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WO2008038599A1 - Axonal regeneration promoter - Google Patents

Axonal regeneration promoter Download PDF

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Publication number
WO2008038599A1
WO2008038599A1 PCT/JP2007/068449 JP2007068449W WO2008038599A1 WO 2008038599 A1 WO2008038599 A1 WO 2008038599A1 JP 2007068449 W JP2007068449 W JP 2007068449W WO 2008038599 A1 WO2008038599 A1 WO 2008038599A1
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Prior art keywords
amino acid
acid sequence
seq
peptide
axon
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PCT/JP2007/068449
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French (fr)
Japanese (ja)
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Toshihide Yamashita
Masayoshi Suda
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National University Corporation Chiba University
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Priority to JP2008536361A priority Critical patent/JPWO2008038599A1/en
Publication of WO2008038599A1 publication Critical patent/WO2008038599A1/en

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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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • 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 an axonal regeneration promoting agent capable of promoting regeneration of nerve cells, in particular, axons of nerve cells of the central nervous system.
  • RGM Repulsive Guidance Molecule
  • RGM Repulsive Guidance Molecule
  • it has a neutralizing activity of inhibitory activity! (For example, see Patent Document 1 and Non-Patent Document 5 below).
  • RGM was first identified as a protein involved in the formation of retinal lid processes in chicken fetuses, and RGM caused repulsion of the optic nerve axon during development (see Non-Patent Documents 6 and 7 below). It is reported that the expression of this protein increases when the spinal cord is damaged (see Non-Patent Document 8 below), and that the expression increases in human brain ischemia and brain trauma (see Non-Patent Document 9 below). It has been tell.
  • Patent Document 1 International Publication WO2005 / 087268 Pamphlet
  • Non-patent literature 1 S. David et al., Science, 214, 931-933, 1981
  • Non-patent literature 2 MS Chen et al., Nature, 403, 434-439, 2000
  • Non-patent document 3 G. Mukhopadhyay et al., Neuron, 13, 757-767, 1994
  • Non-patent document 4 V. Kottis et al., J. Neurochem., 82, 1566-1569, 2002
  • Non-patent document 5 Hata et al., J. Biol. Cell, 173 (1), 47-58, 2005
  • Non-Patent Document 6 Muller et al., Curr Biol., 6 (11), 1497, 1996
  • Non-Patent Document 7 Monnier et al., Nature, 419 (6905), 392, 2002
  • Non-Patent Document 8 Schwab et al., Eur. J. Neurosci., 21, 1569-1576, 2005
  • Non-Patent Document 9 Schwab et al., Arch. Neurol., 62, 1561-1568, 2005 Disclosure of the Invention
  • an object of the present invention is to provide a novel axonal regeneration accelerator that solves the above-mentioned problems.
  • the present inventor has intensively studied the above problem! /, however, has found that a partial peptide derived from the amino acid sequence of the RGM protein can suppress the effect of RGM, and completed the present invention. I came to let you. That is, the axonal regeneration promoter according to one means for solving the above problems contains at least one of the following (a) to (d) as an active ingredient.
  • amino acid sequence according to any one of SEQ ID NOs: 1 to 4, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and the RGMa protein axon growth inhibitory activity Peptides with a function to neutralize [0009]
  • amino acid sequence described in SEQ ID NO: 1 corresponds to a part of the amino acid sequence of human RGMa protein, and more specifically, the 243rd force of the human RGMa protein is the 256th amino acid sequence.
  • amino acid sequence described in SEQ ID NO: 2 corresponds to a part of the amino acid sequence of human RGMa protein, and more specifically from the 326th position.
  • amino acid sequence described in SEQ ID NO: 5 shows the amino acid sequence of human RGMa protein
  • SEQ ID NO: 6 shows the base sequence of the DNA.
  • amino acid sequence set forth in SEQ ID NO: 3 corresponds to a part of the amino acid sequence of rat RGMa protein, and more specifically, the 243rd to 256th amino acid sequences of rat RGMa protein. Corresponds to the second amino acid sequence.
  • amino acid sequence described in SEQ ID NO: 4 corresponds to a part of the amino acid sequence of rat RGMa protein, and more specifically, the 326th position.
  • the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 and the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 are both those of rats. Since it is preserved, it is effective as a human axon regeneration promoter.
  • SEQ ID NO: 7 shows the amino acid sequence of rat RG Ma protein
  • SEQ ID NO: 8 shows the base sequence of the DNA.
  • the axon is preferably a central axon, although not limited thereto.
  • the “central nervous system” refers to the brain and spinal cord, and is not limited to, for example, the corticospinal tract nerve and the spinal thalamic tract nerve.
  • a novel axonal regeneration accelerator can be provided.
  • This axonal regeneration accelerator is particularly effective for regeneration of central nervous system axons. As a result, it is possible to contribute to the treatment of patients with damage to the central nervous system such as the spinal cord.
  • the axon regeneration accelerator (hereinafter referred to as "the axon regeneration accelerator") according to this embodiment is as follows.
  • One of the characteristics is that it contains at least one of the peptides (a) to (d).
