WO2011105527A1 - Promoteur de croissance nerveuse - Google Patents

Promoteur de croissance nerveuse Download PDF

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Publication number
WO2011105527A1
WO2011105527A1 PCT/JP2011/054234 JP2011054234W WO2011105527A1 WO 2011105527 A1 WO2011105527 A1 WO 2011105527A1 JP 2011054234 W JP2011054234 W JP 2011054234W WO 2011105527 A1 WO2011105527 A1 WO 2011105527A1
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shp
antibody
pir
trk receptor
substance
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PCT/JP2011/054234
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English (en)
Japanese (ja)
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俊英 山下
幸 藤田
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国立大学法人大阪大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a nerve growth promoter, and in particular, to a nerve growth promoter containing an inhibitor of SHP-1 or SHP-2 as an active ingredient.
  • the present invention also relates to a method for screening an active ingredient candidate for a nerve growth promoter.
  • the present inventor has conducted basic research and development research that leads to overcoming aftereffects of the nervous system, and has already inhibited at least one of bone morphogenetic protein, bone morphogenetic protein receptor, and signal factor downstream of the receptor.
  • NgR Nogo receptor
  • PIR-B Pigmentar immunoglobulin-like receptor B
  • myelin-derived axon regeneration inhibitor a major histocompatibility complex class I receptor
  • the signaling pathway downstream of PIR-B leading to neurite outgrowth inhibition is unknown. Therefore, it was expected to contribute significantly to the development of new nerve growth promoters by elucidating the signal transduction pathway by binding of PIR-B and myelin-derived axon regeneration inhibitor and finding the key factors. .
  • PirB is a functional receptor for myelin inhibitors of axonal regeneration. Science 322, 967-970 (2008).
  • the present invention elucidates a signal transduction pathway downstream of PIR-B, which is a receptor for an inhibitor of myelin-derived axon regeneration, a novel nerve growth promoter, a pharmaceutical composition for improving sequelae caused by central neuropathy, and nerve growth It is an object of the present invention to provide a method for screening an active ingredient candidate substance for an accelerator.
  • a nerve growth promoter comprising an inhibitor of SHP-1 or SHP-2 as an active ingredient.
  • (A) Substance that inhibits binding of SHP-1 or SHP-2 to Trk receptor B) Substance that inhibits Trk receptor tyrosine phosphatase activity of SHP-1 or SHP-2
  • the substance that inhibits the binding of SHP-1 or SHP-2 to a Trk receptor is any of the following (a) to (f): 2] The nerve growth promoter according to 2.
  • A a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof containing tyrosine at position 490 (b) in the sequence portion other than tyrosine at position 490 of the amino acid sequence represented by SEQ ID NO: 1;
  • Peptide consisting of an amino acid sequence or a partial sequence containing tyrosine at position 532 (d) In the sequence portion other than tyrosine at position 532 of the amino acid sequence represented by SEQ ID NO: 2, 1 to several amino acids are deleted or substituted Or consisting of an added amino acid sequence or a partial sequence containing tyrosine at position 532, and SHP-1 Peptide that binds to SHP-2 (e)
  • a pharmaceutical composition for improving sequelae caused by central nervous disorder comprising the nerve growth promoter according to any one of [1] to [5].
  • Use of SHP-1 and / or SHP-2 for screening an active ingredient of a nerve growth promoter is any one of the following (A) to (C).
  • A Substance that inhibits binding of SHP-1 or SHP-2 to Trk receptor
  • B Substance that inhibits Trk receptor tyrosine phosphatase activity of SHP-1 or SHP-2
  • C SHP- 1 or a substance that inhibits the expression of SHP-2 [10] contacting the test substance with the intracellular domain of Trk receptor and SHP-1 and / or SHP-2, the intracellular domain of Trk receptor and SHP
  • a method for screening a candidate substance for an active ingredient of a nerve growth promoting agent comprising the step of confirming the binding state with -1 or SHP-2.
  • the method includes contacting a test substance with an intracellular domain of a Trk receptor and SHP-1 and / or SHP-2, and confirming a phosphorylation state of the intracellular domain of the Trk receptor.
  • a screening method for a candidate substance for an active ingredient of a nerve growth promoter characterized by the above.
  • a method for improving sequelae due to central nervous disorder comprising a step of administering an effective amount of the nerve growth promoter according to any one of [1] to [5] to a mammal.
  • a novel nerve growth promoter can be provided.
  • the nerve growth promoter is useful for improving sequelae caused by central nervous system disorders.
  • the active ingredient candidate substance of a nerve growth promoter can be obtained by the screening method of the present invention.
  • Immunoprecipitation was performed using a cell lysate of COS-7 cells transiently transfected with HA-tagged full-length TrkA (HA-TrkA FL) expression vector or full-length PIR-B (PIR-B FL) expression vector or both. And (b) shows the results of immunoprecipitation using an anti-HA antibody, (a) shows the results of immunoprecipitation using an anti-PIR-B antibody, and (b) shows the results of immunoprecipitation using an anti-HA antibody.
  • FIG. Cell lysis of COS-7 cells transiently transfected with a protein (HA-TrkB T1) expression vector or full-length PIR-B expression vector added with an HA tag to TrkB lacking most of the intracellular domain
  • FIG. 3 is a diagram showing the results of immunoprecipitation and Western blotting using a liquid as a sample.
  • A shows the results of immunoprecipitation using an anti-PIR-B antibody.
  • B shows the results of immunization using an anti-HA antibody. It is a figure which shows the result of having performed sedimentation.
  • FIG. 6 shows the results of immunoprecipitation (using anti-Myc antibody) and Western blotting using a cell lysate of COS-7 cells transfected as a sample.
  • FIG. 4 is a diagram showing the results of immunoprecipitation and Western blotting using a cell lysate prepared with or without treatment of cerebellar granule cells with MAG-Fc.
  • A shows anti-PIR-B antibody
  • B is a diagram showing the results of immunoprecipitation using an anti-TrkB antibody or a control antibody (anti-IgG antibody).
  • Cerebellar granule cells derived from wild-type (WT) mice and p75KO mice were treated with or without MAG-Fc, and cell lysates prepared as samples were used for immunoprecipitation (using anti-PIR-B antibody) and Western blotting.
