% or more. Hybridization can be performed by a known method such as molecular cloning (A Molecular Laboratory, Third Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001, hereinafter, Molecular Cloning, 3rd Edition). )), Etc. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

  Furthermore, the DNA encoding the HGF protein of the present invention is not limited to the above, and can be used as the DNA encoding the HGF protein of the present invention as long as the expressed protein is a DNA having substantially the same action as the HGF protein. For example, DNA encoding a partial peptide of HGF protein is included in the category of nucleic acid encoding HGF protein of the present invention as long as it encodes a partial peptide having an action as HGF. The DNA encoding the partial peptide of the HGF protein may be any DNA as long as it has a base sequence encoding the partial peptide described above.

  Similarly to the DNA encoding the HGF protein, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Moreover, it can also amplify by direct RT-PCR method using what prepared the mRNA fraction from the cell / tissue mentioned above. Specific examples of DNA encoding the partial peptide of the present invention include (a) DNA having a partial base sequence of DNA having the base sequence represented by SEQ ID NO: 3 or 4, and (b) SEQ ID NO: 3 or 4 A DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to a DNA having a partial base sequence of the DNA having the base sequence represented, and having substantially the same quality of activity as an HGF protein Or a DNA having the partial base sequence of (a) or (b) above.

  The DNA encoding the HGF protein of the present invention can be easily obtained, for example, by a normal hybridization method or PCR method. Specifically, the DNA can be obtained with reference to basic documents such as Molecular Cloning. It can be carried out.

  In addition, the nucleic acid encoding the HGF protein used in the present invention is preferably a nucleic acid encoding an HGF protein derived from a mammal other than a human, although it is preferably used when applied to a human. May be. For example, DNA encoding rat-derived HGF protein is represented by SEQ ID NO: 6.

  Moreover, the RNA encoding the HGF protein used in the present invention can also be used as a nucleic acid encoding the HGF protein according to the present invention as long as it can express the HGF protein or partial peptide by reverse transcriptase. Is within the scope of the present invention. The RNA can also be obtained by known means.

  When HGF protein or a nucleic acid encoding HGF protein is administered to a patient with spinal cord injury, the administration form, administration method, dosage, etc. are the same depending on whether the active ingredient is HGF protein or a nucleic acid encoding HGF protein. Slightly different.

For example, when the active ingredient is HGF protein, various preparation forms such as liquids and solids can be used. Generally, sustained-release preparations (for example, porous preparations) are used together with HGF protein alone or a conventional carrier. Sex sponges, depots, etc.), injections and sprays.
Examples of the sustained release preparation include, for example, a preparation in which a biodegradable polymer is impregnated with HGF protein. By making the HGF protein into a sustained-release preparation, effects such as reduction in the number of administrations, sustained action and reduction of side effects can be expected. The biodegradable polymer used in the sustained-release preparation can be appropriately selected from known biodegradable polymers. For example, a polysaccharide such as starch, dextran or chitosan, a protein such as collagen or gelatin, and the like. Polyamino acids such as polyglutamic acid, polylysine, polyleucine, polyalanine or polymethionine, polylactic acid, polyglycolic acid, lactic acid / glycolic acid polymer or copolymer, polycaprolactone, poly-β-hydroxybutyric acid, polymalic acid, Poly acid anhydride or polyester such as fumaric acid / polyethylene glycol / vinyl pyrrolidone copolymer, polyorthoester or polyalkyl cyanoacrylic acid such as polymethyl-α-cyanoacrylic acid, polyethylene carbonate or polypropylene carbonate Such as polycarbonate. Gelatin is preferable, and porous gelatin sponge is more preferable. As a porous gelatin sponge, what is marketed by the brand name of Spongel (Spongel) and gel foam (Gelfoam) can be used, for example. In order to produce a sustained-release preparation using a porous gelatin sponge, it can be prepared, for example, by impregnating a suitably-sized porous gelatin sponge with a solution of HGF protein.