  • This axon regeneration promoter contains a peptide having a part of the RGMa protein as an active ingredient, which can neutralize the axon growth inhibitory activity of the RGMa protein in neurons. Can be promoted. That is, the axonal regeneration promoter is useful as a pharmaceutical composition for treating a patient who has damaged the central nervous system.
  • the peptide in the present axon regeneration promoter is not limited as long as it exhibits the above-described effects.
  • a peptide consisting of an amino acid sequence in which several amino acids are deleted, substituted or added can also be used.
  • the above-mentioned effects are not lost! /, Substitution to an amino acid having a degree of chemical modification, addition of a polymer or a sugar chain. Substitution with other amino acids.
  • amino acid or a fusion protein that facilitates formulation at least one of the N-terminal side and the C-terminal side of the peptides shown in SEQ ID NOS: 1 to 4 can be added.
  • amino acids which are convenient for expressing the peptide
  • amino acids which are convenient for producing and producing the peptide
  • the present axon regeneration promoter is an ordinary pharmaceutically acceptable carrier, binder, stabilizer, excipient, diluent (eg, distilled water), pH buffering agent. It can contain various preparation ingredients such as (for example, phosphate buffered saline), disintegrants, solubilizers, solubilizers, and isotonic agents.
  • the axon regeneration promoter can be administered orally or parenterally, for example, depending on the form of use.
  • oral administration it is possible to adopt commonly used administration forms such as powders, granules, tablets, capsules, solutions, suspensions, oils, emulsifiers and the like.
  • parenteral administration a commonly used administration form, for example, a method of administering the above-mentioned solution, suspension or the like directly to the injured site, a form of administration by injection or the like can be adopted.
  • the dose of the axonal regeneration promoter is determined by the patient's weight, sex, degree of nerve damage, It can be selected appropriately according to the law.For example, when administered parenterally and administered directly to the site of injury, for example, adults per day, the above peptide should be included in the range of 1 mg to 10 mg per injury site. Is more preferably 5 mg or more and 50 mg or less. In the case of parenteral administration other than this, for example, in the case of injection, it is preferably about 10 times this.
  • the peptide according to the present axon regeneration promoter can be produced by a known method without limitation.
  • it can be produced by an ordinary chemical synthesis method, an enzymatic degradation method of a protein molecule, a gene recombination technique, or the like.
  • the usual chemical synthesis method is not limited, but for example, a solid phase synthesis method using a peptide synthesizer can be preferably used.
  • a solid phase synthesis method using a peptide synthesizer can be preferably used.
  • well-known purification methods such as salting-out, Filtration, reversed-phase chromatography, ion exchange chromatography, affinity chromatography, etc. are preferably used.
  • the gene recombination technique is not limited.
  • a DNA fragment encoding the target amino acid sequence is incorporated into an appropriate expression vector, and microorganisms and animal cells are transformed using this expression vector.
  • a technique for obtaining a peptide having a desired amino acid sequence by culturing the transformed microorganism or animal cell after conversion can be employed.
  • the expression vector used here is not limited, and a well-known one can be employed.
  • plasmids, virus vectors, and the like can be used.
  • the cell extract or the culture supernatant is collected from the cultured medium, and the above-described purification method is used. It is preferable to use it.
  • Custom Peptide Synthesis (3 ⁇ 4igma Genosys ⁇ ⁇ ⁇ ⁇ A peptide consisting of IJ was synthesized.
  • RGMa-expressing cells were prepared using Flp-InSystem (Invitrogen) and following the manufacturer's guidelines.
  • the HA-RGMa fragment containing the signal peptide was prepared from the pSecTag2 vector using two restriction endonucleases and ligated to pcDN A5FRT (Invitrogen).
  • This construct pcDNA5FRT / lg ⁇ leader / HA / RGMa
  • pOG44 were co-transformed into Flp-inCHO cells and grown for 2 weeks in a medium containing Neugromycin B (500 ag / ml; Invitrogen).
  • Cells stably expressing HA-RGMa hereinafter referred to as “RGMa-CHO cells”) were obtained.
  • the expression of HA-RGMa was confirmed by immunodetection using Western blot and immunocytochemistry.
  • Cerebellar granule cells from pup rats 7-9 days after birth were isolated by trypsinization (0.25% trypsin in PBS, 37 ° C, 15 minutes), resuspended in serum-containing medium, Milled and washed 3 times with PBS. Neurons were seeded on confluent monolayers of RGMa-CHO cells or subject CHO cells in chamber slides (Lab Tek II; Nunc). Pepl or Pep2 was added to the cultures at the indicated concentrations (1 M, 4 M, 10 M) for neutralization assays. Cultures were grown in serum-free DMEM / F12 medium for 24 hours.
  • the cells were fixed with 4% (wt / vol) paraformaldehyde and immunostained with a monoclonal antibody (Tujl) (l: 1000; Covance) that recognizes the neuron-specific 13 tubulin III protein.
  • Tujl monoclonal antibody
  • the length of the longest neurite was measured for each ⁇ -tubulin III positive neuron.
  • control indicates the target CHO cell
  • RGM— indicates that RGM protein is expressed (hereinafter referred to as “RGM—CHO cell”)
  • peerp l 1 ⁇ indicates 1 pep l.