  • FIG. 4 is a diagram showing the results of immunoprecipitation and Western blotting using a cell lysate prepared by treating cerebellar granule cells with or without MAG-Fc as a sample, (a) shows anti-PIR-B antibody or As a result of immunoprecipitation using a control antibody (anti-IgG antibody), (b) shows the results of immunoprecipitation using an anti-SHP-2 antibody or a control antibody.
  • COS-7 cells transfected with HA-tagged TrkB expression vector and PIR-B expression vector, or cerebellar granule cells treated with or without MAG-Fc were used as samples for immunoprecipitation ( FIG.
  • FIG. 7 is a diagram showing the results of Western blotting using an anti-TrkB antibody or a control antibody (anti-IgG antibody), (a) shows the results of COS-7 cells, and (b) shows the results of cerebellar granule cells. It is. It is a figure which shows the result of having examined the phosphorylation level of the Trk receptor by the presence or absence of MAG-Fc stimulation using a cerebellar granule cell.
  • FIG. 4 shows the results of examining the phosphorylation level of Trk receptor with or without MAG-Fc stimulation after pretreatment of cerebellar granule cells with SHP inhibitor NSC-87877.
  • FIG. 3 shows the results of examining the phosphorylation level of Trk receptor with or without MAG-Fc stimulation using cerebellar granule cells transfected with SHP-1 or SHP-2 siRNA.
  • A shows SHP-
  • B shows the results of using SHP-2 siRNA. It is a figure which shows the result of having examined the phosphorylation level of the Trk receptor by the presence or absence of MAG-Fc stimulation using the cerebellar granule cell derived from a wild type (WT) mouse
  • FIG. 5 shows the results of measuring the length of neurites with and without MAG-Fc stimulation using cerebellar granule cells transfected with SHP-1 and / or SHP-2 siRNA.
  • FIG. 2 is a graph comparing the lengths of neurites, and (b) is a photomicrograph showing the morphology of a typical cell. It is a figure which shows the result of having measured the length of the neurite by the presence or absence of MAG-Fc stimulation using the cerebellar granule cell pre-processed with K252a. It is a figure which shows the result of having measured the length of the neurite by the presence or absence of MAG-Fc stimulation using the cerebellar granule cell which transfected TrkBsiRNA1, TrkBsiRNA2, or control siRNA.
  • FIG. 5 is a diagram showing the results of measuring the length of neurites with and without MAG-Fc stimulation using cerebellar granule cells pretreated with TAT-TrkA484-497 or TAT-TrkA481-494, (a) shows TAT- The results of TrkA484-497, (b) is a diagram showing the results of TAT-TrkA481-494. It is a figure which shows the result of having examined the phosphorylation level of the Trk receptor by the presence or absence of MAG-Fc stimulation using the cerebellar granule cell derived from a wild type (WT) mouse and a p75KO mouse
  • WT wild type
  • FIG. 4 is a diagram showing the results of immunoprecipitation and Western blotting as samples, (a) shows the results of immunoprecipitation using an anti-PIR-B antibody, and (b) shows the results of immunoprecipitation using an anti-HA antibody. It is a figure which shows the result of having performed.
  • FIG. 6 shows the results of immunoprecipitation (using anti-Myc antibody) and Western blotting using a cell lysate of COS-7 cells transfected as a sample. Cerebellar granule cells were treated with MAG-Fc or Nogo-Fc or OMgp, and cell lysates prepared without treatment were used for immunoprecipitation (using anti-PIR-A / B antibody) and Western blotting. It is a figure which shows a result.
  • FIG. 3 is a view showing the results of immunoprecipitation (using anti-PIR-A / B antibody) and Western blotting using a lysate as a sample.
  • FIG. 3 is a view showing the results of immunoprecipitation (using anti-PIR-A / B antibody) and Western blotting using a lysate as a sample.
  • FIG. 3 is a view showing the results of immunoprecipitation (using anti-PIR-A / B antibody) and Western blotting using a lysate as a sample. It is a figure which shows the result of having performed immunoprecipitation (using an anti-PIR-B antibody) and Western blotting, using a cell lysate prepared with or without treating cerebellar granule cells with MAG-Fc.
  • FIG. 3 shows the results of confirming the expression of SHP-1 and SHP-2 by Western blotting after transfecting SHP-1 siRNA, SHP-2 siRNA or control siRNA into the vitreous of an optic nerve injury model mouse.
  • FIG. 3 is a graph showing the results of comparing and comparing the SHP-1 siRNA, the SHP-2 siRNA or the control siRNA into the vitreous of an optic nerve injury model mouse, and quantifying axonal regeneration from an optic nerve tissue specimen.
  • Nogo MAG and OMgp, which are known as inhibitors of regeneration of the central nervous system of adult mammals, bind to its receptor PIR-B, and neurites (axons) of the central nervous system.
  • PIR-B receptor for the central nervous system
  • neurites axons of the central nervous system.
  • Trk tropomyosin-receptor-kinase
  • the present invention provides a nerve growth promoter containing an inhibitor of SHP-1 or SHP-2 as an active ingredient.
  • the inhibitor of SHP-1 or SHP-2 of the present invention may be a substance that inhibits the function of SHP-1 or SHP-2, and may be any substance that inhibits the expression of SHP-1 or SHP-2.
  • the inhibitor of SHP-1 or SHP-2 may be any one of the inhibitors, but a substance that simultaneously inhibits both SHP-1 and SHP-2 is preferable.
  • SHPs will be referred to as “SHPs”.
  • inhibitors of SHPs include substances that inhibit the binding of SHPs to Trk receptors, substances that inhibit the Trk receptor tyrosine dephosphorylating enzyme activity of SHPs, substances that inhibit the expression of SHPs, and the like. Include low molecular weight compounds, peptides, antibodies, nucleic acids, and the like.
  • Examples of the substance that inhibits the binding between SHPs and the Trk receptor include any of the following peptides (a) to (f).
  • A a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof containing tyrosine at position 490 (b) in the sequence portion other than tyrosine at position 490 of the amino acid sequence represented by SEQ ID NO: 1;
  • Peptide consisting of an amino acid sequence or a partial sequence containing tyrosine at position 532
  • D In the sequence portion other than tyrosine at position 532 of the amino acid sequence represented by SEQ ID NO: 2, 1 to several amino acids are deleted or substituted Or consisting of an added amino acid sequence or a partial sequence containing
  • the “peptide” in the present invention means a peptide in which two or more amino acids are bonded by peptide bonds, and the number of amino acids to be bonded is not limited. That is, the “peptide” in the present invention includes a polypeptide.