  The injection may be either an aqueous injection or an oily injection. In the case of an aqueous injection, according to a known method, for example, an aqueous solvent (water for injection, purified water, etc.) is added to a pharmaceutically acceptable additive such as an isotonic agent (sodium chloride, potassium chloride, glycerin, mannitol, Sorbitol, boric acid, borax, glucose, propylene glycol, etc.), buffer (phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, epsilon Aminocaproic acid buffer), preservatives (methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boro Acid, borax, etc.), thickener (hydroxyethyl acetate) Loin, hydroxypropyl cellulose, polyvinyl alcohol, polyethylene glycol, etc.), stabilizers (sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc.) or pH adjusters (hydrochloric acid, water, etc.) It can be prepared by dissolving the HGF protein in a solution to which sodium oxide, phosphoric acid, acetic acid, etc.) are appropriately added, and then sterilizing by filtration with a filter or the like, and then filling in an aseptic container. Further, a suitable solubilizer such as alcohol (ethanol etc.), polyalcohol (propylene glycol, polyethylene glycol etc.) or nonionic surfactant (polysorbate 80, polyoxyethylene hydrogenated castor oil 50 etc.) may be used. . In the case of an oily injection, for example, sesame oil or soybean oil may be used as the oily solvent, and benzyl benzoate or benzyl alcohol may be used as a solubilizer. The prepared injection is usually filled in an appropriate ampoule or vial. In addition, it is desirable to store liquid preparations such as injections after removing moisture by freezing or lyophilization. The freeze-dried preparation is used by re-dissolving and adding distilled water for injection at the time of use.

  Propellants can also be prepared by routine pharmaceutical methods. When manufactured as a spray, the additive may be any additive as long as it is generally used in inhalation preparations. For example, in addition to a spray, the above-mentioned solvents, preservatives, An agent, an isotonic agent, a pH adjuster and the like are used. As the propellant, a liquefied gas propellant or a compressed gas is used. Examples of the liquefied gas propellant include fluorinated hydrocarbons (alternative chlorofluorocarbons such as HCFC22, HCFC-123, HCFC-134a, and HCFC142), liquefied petroleum, dimethyl ether, and the like. Examples of the compressed gas include soluble gas (such as carbon dioxide gas and nitrous oxide gas) or insoluble gas (such as nitrogen gas).

  As an administration method, for example, when a sustained-release preparation is used, a method of contacting or embedding in a site close to the transplanted olfactory nerve sheath cell or a mucosal tissue containing the cell (for example, the olfactory mucosa) is mentioned. . Examples thereof include a method of directly injecting (such as continuous administration with a sustained-release pump) or spraying into an olfactory nerve sheath cell transplanted with an injection or spray, or a mucosal tissue containing the cell (for example, olfactory mucosa). In the case of injection, it may be administered intrathecal.

  The dose is appropriately selected according to the dosage form, the degree of disease (spinal cord injury) or age, and is usually 1 μg to 500 mg, preferably 10 μg to 50 mg, more preferably 1 mg to 25 mg per dose. is there. Further, the number of administrations is appropriately selected according to the dosage form, the degree of disease, age, etc., and can be administered once or can be continuously administered at certain intervals. In the case of continuous administration, the administration interval may be once a day to once every several months. For example, in the case of administration using a sustained-release preparation or continuous intrathecal administration using a sustained-release pump, the HGF protein gradually increases over time. May be released once every several months.

  On the other hand, when the active ingredient is a nucleic acid encoding HGF protein, its administration form is divided into a case of using a non-viral vector and a case of using a viral vector, and the administration method can be carried out by a conventional method.

  When a non-viral vector is used, it is introduced into a spinal cord injury tissue using a recombinant expression vector into which a conventional gene expression vector is incorporated. Nucleic acid introduction methods to tissues include nucleic acid introduction method using encapsulated liposomes, nucleic acid introduction method using electrostatic liposomes, HVJ-liposome method, method using HVC-E vector, improved HVJ-liposome method, and HVC-E vector. By using the method used, receptor-mediated nucleic acid introduction method, method of transferring a nucleic acid molecule together with a carrier with a particle gun, direct introduction of naked-nucleic acid, introduction method using a positively charged polymer, etc. It can be taken into cells such as cells.

  When a viral vector is used, a detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, simbis virus, Sendai virus, SV40, immunodeficiency virus (HIV) It is possible to introduce a nucleic acid encoding an HGF protein into a cell by introducing the nucleic acid of interest into a DNA virus or RNA virus such as and infecting the cell with a recombinant virus.