  • peerpl 4 ⁇ is added when 4 ⁇ of pep l is added.
  • pep l ⁇ ⁇ ⁇ means pep l is ⁇ ⁇ ⁇ ⁇ p
  • pep2 1 M means pep2 is 1 M ⁇
  • 10 ⁇ indicates the case of adding 1 ⁇ of pep2.
  • the vertical axis is the average length of the longest neurite per neuron, and the data is the average soil S. .. ⁇ . Of three independent experiments.
  • Pep l and Pep2 can potently reverse the inhibitory effect of RGMa in a dose-dependent manner. This indicates that these peptides function as neutralizing reagents for RGMa in vitro, and that these peptides can be used as axon growth promoters by using them as active ingredients. It could be confirmed.
  • the axonal regeneration promoter according to the present invention has industrial applicability as a therapeutic agent for a patient who has damaged the central nervous system.
  • FIG. 1 is a diagram showing the inhibitory effect of RGMa protein by the peptide of the present invention.

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Abstract

Disclosed is a more novel axonal regeneration promoter. The axonal regeneration promoter comprises, as an active ingredient, a peptide which has an amino acid sequence having the deletion, substitution or addition of one or several amino acid residues in the amino acid sequence depicted in any one of SEQ ID NOs:1 to 4 in the Sequence Listing and which can act to neutralize the axon growth inhibition activity of RGMa protein.

Description

明 細 書  Specification
軸索再生促進剤  Axon regeneration promoter
技術分野  Technical field
[0001] 本発明は、神経細胞、特に、中枢神経系の神経細胞の軸索の再生を促進すること ができる軸索再生促進剤に関する。  [0001] The present invention relates to an axonal regeneration promoting agent capable of promoting regeneration of nerve cells, in particular, axons of nerve cells of the central nervous system.
背景技術  Background art
[0002] 脊髄等の中枢神経が交通事故や脳血管障害等により損傷を受けるとしばしば部分 的又は完全な麻痺が起きる。ところが神経機能は失われたまま再生することはできな い。これは末梢神経が再生するのと対照的である。したがって、損傷した中枢神経を 再生させることは医療分野において重要な課題である。  [0002] When the central nervous system such as the spinal cord is damaged by a traffic accident or cerebrovascular disorder, partial or complete paralysis often occurs. However, the nerve function is lost and cannot be regenerated. This is in contrast to the regeneration of peripheral nerves. Therefore, regenerating damaged central nerves is an important issue in the medical field.
[0003] 一方、中枢神経が成人の中枢神経の軸索が末梢神経のグラフトを介して再生し得 るという報告がある（例えば下記非特許文献 1参照）。したがって、成人の中枢神経が 再生しない主な原因は、神経細胞を取り巻く局所的な環境にあることが示唆されてお り、これまでに、中枢神経の再生を阻害する主な阻害物質として、 Nogo、ミエリン結 合糖タンパク質（以下「MAG」と!/、う。 )及び乏突起神経膠細胞—ミエリン糖タンパク 質（以下「OMgp」とレ、う。 )が同定されてレ、る (これらにつ!/、ては例えば下記非特許文 献 2乃至 4参照）。  [0003] On the other hand, there is a report that the central nerve can regenerate the adult central nerve axon via the peripheral nerve graft (for example, see Non-Patent Document 1 below). Thus, it has been suggested that the main cause of adult central nervous system regeneration is the local environment surrounding nerve cells. , And myelin-linked glycoprotein (hereinafter “MAG”! /,) And oligodendrocyte-myelin glycoprotein (hereinafter “OMgp”) are identified For example, see Non-Patent Documents 2 to 4 below).
[0004] また一方で、本発明者らはこれまでに Repulsive Guidance Molecule (以下「R GM」という。）が軸索成長を阻害することを明らかにし、 RGMに対するポリクローナ ル抗体が RGMの軸索成長阻害活性の中和活性を有することを見!/、だした（例えば 下記特許文献 1及び非特許文献 5参照）。 RGMはニヮトリ胎児において網膜蓋体の 突起の形成に関与するタンパク質として最初に同定され、 RGMにより発生期の視神 経軸索の反発がおこること（下記非特許文献 6及び 7参照）、ラットにおいて脊髄が損 傷するとこのタンパク質の発現が増大すること（下記非特許文献 8参照）、ヒトにおい て脳虚血部位や脳外傷の部位で発現が増大すること（下記非特許文献 9参照）が報 告されている。  [0004] On the other hand, the present inventors have clarified that Repulsive Guidance Molecule (hereinafter referred to as "RGM") inhibits axon growth, and that polyclonal antibodies against RGM have a RGM axon growth. It has been found that it has a neutralizing activity of inhibitory activity! (For example, see Patent Document 1 and Non-Patent Document 5 below). RGM was first identified as a protein involved in the formation of retinal lid processes in chicken fetuses, and RGM caused repulsion of the optic nerve axon during development (see Non-Patent Documents 6 and 7 below). It is reported that the expression of this protein increases when the spinal cord is damaged (see Non-Patent Document 8 below), and that the expression increases in human brain ischemia and brain trauma (see Non-Patent Document 9 below). It has been tell.