  • the amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence (accession number: NM_001012331, NP_001012331.1) of human Trk receptor A (hereinafter, Trk receptor A is simply referred to as “TrkA”).
  • the amino acid sequence represented by SEQ ID NO: 2 is the amino acid sequence (accession number: NM_006180, NP_006171.2) of human Trk receptor B (hereinafter, Trk receptor B is simply referred to as “TrkB”).
  • Trk receptor C is simply referred to as “TrkC”.
  • SHP-1 is known to form a complex at the position of tyrosine 490 of human TrkA (Marsh, HN, et al. J Cell Biol 163, 999-1010-10 (2003)), and in human TrkB In tyrosine at position 532 and human TrkC, it is presumed that tyrosine at position 515 is involved in binding to SHPs.
  • a full-length human TrkA or a fragment (partial fragment) containing tyrosine at position 490, a full-length human TrkB or a fragment containing tyrosine at position 532, human TrkC Or a fragment containing tyrosine at position 515 can be preferably used as a peptide that inhibits the binding between SHPs and the Trk receptor.
  • the number of amino acid residues in the fragment is not particularly limited, but is preferably 20 or less, more preferably 15 or less, still more preferably 10 or less, and particularly preferably 5 or less.
  • human TrkA, human TrkB, and human TrkC have binding properties to SHPs, they may have mutations in the sequence portions other than tyrosine at 490, 532, and 515, respectively.
  • the peptides (b), (d), and (f) can also be suitably used as peptides that inhibit the binding between SHPs and the Trk receptor.
  • “one to several amino acids have been deleted, substituted or added” means that the number can be deleted, substituted or added by known mutant peptide production methods such as site-directed mutagenesis (preferably Means that 10 or less, more preferably 7 or less, and still more preferably 5 or less) amino acids are deleted, substituted or added.
  • suitable peptides that inhibit the binding between SHPs and the Trk receptor include, for example, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5. .
  • the amino acid sequence represented by SEQ ID NO: 4 corresponds to positions 481 to 494 of SEQ ID NO: 1
  • the amino acid sequence represented by SEQ ID NO: 5 corresponds to positions 484 to 497 of SEQ ID NO: 1.
  • amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 5 may have the aforementioned mutations as long as they have binding properties with SHPs.
  • non-human mammal Trk receptors and fragments thereof having binding ability to SHPs can also be suitably used as peptides that inhibit the binding of SHPs to Trk receptors.
  • the peptides (a) to (f) above can be obtained by (I) constructing a recombinant expression vector of a desired peptide by a known genetic engineering technique, introducing the peptide into an appropriate host cell, and expressing it as a recombinant protein. Can be manufactured. Alternatively, it can be produced by (II) in vitro transcription / translation system. Alternatively, it can be produced by (III) a solid phase synthesis method (Fmoc method, Boc method) or a liquid phase synthesis method according to a known general peptide synthesis protocol.
  • the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁇ ), an amide (—CONH 2 ), or an ester (—COOR).
  • R in the ester is, 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 group such as naphthyl, for example, benzyl, C 7 - 14 aralkyl such as ⁇ - naphthyl -C 1-2 alkyl group such as a phenyl -C 1-2 alkyl or ⁇ - naphthylmethyl such phenethyl
  • a pivaloyloxymethyl group which is widely used as an oral ester, can be mentioned.
  • the peptide of the present invention has a carboxyl group or a carboxylate other than the C-terminus, these groups may be amidated or esterified.
  • the amino group of the N-terminal methionine residue is a protecting group (for example, a C 1-6 acyl group such as a C 2-6 alkanoyl group such as formyl group, acetyl, etc.) ),
  • the N-terminal side is cleaved in vivo, and the resulting glutamyl group is pyroglutamine oxidized, the substituent on the side chain of the amino acid in the molecule (eg, —OH, —SH, amino group) Imidazole group, indole group, guanidino group, etc.) may be protected with an appropriate protecting group (for example, C 1-6 acyl group such as C 2-6 alkanoyl group such as formyl group, acetyl, etc.).
  • an appropriate protecting group for example, C 1-6 acyl group such as C 2-6 alkanoyl group such as formyl group, acetyl, etc.
  • the peptides (a) to (f) may form a pharmaceutically acceptable salt.
  • the salt include hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, Salts with acids such as oxalic acid, malic acid, citric acid, oleic acid, palmitic acid; salts with alkali or alkaline earth metals such as sodium, potassium, calcium, or aluminum hydroxides or carbonates; And salts with triethylamine, benzylamine, diethanolamine, t-butylamine, dicyclohexylamine, arginine and the like.
  • Examples of the substance that inhibits the binding between SHPs and Trk receptor include, for example, an antibody against SHPs or an antibody against Trk receptor, which inhibits the binding between SHPs and Trk receptor, and Trk receptor tyrosine dephosphorylation of SHPs
  • Examples thereof include antibodies that inhibit oxidase activity.
  • Such an antibody can be prepared by a known method using SHP-1 or a fragment thereof, SHP-2 or a fragment thereof, or Trk receptor or a fragment thereof as an immunogen. What is necessary is just to confirm that the obtained antibody inhibits the coupling
  • the antibody may be a polyclonal antibody or a monoclonal antibody. Further, it may be a complete antibody molecule or an antibody fragment (for example, Fab, F (ab ′) 2 , Fab ′, Fv, scFv, etc.) that can specifically bind to an antigen.
  • a polyclonal antibody can be prepared and obtained, for example, as follows. That is, an antigen is dissolved in PBS, and a mammal (mouse, rat, rabbit, goat, horse, etc.) is immunized with an immunogen mixed with an appropriate amount of a normal adjuvant (for example, Freund's complete adjuvant) if desired.
  • a normal adjuvant for example, Freund's complete adjuvant
  • the immunization method is not particularly limited, for example, a method of subcutaneous injection or intraperitoneal injection once or a plurality of times at appropriate intervals is preferable.
  • blood can be collected from the immunized animal, serum can be separated, and the polyclonal antibody fraction can be purified.
  • a monoclonal antibody can be obtained by fusing immune cells (eg, spleen cells) obtained from the immunized mammal with myeloma cells to obtain a hybridoma, and collecting the antibody from the hybridoma culture.