  The method of applying the drug of the present invention to a patient includes an in vivo method in which the drug is directly introduced into a transplanted olfactory sheath cell or a mucosal tissue containing the cell (for example, the olfactory mucosa), or the drug is transplanted. There is an ex vivo method in which the cells or mucosal tissues are introduced or added to the previously extracted olfactory nerve sheath cells or mucosal tissues containing the cells (for example, olfactory mucosa) and transplanted to the site where the spinal cord is damaged.

  In order to administer the nucleic acid encoding the HGF protein to a patient, conventional methods, for example, separate experimental medicine, basic technology of gene therapy, Yodosha, 1996, separate experimental medicine, gene transfer & expression analysis experimental method, Yodosha, It can be performed according to the method described in 1997, Gene Therapy Research Handbook edited by Japanese Society of Gene Therapy, NTS, 1999, and the like.

  As the preparation form, various known preparation forms (for example, injections, sprays, sustained-release preparations (for example, porous sponges, depots, etc.), microcapsules, etc.) suitable for each of the above administration forms are used. It can be taken. Injections, sprays, sustained-release preparations (for example, porous sponges, depots, etc.) can be prepared in the same manner as for HGF protein. In the case of a microcapsule, for example, a host cell into which a nucleic acid encoding HGF protein or an expression plasmid containing a nucleic acid encoding HGF protein is introduced as a core substance is used as a known method (for example, coacervation method, interface It can be produced as fine particles having a diameter of about 1 to 500 μm, preferably about 100 to 400 μm by covering with a coating material according to a polymerization method or a double nozzle method. Coating materials include carboxymethyl cellulose, cellulose acetate phthalate, ethyl cellulose, alginic acid or its salt, gelatin, gelatin gum arabic, nitrocellulose, polyvinyl alcohol or hydroxypropyl cellulose, polylactic acid, polyglycolic acid, chitosan-alginate, cellulose sulfate -Film-forming polymers such as poly (dimethyldiallyl) ammonium chloride, hydroxyethyl methacrylate-methyl methacrylate, chitosan-carboxymethylcellulose, alginate-polylysine-alginate, and the like.

The nucleic acid content and dosage in the preparation can be adjusted as appropriate depending on the disease to be treated, the age, weight, etc. of the patient, but the dosage is usually HCV-HGF (herpes simplex virus-1-derived vector and HGF Converted into a protein-encoding nucleic acid) of 1.5 × 10 ⁶ pfu to 1.5 × 10 ¹² pfu, preferably 1.5 × 10 ⁷ pfu to 1.5 × 10 ¹⁰ pfu This is preferably administered once every several days to several months.

  The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[Test method]
(Extraction and preparation of olfactory and respiratory mucosa)
Eight week old Sprague-Dawley (SD) female rats were anesthetized by intraperitoneal administration of Nembutal 500 μg / 100 g, and the rat nasal cavity was vertically divided in the head direction to expose the nasal septum. The olfactory mucosa containing olfactory nerve sheath cells exists in the upper caudal side of the nasal septum and is yellowish. The respiratory mucosa that does not contain olfactory nerve sheath cells present on the rostral side of this is a grayish white tone and a slightly lighter tone than the olfactory mucosa. The olfactory mucosa and respiratory mucosa were respectively removed and cut into 1 mm squares.

(Preparation of animals with spinal cord injury)
Other homogenous rats were anesthetized by intraperitoneal administration of Nembutal 100 μg / 100 g, and the rectal temperature was maintained at 37 ± 0.5 ° C. using an incubator. The rat was subjected to laminectomy at the 8th-9th thoracic vertebrae level to expose the spinal cord (see FIG. 3A). The exposed spinal cord was extracted 2 mm in width, confirmed to the ventral dura mater, and was completely cut off 2 mm in width (see B in FIG. 3). In this manner, a spinal cord injury animal having a 2 mm wide spinal excision space was prepared.

(Transplantation of olfactory mucosa and HGF protein administration)
The spinal excision cavity of 2 mm width of spinal cord injured animals (5 animals per group) was filled until the 2 mm wide gap created in the spinal cord was filled with the above-mentioned minced olfactory mucosa (see C in FIG. 3). Porous gelatin sponge of 2 mm × 3 mm × 1 mm size (trade name: Gelfoam, Pharmacia) impregnated with 30 μg (100 μL) of the 5-residue-deleted recombinant human HGF protein shown in SEQ ID NO: 2 on the filled olfactory mucosa・ Launched and up John).
As a control group, after filling the 2 mm wide spinal excision cavity of spinal cord injured animals (5 animals per group) with the above-mentioned minced olfactory mucosa or respiratory mucosa until the 2 mm wide gap created in the spinal cord is filled. I used a closed one.