[0005] 特許文献 1：国際公開 WO2005/087268号パンフレット 非特許文献 1 : S . David et al. 、 Science, 214、 931 - 933, 1981 非特許文献 2 : M. S. Chen et al. 、 Nature, 403、 434— 439、 2000 [0005] Patent Document 1: International Publication WO2005 / 087268 Pamphlet Non-patent literature 1: S. David et al., Science, 214, 931-933, 1981 Non-patent literature 2: MS Chen et al., Nature, 403, 434-439, 2000
非特許文献 3 : G. Mukhopadhyay et al. 、 Neuron, 13、 757— 767、 1994 非特許文献 4 : V. Kottis et al. 、J. Neurochem. 、 82、 1566— 1569、 2002 非特許文献 5 : Hata et al. 、J. Biol. Cell, 173 (1)、 47— 58、 2005  Non-patent document 3: G. Mukhopadhyay et al., Neuron, 13, 757-767, 1994 Non-patent document 4: V. Kottis et al., J. Neurochem., 82, 1566-1569, 2002 Non-patent document 5: Hata et al., J. Biol. Cell, 173 (1), 47-58, 2005
非特許文献 6 : Muller et al. 、 Curr Biol. 、 6 (11)、 1497、 1996  Non-Patent Document 6: Muller et al., Curr Biol., 6 (11), 1497, 1996
非特許文献 7 : Monnier et al. 、 Nature, 419 (6905)、 392、 2002  Non-Patent Document 7: Monnier et al., Nature, 419 (6905), 392, 2002
非特許文献 8 : Schwab et al. , Eur. J. Neurosci. 、 21、 1569— 1576、 2005 非特許文献 9 : Schwab et al. 、 Arch. Neurol. 、 62、 1561 - 1568, 2005 発明の開示  Non-Patent Document 8: Schwab et al., Eur. J. Neurosci., 21, 1569-1576, 2005 Non-Patent Document 9: Schwab et al., Arch. Neurol., 62, 1561-1568, 2005 Disclosure of the Invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0006] しかしながら、上記 RGMに対するポリクローナル抗体は軸索再生促進剤として有 用であるものの、治療において局所的に投与せざるを得ないといった制限もあり、より 新規な軸索再生促進剤を見出すことが望まれている。 [0006] However, although the above polyclonal antibody against RGM is useful as an axon regeneration promoter, there is a limitation that it must be administered locally in the treatment, and thus a new axon regeneration promoter is found. Is desired.
[0007] そこで本発明は、上記課題を解決する新規な軸索再生促進剤を提供することを目 的とする。 [0007] Accordingly, an object of the present invention is to provide a novel axonal regeneration accelerator that solves the above-mentioned problems.
課題を解決するための手段  Means for solving the problem
[0008] 本発明者は、上記課題につき鋭意検討を行って!/、たところ、 RGMタンパク質のアミ ノ酸配列に由来する部分的なペプチドが RGMの効果を抑制できることを見出し、本 発明を完成させるに至った。即ち、上記課題を解決する一手段に係る軸索再生促進 剤は、以下（a)乃至（d)の少なくとも!/、ずれかのペプチドを有効成分として含有する。 [0008] The present inventor has intensively studied the above problem! /, However, has found that a partial peptide derived from the amino acid sequence of the RGM protein can suppress the effect of RGM, and completed the present invention. I came to let you. That is, the axonal regeneration promoter according to one means for solving the above problems contains at least one of the following (a) to (d) as an active ingredient.
(a)配列番号 1に記載のアミノ酸配列からなるペプチド  (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1
(b)配列番号 2に記載のアミノ酸配列からなるペプチド  (b) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2
(c)配列番号 3に記載のアミノ酸配列からなるペプチド  (c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3
(d)配列番号 4に記載のアミノ酸配列からなるペプチド  (d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4
(e)配列番号 1乃至 4のいずれかに記載のアミノ酸配列において、 1若しくは数個の アミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、 RGMaタンパ ク質の軸索成長阻害活性を中和する機能を有するペプチド [0009] ここで、配列番号 1に記載のアミノ酸配列は、ヒトの RGMaタンパク質のアミノ酸配列 の一部に相当するものであり、より具体的にはヒトの RGMaタンパク質の第 243番目 力 第 256番目のアミノ酸配列に相当する。また、配列番号 2に記載のアミノ酸配列 も上記配列番号 1に記載のアミノ酸配列と同様、ヒトの RGMaタンパク質のアミノ酸配 列の一部に相当するものであり、より具体的には第 326番目から第 341番目のァミノ 酸配列に相当する。なお配列番号 5に、ヒトの RGMaタンパク質のアミノ酸配列を、配 列番号 6にその DNAの塩基配列を示しておく。 (e) an amino acid sequence according to any one of SEQ ID NOs: 1 to 4, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and the RGMa protein axon growth inhibitory activity Peptides with a function to neutralize [0009] Here, the amino acid sequence described in SEQ ID NO: 1 corresponds to a part of the amino acid sequence of human RGMa protein, and more specifically, the 243rd force of the human RGMa protein is the 256th amino acid sequence. It corresponds to the amino acid sequence of Similarly to the amino acid sequence described in SEQ ID NO: 1, the amino acid sequence described in SEQ ID NO: 2 corresponds to a part of the amino acid sequence of human RGMa protein, and more specifically from the 326th position. Corresponds to the 341st amino acid sequence. SEQ ID NO: 5 shows the amino acid sequence of human RGMa protein, and SEQ ID NO: 6 shows the base sequence of the DNA.