  • an antibody gene can be cloned from a hybridoma, incorporated into an appropriate vector, introduced into a host cell, and a recombinant monoclonal antibody can be produced using gene recombination techniques. Furthermore, it can also be prepared using a phage display method.
  • the antibody is preferably a human chimeric antibody or a humanized antibody.
  • a human chimeric antibody refers to an antibody comprising a heavy chain variable region and a light chain variable region of an antibody derived from a non-human animal, and a heavy chain constant region and a light chain constant region of a human antibody.
  • a humanized antibody is obtained by grafting a CDR (complementarity determining region) of an antibody derived from a non-human animal into a CDR of a human antibody, and is also referred to as a CDR-grafted antibody, a reconstituted antibody, or the like.
  • the FR (framework region) of the humanized antibody is selected so that CDR forms a favorable antigen-binding site.
  • amino acid sequence of FW in the variable region of the antibody may be substituted so that the CDR of the humanized antibody forms an appropriate antigen-binding site.
  • the amino acid sequence of the constant region of a human antibody can be obtained from a known database (Protein Data Bank etc.).
  • SHPs examples include siRNA (short interfering RNA), shRNA (short hairpin RNA), antisense oligonucleotides, and the like of the SHP-1 gene or SHP-2 gene.
  • SHP-1 gene examples include a human SHP-1 gene (accession number: NM_002831) consisting of the base sequence shown in SEQ ID NO: 7, and a mouse SHP-1 gene (accession number) consisting of the base sequence shown in SEQ ID NO: 8.
  • NM_013545 human SHP-2 gene consisting of the base acid sequence shown in SEQ ID NO: 9 (accession number: NM_002834), mouse SHP-2 gene consisting of the base acid sequence shown in SEQ ID NO: 10 (accession number: NM_011202) ) And the like, but is not limited thereto.
  • the base sequences of SHP genes derived from various organisms can be easily obtained from known databases (GenBank etc.).
  • An siRNA is a double-stranded RNA having a length of about 20 bases (for example, about 21 to 23 bases) or less, and by expressing such siRNA in a cell, a gene (this In the invention, the expression of the SHP-1 gene or SHP-2 gene) can be suppressed.
  • shRNA is a single-stranded RNA that contains a partially palindromic base sequence, so that it has a double-stranded structure in the molecule and a short hairpin structure having a protruding portion at the 3 ′ end. It refers to the above molecules.
  • shRNA is introduced into the cell, it is degraded into a length of about 20 bases (typically, for example, 21 bases, 22 bases, 23 bases) in the cell and becomes a target in the same manner as siRNA.
  • Expression of a gene in the present invention, SHP-1 gene or SHP-2 gene
  • the siRNA and shRNA may be in any form as long as it can suppress the expression of the SHP-1 gene or the SHP-2 gene.
  • siRNA or shRNA can be artificially chemically synthesized.
  • antisense and sense RNA can be synthesized in vitro from template DNA using T7 RNA polymerase and T7 promoter.
  • the antisense oligonucleotide may be any nucleotide that is complementary to or hybridizes to a continuous 5 to 100 nucleotide sequence in the DNA sequence of the SHP-1 gene or SHP-2 gene. It may be. Moreover, it may be modified as long as the function is not hindered.
  • Antisense oligonucleotides can be synthesized by conventional methods, for example, can be easily synthesized by a commercially available DNA synthesizer.
  • the present invention provides a pharmaceutical composition for ameliorating sequelae caused by central nervous system disorders, comprising the nerve growth promoter of the present invention.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for ameliorating sequelae caused by a central nervous system disorder containing an inhibitor of SHP-1 or SHP-2 as an active ingredient.
  • central nervous system disorders include cerebrovascular disorders, brain trauma, spinal cord injury, and the like.
  • cerebrovascular disorder include stroke, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and the like.
  • sequelae caused by central nervous system disorders include motor dysfunction, sensory dysfunction, and language impairment.
  • the pharmaceutical composition of the present invention can be formulated by using an inhibitor of SHPs as an active ingredient and appropriately blending a pharmaceutically acceptable carrier or additive.
  • oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions; parenterals such as injections, infusions, suppositories, ointments, patches, etc. can do.
  • What is necessary is just to set suitably about the mixture ratio of a carrier or an additive based on the range normally employ
  • Carriers or additives that can be blended are not particularly limited.
  • various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; excipients, binders, pH adjusters, disintegrants, absorption
  • Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.
  • the inhibitor of SHPs is a peptide or antibody
  • a parenteral route of administration such as intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous Or it is preferable to administer locally.
  • injections or infusions containing antibodies can be used as solutions, suspensions or emulsions.
  • the solvent include distilled water for injection, physiological saline, glucose solution and isotonic solution (for example, solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.) Can be used.
  • this injection or infusion may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, preservatives, pH adjusters and the like.
  • a stabilizer for example, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, EDTA sodium, sodium citrate, dibutylhydroxytoluene, etc. should be used. Can do.
  • solubilizers include alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate 80 (registered trademark), HCO-50, etc.) Etc.
  • alcohols eg, ethanol
  • polyalcohols eg, propylene glycol, polyethylene glycol, etc.
  • nonionic surfactants eg, polysorbate 80 (registered trademark), HCO-50, etc.
  • Etc. can be used.
  • the suspending agent for example, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used.
  • emulsifier for example, gum arabic, sodium alginate, tragacanth and the like can be used.
  • the soothing agent for example, benzyl alcohol, chlorobutanol, sorbitol and the like can be used.
  • the buffer for example, phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, and the like can be used.
  • Preservatives include, for example, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, boron Sand or the like can be used.
  • preservative for example, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
  • pH adjuster for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid and the like can be used.
  • the inhibitor of SHPs is a nucleic acid (siRNA, shRNA, antisense oligonucleotide, etc.)
  • it can be administered in the form of a non-viral vector or a viral vector.
  • a non-viral vector form a method for introducing nucleic acid molecules using liposomes (liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.), microinjection method, gene gun (Gene Gun ), A method of transferring a nucleic acid molecule into a cell together with a carrier (metal particle) can be used.
  • a viral vector such as a recombinant adenovirus or a retrovirus
  • DNA expressing siRNA or shRNA is added to DNA viruses or RNA viruses such as detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, poliovirus, Sindbis virus, Sendai virus, SV40, etc.
