JPH07206700A - Agent for suppressing death of nerve cell - Google Patents

Abstract

PURPOSE:To obtain an agent for suppressing death of nerve cell containing a specific peptide as an active component, having action to suppress the death of nerve cell caused by the exposure to high-concentration glutamic acid, free from restriction on the administration timing of the drug and useful as an agent for the prevention and treatment of dementia, etc., induced by hypoxic disorder, etc. CONSTITUTION:This agent for suppressing the death of nerve cell contains a peptide having the amino acid sequence of formula as an active component. Conventional agent (e.g. glutamic acid receptor antagonist) for preventing the death of nerve cell caused by glutamic acid is ineffective unless the agent is administered before the contact of the nerve cell with glutamic acid and, accordingly, the administration timing is restricted. In contrast with the conventional agent, this suppressing agent is effective even by administering after the contact with glutamic acid to eliminate the restriction on the administration timing and, accordingly, the agent is useful in practical field.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a nerve cell death inhibitor. Specifically, the present invention relates to a nerve cell death inhibitor containing a known peptide known to exhibit neurotrophic factor-like activity as an active ingredient.

[0002]

2. Description of the Related Art Death of nerve cells in the brain gradually progresses after adulthood due to natural aging or pathological causes and causes senile dementia, which is a major problem in the aging society of Japan. The cause of the death of these nerve cells is cerebrovascular disorder, such as cerebral ischemia associated with cerebral thrombosis and cerebral hemorrhage, hypoxic injury causes excessive release of glutamate, and this excess glutamate induces death of nerve cells. Is believed to be. It is known that glutamate-induced nerve cell death is prevented by antagonists of glutamate receptors, calcium channel blockers, calcium chelator, and the like. It is expected that prevention of cerebral nerve cell death will lead to a fundamental treatment for senile dementia, which is currently only symptomatic or palliative.

On the other hand, the brain is composed of many nerve cells and glial cells, which interact with each other to express and maintain complex functions. Neurotrophic factors are important factors involved in the differentiation of nerve cells and maintenance of their functions.
There are proteinaceous factors collectively called (neurotrophic factors), and it is considered that when the brain is injured, the production of such neurotrophic factors is increased to perform some repair. In recent years, in the central nervous system, nerve growth factor (NG
F) and several neurotrophic factors that make up the family have been discovered (Neuron Advancement, Vol. 34, No. 4, 515-515).
523, August 1990).

The present inventors previously proposed a novel peptide having neurotrophic factor activity in order to elucidate the mechanism of functional expression, maintenance and repair of the brain (Japanese Patent Laid-Open No. Hei 5 (1999) -58).
-1098).

[0005]

However, conventional drugs that prevent neuronal cell death caused by glutamate are not effective unless the drug is administered before the nerve cells come into contact with glutamate. There was a timely limitation on drug administration, and it was mentioned as a problem for practical use.

[0006]

In order to solve the above problems, the present inventors have searched various substances, and as a result, the novel peptide previously proposed by the present inventors showed that the differentiation of nerve cells and their functions. The inventors have found that not only the neurotrophic factor activity involved in the maintenance of erythrocyte but also the activity of suppressing nerve cell death due to exposure to high-concentration glutamate, the present invention has been completed.

That is, the gist of the present invention is to provide SEQ ID NO: 1 in the sequence listing.
A neuronal cell death inhibitor comprising the peptide represented by the amino acid sequence described in 1. as an active ingredient. Hereinafter, the present invention will be described in detail. A peptide serving as an active ingredient in the neuronal cell death inhibitor of the present invention (hereinafter referred to as "the present peptide").
Consists of 13 amino acid residues described in SEQ ID NO: 1 in the sequence listing. Such a peptide is described as a peptide having neurotrophic factor-like activity in JP-A-5-1098, and can be easily produced according to the method described in that publication.