[0010] ここで、配列番号 3に記載のアミノ酸配列は、ラットの RGMaタンパク質のアミノ酸配 列の一部に相当するものであり、より具体的にはラットの RGMaタンパク質の第 243 番目から第 256番目のアミノ酸配列に相当する。また、配列番号 4に記載のアミノ酸 配列も上記配列番号 3に記載のアミノ酸配列と同様、ラットの RGMaタンパク質のアミ ノ酸配列の一部に相当するものであり、より具体的には第 326番目から第 341番目の アミノ酸配列に相当する。なお配列番号 3に記載のアミノ酸配列からなるペプチド、配 列番号 4に記載のアミノ酸配列からなるペプチドはいずれもラットのものである力 S、RG Mタンパク質についてはラットとヒトとの間で高度に保存されたものであるため、ヒトの 軸索再生促進剤として効果を発揮することができる。なお配列番号 7に、ラットの RG Maタンパク質のアミノ酸配列を、配列番号 8にその DNAの塩基配列を示しておく。  Here, the amino acid sequence set forth in SEQ ID NO: 3 corresponds to a part of the amino acid sequence of rat RGMa protein, and more specifically, the 243rd to 256th amino acid sequences of rat RGMa protein. Corresponds to the second amino acid sequence. Similarly to the amino acid sequence described in SEQ ID NO: 3, the amino acid sequence described in SEQ ID NO: 4 corresponds to a part of the amino acid sequence of rat RGMa protein, and more specifically, the 326th position. To 341th amino acid sequence. The peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 and the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 are both those of rats. Since it is preserved, it is effective as a human axon regeneration promoter. SEQ ID NO: 7 shows the amino acid sequence of rat RG Ma protein, and SEQ ID NO: 8 shows the base sequence of the DNA.
[0011] また、本手段において、限定されるわけではないが、軸索は中枢神経系の軸索で あることが好ましい。ここで、「中枢神経系」とは脳及び脊髄をいい、限定されるわけで はないが、例えば皮質脊髄路の神経、脊髄視床路の神経が該当する。  [0011] In this means, the axon is preferably a central axon, although not limited thereto. Here, the “central nervous system” refers to the brain and spinal cord, and is not limited to, for example, the corticospinal tract nerve and the spinal thalamic tract nerve.
発明の効果  The invention's effect
[0012] 以上により、新規な軸索再生促進剤を提供することができる。本軸索再生促進剤は 特に、中枢神経系の軸索の再生に有効である。この結果、脊髄等の中枢神経系に 損傷を受けた患者の治療に寄与することができる。  [0012] As described above, a novel axonal regeneration accelerator can be provided. This axonal regeneration accelerator is particularly effective for regeneration of central nervous system axons. As a result, it is possible to contribute to the treatment of patients with damage to the central nervous system such as the spinal cord.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0013] 本実施形態に係る軸索再生促進剤（以下「本軸索再生促進剤」という。）は、以下の [0013] The axon regeneration accelerator (hereinafter referred to as "the axon regeneration accelerator") according to this embodiment is as follows.
(a)乃至（d)の少なくともいずれかのペプチドを含有することを特徴の一つとする。 (a)配列番号 1に記載のアミノ酸配列からなるペプチド (b)配列番号 2に記載のアミノ酸配列からなるペプチド One of the characteristics is that it contains at least one of the peptides (a) to (d). (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2
(c)配列番号 3に記載のアミノ酸配列からなるペプチド  (c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3
(d)配列番号 4に記載のアミノ酸配列からなるペプチド  (d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4
[0014] 本軸索再生促進剤は、 RGMaタンパク質の一部を有するペプチドを有効成分とし ており、これによりニューロンにおける RGMaタンパク質の軸索成長阻害活性を中和 することが可能であり、軸索の再生を促進することができる。即ち、本軸索再生促進 剤は、中枢神経系を損傷した患者を治療するための医薬組成物として有用である。  [0014] This axon regeneration promoter contains a peptide having a part of the RGMa protein as an active ingredient, which can neutralize the axon growth inhibitory activity of the RGMa protein in neurons. Can be promoted. That is, the axonal regeneration promoter is useful as a pharmaceutical composition for treating a patient who has damaged the central nervous system.