(Motor function evaluation method)
Two independent individuals unrelated to the present invention performed hindlimb motor function assessments every week up to 8 weeks after surgery using the BBB score (Basso et al., Journal of Neurotrauma, 12, 1-21, 1995).

[Test results]
The test results are shown in FIGS. FIG. 4 shows a comparison between the control group (OM) for olfactory mucosa transplantation and the control group (RM) for respiratory mucosa transplantation, and FIG. 5 shows the control group (OM) for olfactory mucosa transplantation and the HGF administration group ( OM + HGF).
From the results of FIG. 4, it can be seen that in the rat spinal cord transection model, the control group of olfactory mucosa transplantation significantly improved hindlimb motor function compared to the control group of respiratory mucosal transplantation. In addition, it can be seen from FIG. 5 that further improvement of hindlimb motor function was achieved by administering HGF during olfactory mucosa transplantation.

  For spinal cord injury, HGF protein or a nucleic acid encoding the same is administered when regenerative treatment for reconstructing the spinal cord by transplanting olfactory nerve sheath cells or mucosal tissue containing the cells (for example, olfactory mucosa) to the damaged site By doing so, the effect of regenerative treatment can be remarkably enhanced.

FIG. 1 is a schematic diagram showing the positions of human olfactory nerve sheath cells. FIG. 2 is a schematic diagram showing a cross-section of a human nose part and a location where an olfactory mucosa is removed. FIG. 3 shows a photograph (A) in which the spinal cord of a rat is exposed, a photograph (B) in which the spinal cord of the rat is completely cut off at a width of 2 mm, and a site where the rat olfactory mucosa is transplanted to the site of spinal cord injury that has been completely cut off. A photograph (C) is shown. FIG. 4 shows the result of comparison between the olfactory mucosa transplant group (OM) and the respiratory mucosa transplant group (RM) for the motor function evaluation of the rat spinal cord transection model in the examples. FIG. 5 shows the results of comparing the HGF administration group (OM + HGF) and the HGF non-administration group (OM) for the motor function evaluation of the rat spinal cord transection model in which olfactory mucosa transplantation was performed in the Examples.

Claims (11)

  A potentiating agent for regenerative treatment in which olfactory sheath cells or mucosal tissues containing the cells are transplanted into a spinal cord injury patient to reconstruct the spinal cord, comprising HGF protein or a nucleic acid encoding HGF protein as an active ingredient An effect enhancer.   The effect enhancer according to claim 1, wherein the active ingredient is HGF protein.   A protein in which the HGF protein comprises the amino acid sequence represented by SEQ ID NO: 1 or 2, or a protein comprising the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or 2, and acts as HGF The effect enhancer according to claim 2, wherein   The effect enhancer according to any one of claims 1 to 3, for local application to a spinal cord injury site.   The effect enhancer according to any one of claims 1 to 4, which is a dosage form of a sustained-release preparation.   6. The effect enhancer according to claim 5, wherein the sustained-release preparation is a preparation in which a porous gelatin sponge is impregnated with HGF protein.   The effect enhancer according to claim 5, wherein the sustained-release preparation is an injection that can be intermittently administered using a pump.   The effect enhancer according to any one of claims 1 to 7, wherein the mucosal tissue containing olfactory nerve sheath cells is the olfactory mucosa.   The effect enhancer according to claim 8, wherein the olfactory mucosa is an autologous olfactory mucosa.   Spinal cord regeneration treatment, characterized by administering HGF protein or a nucleic acid encoding HGF protein to the patient when regenerating the spinal cord by transplanting olfactory sheath cells or mucosal tissue containing the cells into a spinal cord injury patient. Law. Use of HGF protein or a nucleic acid encoding HGF protein for producing an effect enhancing agent in regenerative treatment for transplanting olfactory sheath cells or a mucosal tissue containing the cells to a spinal cord injury patient to reconstruct the spinal cord.