  • a gene can be introduced into a cell or tissue by introducing and infecting the cell or tissue with this recombinant virus.
  • the dosage of the pharmaceutical composition of the present invention is appropriately determined in consideration of the purpose, the severity of the disease, the patient's age, weight, sex, medical history, type of active ingredient, and the like.
  • an average human having a body weight of about 65 to 70 kg is used as a subject, it is preferably about 0.02 to 4000 mg, more preferably about 0.1 to 200 mg per day.
  • the total daily dose may be a single dose or divided doses.
  • the present invention includes a method for improving sequelae caused by a central nervous disorder, which comprises the step of administering an effective amount of the nerve growth promoter of the present invention.
  • the present invention includes the use of the nerve growth promoter of the present invention described above for producing a pharmaceutical composition for improving sequelae caused by central nervous system disorders.
  • the present invention includes the nerve growth promoter of the present invention described above for use in improving sequelae caused by central nervous system disorders.
  • the novel findings regarding the signal transduction pathway found by the present inventors have revealed that inhibitors of SHP-1 or SHP-2 are useful as active ingredients of nerve growth promoters. That is, it was revealed that SHP-1 and / or SHP-2 can be used for screening an active ingredient of a nerve growth promoter. Accordingly, the present invention includes the use of SHP-1 and / or SHP-2 for screening active ingredients of nerve growth promoters.
  • the inhibitor of SHP-1 or SHP-2 that can be an active ingredient of a nerve growth promoter is not particularly limited, and examples thereof include the following (A) to (C).
  • A Substance that inhibits binding of SHP-1 or SHP-2 to Trk receptor
  • B Substance that inhibits Trk receptor tyrosine phosphatase activity of SHP-1 or SHP-2
  • C SHP- Substances that inhibit the expression of 1 or SHP-2
  • the present invention comprises a step of contacting a test substance with an intracellular domain of a Trk receptor and SHP-1 and / or SHP-2, and a binding state between the intracellular domain of the Trk receptor and SHP-1 or SHP-2
  • a method for screening a candidate substance for an active ingredient of a nerve growth promoting agent comprising the step of: By this screening method, a substance that inhibits the binding between SHP-1 or SHP-2 and the Trk receptor can be screened.
  • the present invention also comprises a step of contacting a test substance, the intracellular domain of the Trk receptor, and SHP-1 and / or SHP-2, and a step of confirming the phosphorylation state of the intracellular domain of the Trk receptor.
  • the screening method of the active ingredient candidate substance of the nerve growth promoter characterized by including is provided. By this screening method, a substance that inhibits the Trk receptor tyrosine phosphatase activity of SHP-1 or SHP-2 can be screened.
  • the method for bringing the test substance into contact with the intracellular domain of the Trk receptor and SHP-1 and / or SHP-2 is not particularly limited.
  • a reaction system containing (1) Trk receptor or a fragment containing the intracellular domain of Trk receptor and (2) SHP-1 or SHP-2 or both is prepared, and a test substance is added thereto The method of doing is mentioned.
  • the contact time and the contact temperature are not particularly limited and may be appropriately selected. Moreover, it is preferable to provide a control group that does not contact the test substance.
  • the intracellular domain of the Trk receptor used in the screening method of the present invention may be any as long as it contains the intracellular domain of the Trk receptor, and a full-length Trk receptor or a fragment containing the intracellular domain of the Trk receptor is preferable. Can be used.
  • the Trk receptor A has positions 434 to 790 of SEQ ID NO: 1
  • the Trk receptor B has positions 455 to 838 of SEQ ID NO 2
  • the Trk receptor C has positions 454 to 838 of SEQ ID NO 3.
  • Position 839 corresponds to the intracellular domain.
  • the Trk receptor or the fragment containing the intracellular domain of the Trk receptor, SHP-1 and SHP-2 used in the screening method of the present invention can be obtained using, for example, a known genetic recombination technique.
  • the method for confirming the binding state between the intracellular domain of the Trk receptor and SHP-1 or SHP-2 is not particularly limited, and a known method may be appropriately selected and used.
  • the ELISA method can be suitably used.
  • the method for confirming the phosphorylation state of the intracellular domain of the Trk receptor is not particularly limited, and a known method may be appropriately selected and used.
  • the phosphorylation state of the intracellular domain of the Trk receptor can be easily confirmed by using a commercially available anti-phosphotyrosine antibody.
  • a substance that inhibits the binding between the intracellular domain of the Trk receptor and SHP-1 or SHP-2 is promoted for nerve growth. It can be selected as an active ingredient candidate substance of the agent. Further, as a result of confirming the phosphorylation state of the intracellular domain of the Trk receptor, a substance that inhibits the dephosphorylation of the intracellular domain of the Trk receptor can be selected as a candidate substance for an active ingredient of a nerve growth promoter. Inhibition does not require complete inhibition, and binding activity to the intracellular domain of Trk receptor inherent to SHP-1 or SHP-2, or dephosphorylation of the intracellular domain of Trk receptor Any substance that attenuates oxidation activity may be used.
  • test substance Based on the binding activity level or dephosphorylation activity level of the control not in contact with the test substance, if the test substance has reduced these activity levels, determine that it is a candidate substance for the active ingredient of nerve growth promoter. Can do.
  • the present invention includes a step of contacting a test substance with a cell capable of confirming the expression level of SHP-1 and / or SHP-2, and an expression level of SHP-1 and / or SHP-2 in the cell in contact with the test substance And a method of screening a candidate substance for an active ingredient of a nerve growth promoting agent, comprising comparing the expression level of SHP-1 and / or SHP-2 in a cell not in contact with the test substance provide.
  • a substance that inhibits the expression of SHP-1 or SHP-2 can be screened.
  • Cells capable of confirming the expression level of SHP-1 and / or SHP-2 include cell lines expressing SHP-1 and / or SHP-2, and expressing SHP-1 and / or SHP-2. And cells into which a vector for a reporter assay of SHP-1 and / or SHP-2 has been introduced.
  • Cell lines expressing SHP-1 and / or SHP-2 include, but are not limited to, COS-7, HEK293, Hela, PC12 and the like.
  • Examples of nerve cells expressing SHP-1 and / or SHP-2 include cerebral cortical cells, hippocampal cells, cerebellar cells and the like, and these primary cultured cells can be preferably used.