[0015] 本軸索再生促進剤におけるペプチドは、上記の作用効果を奏する限りにおいて限 定されず、配列番号 1乃至 4に記載されたアミノ酸配列からなるペプチド以外に、これ らのアミノ酸配列に 1又は数個のアミノ酸を欠失、置換又は付加させたアミノ酸配列か らなるペプチドも採用可能である。なお、限定されるわけではないが、例えば置換とし ては、上記の作用効果が失われな!/、程度の化学的な修飾が施されたアミノ酸への置 換、ポリマーや糖鎖が付加されたアミノ酸への置換が挙げられる。また、限定されるわ けではないが、付加としては、例えば配列番号 1乃至 4に記載のペプチドの N末端側 又は C末端側の少なくとも一方に、製剤化を容易にするアミノ酸、融合タンパク質とし てペプチドを発現させるのに便利なアミノ酸、ペプチドの製造及び生成に便利なアミ ノ酸、を 1個又は数個付加することが挙げられる。  [0015] The peptide in the present axon regeneration promoter is not limited as long as it exhibits the above-described effects. In addition to the peptide consisting of the amino acid sequences described in SEQ ID NOs: 1 to 4, 1 Alternatively, a peptide consisting of an amino acid sequence in which several amino acids are deleted, substituted or added can also be used. Although not limited, for example, as a substitution, the above-mentioned effects are not lost! /, Substitution to an amino acid having a degree of chemical modification, addition of a polymer or a sugar chain. Substitution with other amino acids. Further, although not limited thereto, for example, as an amino acid or a fusion protein that facilitates formulation, at least one of the N-terminal side and the C-terminal side of the peptides shown in SEQ ID NOS: 1 to 4 can be added. The addition of one or several amino acids, which are convenient for expressing the peptide, and amino acids, which are convenient for producing and producing the peptide, are mentioned.
[0016] 本軸索再生促進剤は、上記記載のペプチドの他、薬学的に許容される通常の担体 、結合剤、安定化剤、賦形剤、希釈剤 (例えば蒸留水）、 pH緩衝剤 (例えばリン酸緩 衝生理食塩水）、崩壊剤、可溶化剤、溶解補助剤、等張剤等の各種調剤用配合剤 成分を含有することができる。  [0016] In addition to the above-mentioned peptides, the present axon regeneration promoter is an ordinary pharmaceutically acceptable carrier, binder, stabilizer, excipient, diluent (eg, distilled water), pH buffering agent. It can contain various preparation ingredients such as (for example, phosphate buffered saline), disintegrants, solubilizers, solubilizers, and isotonic agents.
[0017] 本軸索再生促進剤は、その使用形態に応じて、例えば、経口的に又は非経口的に 投与すること力 Sできる。経口的な投与としては、通常用いられる投与形態、例えば粉 末、顆粒、錠剤、カプセル剤、液剤、懸濁液、油剤、乳化剤等の投与形態を採用す ること力 Sできる。非経口的な投与としては、通常用いられる投与形態、例えば上記の 液剤、懸濁液等にしたものを直接損傷部位に投与する方法、注射等により投与する 形態を採用することができる。  [0017] The axon regeneration promoter can be administered orally or parenterally, for example, depending on the form of use. For oral administration, it is possible to adopt commonly used administration forms such as powders, granules, tablets, capsules, solutions, suspensions, oils, emulsifiers and the like. As parenteral administration, a commonly used administration form, for example, a method of administering the above-mentioned solution, suspension or the like directly to the injured site, a form of administration by injection or the like can be adopted.
[0018] 本軸索再生促進剤の投与量は、患者の体重、性別、神経損傷の程度、投与の方 法に応じて適宜選択されうるが、例えば非経口投与であって、損傷部位に直接投与 する場合、例えば成人 1日、 1損傷部位あたり上記ペプチドが lmg以上 lOOmg以下 の範囲で含まれていることが好ましぐより好ましくは 5mg以上 50mg以下である。な お、これ以外の非経口投与の場合、例えば注射の場合、この 10倍程度であることが 好ましい。 [0018] The dose of the axonal regeneration promoter is determined by the patient's weight, sex, degree of nerve damage, It can be selected appropriately according to the law.For example, when administered parenterally and administered directly to the site of injury, for example, adults per day, the above peptide should be included in the range of 1 mg to 10 mg per injury site. Is more preferably 5 mg or more and 50 mg or less. In the case of parenteral administration other than this, for example, in the case of injection, it is preferably about 10 times this.
[0019] また、本軸索再生促進剤に係るペプチドは、限定されることなく周知の方法により作 成すること力 Sできる。例えば、通常の化学的合成法、タンパク分子の酵素的分解法、 遺伝子組換技術等により製造することができる。  [0019] In addition, the peptide according to the present axon regeneration promoter can be produced by a known method without limitation. For example, it can be produced by an ordinary chemical synthesis method, an enzymatic degradation method of a protein molecule, a gene recombination technique, or the like.
[0020] 通常の化学的合成法としては、限定されるわけではないが、例えば、ペプチド合成 機を使用した固相合成法を好ましく用いることができる。また、限定されるわけではな いがこの方法により得たペプチドに対しては精製を行うことが好ましぐ限定されるわ けではないが、周知の精製方法、例えば、塩析法、限外ろ過法、逆相クロマトグラフィ 一法、イオン交換クロマトグラフィー法、ァフィ二ティークロマトグラフィー法等を好まし く用いること力 Sでさる。  [0020] The usual chemical synthesis method is not limited, but for example, a solid phase synthesis method using a peptide synthesizer can be preferably used. In addition, although not limited, it is not preferable to purify the peptide obtained by this method. However, well-known purification methods such as salting-out, Filtration, reversed-phase chromatography, ion exchange chromatography, affinity chromatography, etc. are preferably used.
