WO2008038599A1

WO2008038599A1 - Axonal regeneration promoter

Info

Publication number: WO2008038599A1
Application number: PCT/JP2007/068449
Authority: WO
Inventors: Toshihide Yamashita; Masayoshi Suda
Original assignee: National University Corporation Chiba University
Priority date: 2006-09-25
Filing date: 2007-09-21
Publication date: 2008-04-03
Also published as: JPWO2008038599A1

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

Specification

Axon regeneration promoter

Technical field

[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.

Background art

[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] 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] 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] 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

Problems to be solved by the invention

[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] Accordingly, an object of the present invention is to provide a novel axonal regeneration accelerator that solves the above-mentioned problems.

Means for solving the problem

[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) 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) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3

(d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4

(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.

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] 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.

The invention's effect

[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.

BEST MODE FOR CARRYING OUT THE INVENTION

[0013] 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). (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) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3

(d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4

[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] 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] 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] 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] 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] 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] 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] 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] (Experimental example)

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) Peptide synthesis

Custom Peptide Synthesis (¾igma Genosys ペプチド A peptide consisting of IJ was synthesized.

[table 1]

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, lower i '«p S j and: total)

[0024] (2) RGMa— Generation of CHO cells

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) Axon Growth Atsey

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] 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] 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.

Industrial applicability

[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.

Brief Description of Drawings

[0029] FIG. 1 is a diagram showing the inhibitory effect of RGMa protein by the peptide of the present invention.

Claims

The scope of the claims

[1] An axonal regeneration promoter containing at least one of the following (a) to (d) as an active ingredient:!

(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) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3

(d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4

(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. The axonal regeneration promoter according to claim 1, wherein the axon is a central nervous system axon.