  • a method for bringing a cell capable of confirming the expression level of SHP-1 and / or SHP-2 into contact with a test substance is not particularly limited, and examples thereof include a method of culturing cells using a culture solution to which a test substance is added. It is done. Alternatively, the contact may be made in vivo (see Example 1 (7-1), FIG. 29).
  • the expression level can be expressed by the protein level of SHP-1 and / or SHP-2, the mRNA level of SHP-1 and / or SHP-2, the reporter expression level of the reporter assay, and the like.
  • the amount of protein, the amount of mRNA, the amount of reporter expression, etc. can be quantified by appropriately selecting a known method. Comparing the expression level of SHP-1 and / or SHP-2 in the cells in contact with the test substance and the expression level of SHP-1 and / or SHP-2 in the cells not in contact with the test substance, If the expression level of SHP-1 and / or SHP-2 in the contacted cells is reduced, the test substance can be determined as a candidate substance for an active ingredient of a nerve growth promoter.
  • a test substance that reduces the expression level to 50% or less, more preferably a test substance that reduces the expression level to 25% or less is determined as an active ingredient candidate substance for a nerve growth promoter.
  • Example 1 [Experimental materials and experimental methods]
  • Animals C57BL / 6J mice (8 weeks old) were purchased from Charles River Laboratories and bred at the Osaka University graduate School of Medicine / Animal Experiment Facility.
  • PIR-B ⁇ / ⁇ mice are described in Ujike et al. (Ujike, A., et al. Impaired dendritic cell maturation and increased T (H) 2 responses in PIR-B ( ⁇ / ⁇ ) mice. Nat Immunol 3, 542-548. (2002).)
  • a C57BL / 6J mouse strain carrying the third exon disruption of the target p75 gene was used. This strain was originally obtained from the Jackson Laboratory. All experimental procedures were approved by the organizing committee of Osaka University.
  • TAT-TrkA peptide 481-494 (TAT-QGHIIENPQYFSDA: SEQ ID NO: 4), TAT-TrkA peptide 484-497 (TAT-IIENPQYFSDACVH: SEQ ID NO: 5) and TAT fusion control peptide (TAT-HVCAESFYQPNEII: SEQ ID NO: 6) Synthesized (Sigma-Aldrich).
  • CHO cells that stably secrete human MAG-Fc were provided by Mr. Endo of Kobe University. The cells were cultured using a serum-free medium. After culturing for 3 days, the medium was collected, and MAG-Fc was purified using protein A sepharose beads.
  • anti-HA antibody HA-7, 1: 5000, Sigma-Aldrich
  • anti-c-Myc antibody 9E10, 1: 1000, Santa Cruz Biotechnology
  • biotin-labeled anti-TrkB antibody 1: 2500, R & D systems
  • Anti-SHP-1 antibody 1: 1000, BD Transduction Laboratories
  • anti-SHP-2 antibody 1: 1000, BD Transduction Laboratories
  • anti- ⁇ tubulin antibody (1: 1000, Santa Cruz Biotechnology
  • anti-neutral Class III ⁇ tubulin antibody 1: 5000, Covance Laboratories
  • anti-p75 antibody (1: 1000, Promega)
  • anti-SHP-1 antibody As polyclonal antibodies, anti-SHP-1 antibody, anti-SHP-2 antibody, anti-Trk antibody, anti-TrkB antibody, anti-PIR-B antibody (above 1: 1000, Santa Cruz Biotechnology)
  • Anti-PIR-B antibody (1 mg / mL, R & D systems) and anti-PIR-A / B antibody (1: 1000, BD Pharmingen) were used.
  • HRP-labeled anti-mouse, anti-rabbit, anti-rat IgG secondary antibody Santa Cruz Biotechnology
  • streptavidin peroxidase (Roche Applied Science)
  • Alexa488 or 568-labeled anti-mouse IgG goat antibody (Molecular Probes) were used.
  • Plasmid and siRNA DNA encoding full-length p75 with an HA tag or Myc tag added to the N-terminus was subcloned into pcDNA3 vector (Invitrogen) (Yamashita, T., et al. J Cell Biol 157, 565-570 (2002)). The extracellular and intracellular domains of p75 were prepared from p75-HA.
  • DNA encoding PIR-B was subcloned into pcDNA3.1 Zero (+) vector (Endo, S., et al. Proc Nat Acad Sci USA 105, 14515-14520 (2008)).
  • the expression vector for TrkB was provided by Barde YA (Bibel, M., Hoppe, E.
  • TrkB A mammalian cell expression vector for TrkB was purchased from Addgene. The following siRNAs were used for knockdown experiments. For mouse SHP-1, for mouse SHP-2 (ON-TARGET plus SMARTpool, Thermo Fisher Scientific, Dharmacon Products) and for mouse NTRK2 (stealth siRNA, Invitrogen). ON-TARGET plus SMARTpool is a mixture of four types of double-stranded siRNA.
  • COS-7 cells were cultured using DMEM (Invitrogen) containing 10% fetal bovine serum.
  • DMEM Invitrogen
  • Lipofectamine 2000 Invitrogen
  • Cells were lysed 24-48 hours after transfection and subjected to immunoprecipitation.
  • Primary dissociation culture of cerebellar granule cells (CGNs) derived from 7-day-old mice was performed as described by Hata et al. (Hata, K., et al. J Cell Biol 184, 737-750 (2009)).
  • the mouse cerebellum was digested with 0.25% trypsin (Gibco / Invitrogen) and DNaseI (Takara) for 15 minutes at 37 ° C., and then the cells were gently dissociated.
  • DMEM / F12 containing 10% fetal bovine serum was added and the cells were centrifuged at 1000 rpm for 3 minutes.
  • the obtained CGNs were seeded on a poly-L-lysine-coated dish and maintained at 37 ° C. under 5% CO 2 condition using DMEM / F12 to which B27 (Invitrogen) was added. After 24 hours, the medium was changed to DMEM / F12 containing 0.1% BSA. After 24 hours of incubation, CGNs were collected and subjected to immunoprecipitation or Western blotting.
  • the cells were mixed with 500 ⁇ L of DMEM / F12 containing 10% fetal calf serum pre-warmed, and the cell suspension was seeded on a poly-L-lysine-coated dish.