[0021] 遺伝子組換技術としては、限定されるわけではないが、例えば、 目的とするアミノ酸 配列をコードする DNA断片を適当な発現ベクターに組み込み、この発現ベクターを 用いて微生物や動物細胞を形質転換し、この形質転換された微生物や動物細胞を 培養することによって目的のアミノ酸配列からなるペプチドを得る技術が採用できる。 なお、ここで用いられる発現ベクターとしては、限定されることなく周知のものを採用 すること力 Sでき、例えばプラスミド、ウィルスベクター等を用いることができる。また、限 定されるわけではないがこの方法により得たペプチドに対しては精製を行うことが好 ましぐ例えば、培養した培地から細胞抽出液又は培養上清を回収し、上記した精製 法を用いることが好ましい。  [0021] The gene recombination technique is not limited. For example, a DNA fragment encoding the target amino acid sequence is incorporated into an appropriate expression vector, and microorganisms and animal cells are transformed using this expression vector. A technique for obtaining a peptide having a desired amino acid sequence by culturing the transformed microorganism or animal cell after conversion can be employed. The expression vector used here is not limited, and a well-known one can be employed. For example, plasmids, virus vectors, and the like can be used. Although not limited, it is preferable to purify the peptide obtained by this method. For example, the cell extract or the culture supernatant is collected from the cultured medium, and the above-described purification method is used. It is preferable to use it.
[0022] (実験例）  [0022] (Experimental example)
本軸索再生促進剤の効果について実験を行い確認した。以下説明する。もちろん 、本発明が以下に示す実験例に狭く限定されるものではないことは言うまでもない。  Experiments were conducted to confirm the effect of this axon regeneration accelerator. This will be described below. Of course, it goes without saying that the present invention is not limited to the following experimental examples.
[0023] (1)ペプチドの合成 [0023] (1) Peptide synthesis
Custom Peptide Synthesis (¾igma Genosysパこより ^こ不すア^ノ酸目 歹 IJからなるペプチドを合成した。 Custom Peptide Synthesis (¾igma Genosys ペ プ チ ド A peptide consisting of IJ was synthesized.
[表 1]  [table 1]
P * ASa Asp S S Ly¾ Asn GSy C¾y Asp i.ys His Giy Ai¾ 凝錄 3、 以下 ί .¾ « p Λ \ と: )P * ASa Asp SS Ly¾ Asn GSy C¾y Asp i.ys His Giy Ai¾ Aggregate 3, ί .¾ « p Λ \ and:)
Asp Gin Αί.¾ ί¾；: ΛΓ¾ Aifi A Qin Pw Arg Arg (iS ! 4、 下 i' « p S j と:総) Asp Gin Αί.¾ ί¾ ;: ΛΓ¾ Aifi A Qin Pw Arg Arg (iS! 4, lower i '«p S j and: total)
[0024] (2) RGMa— CHO細胞の生成 [0024] (2) RGMa— Generation of CHO cells
次に、 Flp— InSystem (Invitrogen)を用い、かつ、この製造元の指針に従い、 R GMa発現細胞を作成した。 2種類の制限エンドヌクレアーゼを用いて pSecTag2ベ クタ一からシグナルペプチドを含む HA—RGMaフラグメントを調製し、これを pcDN A5FRT (Invitrogen)にライゲーシヨンした。このコンストラクト（pcDNA5FRT/lg κ leader/HA/RGMa)および pOG44を Flp— inCHO細胞にコトランスフエクショ ンし、ノヽィグロマイシン B (500 a g/ml ; Invitrogen)を含む培地で 2週間増殖させ た後、 HA— RGMaを安定に発現する細胞（以下「RGMa— CHO細胞」という。）を 得た。なお、 HA— RGMaの発現はウェスタンブロットおよび免疫細胞化学を用いて 免疫的に検出することにより確認した。  Next, RGMa-expressing cells were prepared using Flp-InSystem (Invitrogen) and following the manufacturer's guidelines. The HA-RGMa fragment containing the signal peptide was prepared from the pSecTag2 vector using two restriction endonucleases and ligated to pcDN A5FRT (Invitrogen). This construct (pcDNA5FRT / lgκ leader / HA / RGMa) and pOG44 were co-transformed into Flp-inCHO cells and grown for 2 weeks in a medium containing Neugromycin B (500 ag / ml; Invitrogen). Cells stably expressing HA-RGMa (hereinafter referred to as “RGMa-CHO cells”) were obtained. The expression of HA-RGMa was confirmed by immunodetection using Western blot and immunocytochemistry.
[0025] (3)軸索成長アツセィ  [0025] (3) Axon Growth Atsey
出生後 7— 9日（P7— 9)の仔ラットからの小脳顆粒細胞をトリプシン処理（PBS中 0 . 25%トリプシン、 37°C、 15分間）により分離し、血清含有培地に再懸濁し、粉砕し、 PBSで 3回洗浄した。チャンバースライド（Lab Tek II； Nunc)中の RGMa— CHO 細胞又は対象 CHO細胞のコンフルェント単層上にニューロンを播種した。中和アツ セィのために Peplまたは Pep2をそれぞれ示される濃度（1 M、 4 M、 10〃 M) で培養物に加えた。培養物を無血清 DMEM/F12培地で 24時間成長させた。次 に、細胞を 4% (wt/vol)パラホルムアルデヒドで固定し、ニューロン特異的 13チュー ブリン III蛋白質を認識するモノクローナル抗体（Tujl ) ( l : 1000 ; Covance)を用い て免疫染色した。次に、各 βチューブリン IIIポジティブのニューロンについて、最長 の神経突起の長さを測定した。  Cerebellar granule cells from pup rats 7-9 days after birth (P7-9) were isolated by trypsinization (0.25% trypsin in PBS, 37 ° C, 15 minutes), resuspended in serum-containing medium, Milled and washed 3 times with PBS. Neurons were seeded on confluent monolayers of RGMa-CHO cells or subject CHO cells in chamber slides (Lab Tek II; Nunc). Pepl or Pep2 was added to the cultures at the indicated concentrations (1 M, 4 M, 10 M) for neutralization assays. Cultures were grown in serum-free DMEM / F12 medium for 24 hours. Next, the cells were fixed with 4% (wt / vol) paraformaldehyde and immunostained with a monoclonal antibody (Tujl) (l: 1000; Covance) that recognizes the neuron-specific 13 tubulin III protein. Next, the length of the longest neurite was measured for each β-tubulin III positive neuron.
[0026] この結果を図 1に示す。なお図中" control"は対象 CHO細胞の場合、 "RGM— " は RGMタンパク質が発現している場合（以下「RGM— CHO細胞」という。）、" pep l 1 μ Μ"は pep lを 1 μ Μ加えた場合、 "pepl 4 μ Μ"は pep lを 4 μ Μ加えた場合、 "pep l Ι Ο Μ"は pep lを Ι Ο Μカロえた場合、" pep2 1 M"は pep2を 1 Mカロ えた場合、 "pep2 4 μ Μ"は pep2を 4 μ Μカロえた場合、 "pep2 10 μ Μ"は pep2を 1 β Μ加えた場合、をそれぞれ示している。なお、縦軸はニューロンあたりの最長の 神経突起の平均長さで表され、データは 3回の独立した実験の平均土 S . Ε. Μ.で ある。 [0026] The results are shown in FIG. In the figure, “control” indicates the target CHO cell, “RGM—” indicates that RGM protein is expressed (hereinafter referred to as “RGM—CHO cell”), “pep l 1 μΜ” indicates 1 pep l. When μΜ is added, “pepl 4 μΜ” is added when 4 μΜ of pep l is added. "pep l Ι Ο Μ" means pep l is Ι Ο Μ Μ p "pep2 1 M" means pep2 is 1 M 、 “10 μΜ” indicates the case of adding 1 βΜ of pep2. The vertical axis is the average length of the longest neurite per neuron, and the data is the average soil S. .. Μ. Of three independent experiments.
[0027] 図 1に示されるように、 Pep l及び Pep2は、用量依存的に RGMaの阻害効果を有 意に逆転させること力できる。このことは、これらのペプチドがインビトロで RGMaに対 して中和試薬として機能していることを示しており、これらペプチドを有効成分とする ことで軸索成長促進剤として利用可能であることが確認できた。  [0027] As shown in FIG. 1, Pep l and Pep2 can potently reverse the inhibitory effect of RGMa in a dose-dependent manner. This indicates that these peptides function as neutralizing reagents for RGMa in vitro, and that these peptides can be used as axon growth promoters by using them as active ingredients. It could be confirmed.
産業上の利用可能性  Industrial applicability
[0028] 本発明に係る軸索再生促進剤は、中枢神経系を損傷した患者のための治療剤とし て産業上の利用可能性がある。 [0028] The axonal regeneration promoter according to the present invention has industrial applicability as a therapeutic agent for a patient who has damaged the central nervous system.
図面の簡単な説明  Brief Description of Drawings
[0029] [図 1]図 1は，本発明のペプチドによる RGMaタンパク質の阻害効果を示す図である  [0029] FIG. 1 is a diagram showing the inhibitory effect of RGMa protein by the peptide of the present invention.

Claims

請求の範囲 The scope of the claims
[1] 以下（a)乃至（d)の少なくとも!/、ずれかのペプチドを有効成分として含有する軸索 再生促進剤。  [1] An axonal regeneration promoter containing at least one of the following (a) to (d) as an active ingredient:!
(a)配列番号 1に記載のアミノ酸配列からなるペプチド  (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1
(b)配列番号 2に記載のアミノ酸配列からなるペプチド  (b) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2
(c)配列番号 3に記載のアミノ酸配列からなるペプチド  (c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3
(d)配列番号 4に記載のアミノ酸配列からなるペプチド  (d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4
(e)配列番号 1乃至 4のいずれかに記載のアミノ酸配列において 1若しくは数個のァ ミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、 RGMaタンパク 質の軸索成長阻害活性を中和する機能を有するペプチド  (e) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 to 4, and the RGMa protein has an axon growth inhibitory activity Peptides with a function to neutralize
[2] 前記軸索は、中枢神経系の軸索である請求項 1記載の軸索再生促進剤。  2. The axonal regeneration promoter according to claim 1, wherein the axon is a central nervous system axon.
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