  • the cells were left in an incubator, and after 3 hours, the medium was replaced with fresh DMEM / F12 containing B27, and the culture was continued for 48 to 72 hours. Thereafter, the medium was replaced with serum-free medium and subjected to immunoprecipitation or Western blotting. Alternatively, the medium was removed and seeded on a poly-L-lysine coated dish and subjected to a neurite outgrowth test.
  • Immunoprecipitation Cells were washed with ice-cold PBS and lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5-1% NP-40, 10 mM NaF) containing protease inhibitor cocktail (Roche Diagnostics) on ice. , 1 mM Na 3 VO 4 ), and centrifuged at 4 ° C., 15000 rpm for 10 minutes. The supernatant was incubated with the desired antibody at 4 ° C. for 2 hours or overnight.
  • lysis buffer 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5-1% NP-40, 10 mM NaF
  • protease inhibitor cocktail Roche Diagnostics
  • Neurite outgrowth test was performed according to the method of Hata et al. (Hata, K., et al. J Cell Biol 173, 47-58 (2006)). Specifically, after treatment with MAG-Fc dissolved in DMEM / F12 or control (DMEM / F12 only) for 24 hours, cerebellar granule cells (CGNs) were fixed with 4% (w / v) paraformaldehyde to recognize TuJ1. Immunostained with monoclonal antibodies.
  • cholera toxin ⁇ subunit conjugated with Alexa Fluor (trade name, 2 ⁇ g / ⁇ L, Invitrogen) was injected into the vitreous using a glass needle.
  • CTB cholera toxin ⁇ subunit conjugated with Alexa Fluor
  • the mice were euthanized by overanesthesia, refluxed with 4% PFA, and the eyeballs were removed.
  • the remaining eye cup with the nerve portion after removal of the lens and vitreous was postfixed with 4% PFA at 4 ° C. for 12 hours.
  • the eyecup was dehydrated overnight at 4 ° C. using a 10-30% sucrose solution and immersed in Tissue Tech OCT Compound (trade name).
  • the tissue was frozen with dry ice and a continuous section (16 ⁇ m) was prepared using a cryostat and collected on a MAS-coated slide glass.
  • FIG. 1 (A) shows the result of immunoprecipitation using an anti-PIR-B antibody
  • (b) shows the result of immunoprecipitation using an anti-HA antibody.
  • HA-TrkB was deposited on the immunoprecipitation complex using anti-PIR-B antibody (see FIG. 1 (a)), and the immunoprecipitation complex using anti-HA antibody was PIR-B was precipitated (see FIG. 1 (b)).
  • FIG. 3 (a) shows the result of immunoprecipitation using an anti-PIR-B antibody
  • FIG. 3 (b) shows the result of immunoprecipitation using an anti-HA antibody.
  • HA-TrkB T1 was not deposited on the immunoprecipitation complex using the anti-PIR-B antibody (see FIG. 3 (a)), and PIR-B was not deposited on the immunoprecipitation complex using the anti-HA antibody ( (Refer FIG.3 (b)).
  • CGNs Cerebellar granule cells derived from 7-day-old mice were treated with MAG-Fc (25 ⁇ g / mL) for 15 minutes, or a cell lysate was prepared without treatment and immunoprecipitation was performed.
  • anti-PIR-B antibody, anti-TrkB antibody or control antibody anti-IgG antibody
  • Western blotting of the cell lysate was performed using the same detection antibody. The results are shown in FIGS. 5 (a) and (b).
  • (A) shows the result of immunoprecipitation using an anti-PIR-B antibody
  • (b) shows the result of immunoprecipitation using an anti-TrkB antibody or a control antibody.
  • TrkB was precipitated on the immunoprecipitation complex using the anti-PIR-B antibody only in the cells treated with MAG-Fc.
  • PIR-B was deposited on the immunoprecipitation complex using anti-TrkB antibody only in the cells treated with MAG-Fc. From these results, it was shown that PIR-B interacts with TrkB in a ligand-dependent manner.
  • COS-7 cells transfected with a full-length PIR-B (PIR-B FL) expression vector and a full-length p75 (p75 FL-HA) expression vector to which an HA tag has been added are expressed as MAG-Fc (25 ⁇ g / mL).
  • the cell lysate was prepared with or without treatment for 15 minutes and immunoprecipitated.
  • Anti-SHP-2 antibody was used for immunoprecipitation, and detection was performed with anti-PIR-B antibody and anti-SHP-2 antibody.
  • Western blotting of cell lysate was performed using anti-PIR-B antibody, anti-SHP-2 antibody and anti-HA antibody as detection antibodies. The results are shown in FIG. As can be seen from FIG. 7, PIR-B was precipitated in the immunoprecipitation complex using anti-SHP-2 antibody in cells not treated with MAG-Fc. In addition, the binding of PIR-B and SHP-2 was enhanced by MAG-Fc treatment.
  • CGNs cerebellar granule cells
  • MAG-Fc 25 ⁇ g / mL
  • cell lysates were prepared and immunoprecipitated.
  • anti-PIR-B antibody, anti-SHP antibody or control antibody anti-IgG antibody
  • detection was performed with anti-PIR-B antibody and anti-SHP-2 antibody, respectively.
  • Western blotting of the cell lysate was performed using the same detection antibody. The results are shown in FIGS. 8 (a) and (b).
  • FIG. 8 (A) shows the results of immunoprecipitation using anti-PIR-B antibody or control antibody
  • (b) shows the results of immunoprecipitation using anti-SHP-2 antibody or control antibody.
  • SHP-2 was precipitated in the immunoprecipitation complex using anti-PIR-B antibody in cells not treated with MAG-Fc.
  • the binding of PIR-B and SHP-2 was enhanced by MAG-Fc treatment.
  • PIR-B was deposited on the immunoprecipitation complex using anti-SHP-2 antibody in cells not treated with MAG-Fc.
  • the binding of PIR-B and SHP-2 was enhanced by MAG-Fc treatment.
  • TrkB was involved in the formation of PIR-B-SHP-2 complex.
  • Prepare cell lysate by treating COS-7 cells transfected with HA-tagged TrkB expression vector and PIR-B expression vector or CGNs with MAG-Fc (25 ⁇ g / mL) for 15 minutes. And immunoprecipitation was performed.
  • anti-TrkB antibody or control antibody anti-IgG antibody
  • detection was performed with anti-SHP-2 antibody and anti-TrkB antibody.
  • Western blotting of the cell lysate was performed using the same detection antibody. The results are shown in FIGS. 9 (a) and (b).
  • (A) is the result of COS-7 cells
  • (b) is the result of CGNs.
  • SHP-2 was precipitated on the immunoprecipitation complex using anti-TrkB antibody in COS-7 cells not treated with MAG-Fc.
  • the binding of PIR-B and SHP-2 was enhanced by MAG-Fc treatment.
  • PIR-B was deposited on the immunoprecipitation complex using anti-SHP-2 antibody in cells not treated with MAG-Fc.
  • the binding of PIR-B and SHP-2 was enhanced by MAG-Fc treatment. From the above results, it was revealed that SHP-2 is attracted to the PIR-B-TrkB receptor complex after MAG stimulation.
  • NSC-87877 (8-hydroxy-7-
  • the experiment was conducted using (6-sulfonaphthalen-2-yl) diazenyl-quinoline-5-sulfonic acid). That is, CGNs was pretreated with NSC-87877 for 3 hours, and then the same experiment as in (3-1) was performed. The results are shown in FIG. As is clear from FIG. 12, by performing pretreatment with NSC-87877, dephosphorylation of the Trk receptor was not induced by MAG-Fc treatment.
  • TrkBsiRNA1 or TrkBsiRNA2 or control siRNA was transfected into CGNs, and cultured for 24 hours in the presence or absence of MAG-Fc using poly-L-lysine-coated dishes. The length of the longest neurite of each cell was measured, and the average value was obtained. In addition, the expression level of TrkB was confirmed by Western blotting. The results are shown in FIG. As is clear from FIG. 17, neurite growth of CGNs was significantly blocked by knocking down TrkB. In addition, the effect of MAG-Fc disappeared. This result indicates that MAG decreases the steady-state activity of TrkB and inhibits neurite outgrowth of CGNs.
  • N-terminal protein transduction domain (11 amino acids) of HIV-derived TAT protein is fused to synthesize TAT-TrkA481-494 and TAT-TrkA484-497. used.
  • FIG. 22 (a) shows the result of immunoprecipitation using an anti-PIR-B antibody
  • FIG. 22 (b) shows the result of immunoprecipitation using an anti-HA antibody.
  • p75 FL-HA was deposited on the immunoprecipitation complex using anti-PIR-B antibody (see FIG. 22 (a)), and the immunoprecipitation complex using anti-HA antibody.
  • PIR-B was deposited on the substrate (see FIG. 22B). From these results, it was confirmed that PIR-B interacts with p75.
  • FIG. 23 (A) shows the result of immunoprecipitation using an anti-PIR-B antibody
  • (b) shows the result of immunoprecipitation using an anti-HA antibody.
  • p75 ECD + TM-HA was precipitated on the immunoprecipitation complex using anti-PIR-B antibody (see FIG. 23 (a)), and immunoprecipitation complex using anti-HA antibody.
  • PIR-B was deposited on the substrate (see FIG. 23B).
  • TrkB FL-HA and p75 FL-Myc were precipitated in the immunoprecipitation complex using the anti-PIR-A / B antibody in the cells transfected with the three expression vectors. Similar results were obtained when the TrkB FL-HA expression vector was used instead of the TrkB FL-HA expression vector (see FIG. 27).
  • FIG. 31 shows the results of quantifying the number of CTB-labeled axons extending 0.2, 0.5, and 1.0 mm from the end of the damaged site.
  • mice transfected with SHP-1 siRNA and SHP-2 siRNA respectively, significantly increased axon regeneration compared to mice transfected with control siRNA. Promoted.
  • the number of mice used was 6 control siRNAs, 6 SHP-1 siRNAs, and 7 SHP-2 siRNAs.
  • the scale bar represents 200 ⁇ m
  • * represents the damaged site.
  • * indicates P ⁇ 0.05
  • ** indicates P ⁇ 0.01.
  • TrkB full length 5 ⁇ g / mL was added to a 96-well ELISA plate, incubated at 4 ° C. for 16 hours, and immobilized. After adding a test substance and SHP-1 to each well and reacting for 2 hours, the well was washed, and an anti-SHP-1 antibody (1 ⁇ g / mL) and an HRP-labeled anti-rabbit IgG secondary antibody (1: 1000) were added. To confirm the binding state between TrkB and SHP-1. A test substance added to a well showing a binding state of 50% or less compared with the binding state of the control (no test substance added) was selected as an active ingredient candidate substance of a nerve growth promoter.
  • TrkB full length 5 ⁇ g / mL was added to a 96-well ELISA plate, incubated at 4 ° C. for 16 hours, and immobilized. After adding a test substance and SHP-1 to each well and reacting for 2 hours, the well was washed, and an anti-phosphotyrosine antibody (1 ⁇ g / mL) and an HRP-labeled anti-mouse IgG secondary antibody (1: 1000) were added. Was used to confirm the phosphorylation state of TrkB. Compared to the phosphorylated state (dephosphorylated) of the control (no test substance added), the test substance added to the well showing a dephosphorylated state of 50% or less was selected as an active ingredient candidate substance of the nerve growth promoter. .
  • test substance was added to a medium (DMEM / F12) of mouse-derived cerebellar granule cells (CGNs) and cultured at 37 ° C. for 72 hours. After completion of the culture, a cell lysate was prepared and subjected to Western blotting. For detection, anti-SHP-1 antibody and anti-SHP-2 antibody were used.
  • the test substance added to the well whose expression level was reduced to 50% or less compared with the control (no test substance addition) SHP-1 and SHP-2 expression levels was selected as a candidate substance for the active ingredient of nerve growth promoter. .

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Abstract

La présente invention concerne : (A) une substance inhibant la liaison entre SHP-1 ou SHP-2 et un récepteur Trk ; (B) une substance inhibant l'activité récepteur Trk tyrosine phosphatase de SHP-1 ou SHP-2 ; et (C) une substance inhibant l'expression de SHP-1 or SHP-2. Chacune des substances (A)-(C) décrites ci-avant peut être employée en tant que principe actif d'un promoteur de croissance nerveuse. La présente invention concerne également un promoteur de croissance nerveuse contenant l'une des substances (A)-(C) décrites ci-avant au titre de principe actif et employé comme produit pharmaceutique dans le soulagement des séquelles de troubles du système nerveux central comme les troubles vasculaires cérébraux, les lésions cérébrales traumatiques et les lésions de la moelle épinière.
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