When the neuronal cell death inhibitor of the present application is used as a pharmaceutical composition, a pharmaceutically acceptable solid or liquid carrier such as an excipient, a diluent or other auxiliary agent is appropriately added. In addition, it can be formulated. The dosage form can be administered orally or parenterally. The amount of the active ingredient in the composition is appropriately determined depending on the age, pathological condition, symptom and the like. For example, when the peptide is allowed to act on the culture solution of nerve cells, the nerve concentration is extremely low at about 1 to 10 ng / ml. Cell death can be suppressed.

The neuronal cell death inhibitor of the present invention not only exhibits an excellent neuronal cell death inhibitory action, but is also known as a glutamate receptor which is conventionally known to prevent neuronal cell death caused by glutamate. Antagonists, calcium channel blockers, calcium chelator, etc.
Inhibition of the nerve cell death of the present invention comprising the peptide as an active ingredient, whereas the effect is not observed unless the drug is administered before the nerve cells come into contact with glutamate The agent is effective even if it is administered after being brought into contact with glutamic acid, and is practically useful because there is no time limitation of drug administration.

[0010]

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples unless it exceeds the gist. Example 1 Measurement of inhibitory action on nerve cell death The present peptide used to measure the inhibitory action on nerve cell death was obtained by the method described in JP-A-5-1098.
In addition, the nerve cells used for the measurement were 1.5 to 1.5 in a glass dish having a diameter of 15 mm, coated with polyethyleneimine, of hippocampal nerve cells obtained by treating papilla with 18-day pregnant embryos of Wistar rats. 2.5 x 10 ⁵
Seeding at a density of cells / cm ² 5% fetal bovine serum and 5%
It was obtained by culturing for 7 days in Dulbecco's modified Eagle's (DME) medium supplemented with heat-inactivated horse serum.

The obtained nerve cells were photographed and recorded under a microscope at an appropriate portion of the cultured cells before the test was started. After that, the nerve cells were sequentially treated in the following medium under the temperature condition of 37 ° C. 1) Serum-free DME medium (5 minutes) 2) Serum-free DME medium supplemented with 1.2 mM calcium chloride and 1 mM glutamate monosodium salt (30 minutes) 3) To wash out glutamate Serum-free DME medium (5 minutes) 4) Serum-containing DME medium (24 hours) At this time, in order to measure the action of the peptide, 50 ng / ml of the peptide was added to each of the media 1) to 4) in advance. I'll do it. In addition, 1.
System without addition of 2 mM calcium chloride and 1 mM glutamic acid monosodium salt, and media 1) -4)
It was also carried out for a system in which the present peptide was not added to any of the above.

After the treatment in the medium 4) for 24 hours in each system, the same field of view as before the treatment was photographed,
The number of surviving nerve cells was counted, and the survival rate was calculated from the counting results before and after the start of treatment. The experiment was repeated 4 times in independent dishes and the Mean ± SE was calculated. The results are shown in Figure 1. From this result, it was found that about 60% of the neuronal cell death was suppressed in the system in which the peptide (50 ng / ml) was added to the media 1) to 4).

Example 2 Effect of timing of addition of the peptide on inhibitory effect In Example 1, instead of adding the peptide to each of the media 1) to 4), 0, 30,
After 90 and 270 minutes, 50 ng / ml of this peptide was added. The results are shown in Fig. 1. It was found that the present peptide is effective in suppressing neuronal cell death even after administration of 270 minutes (4 hours and a half) after neuronal cell exposure to glutamate.

Example 3 Effect of the Concentration of the Present Peptide on the Inhibitory Action In Example 1, instead of adding 50 ng / ml of the present peptide to each of the mediums 1) to 4), each of the mediums 1) to 4) had a concentration of 0. Neurons were treated with the addition of 1, 1.0, 10, 100 ng / ml. Results are shown in FIG. From this result, it was found that this peptide halves the neuronal cell death due to glutamate even at a low concentration of about 1 ng / ml.

[0015]

INDUSTRIAL APPLICABILITY The nerve cell death inhibitor of the present invention has no timely limitation of drug administration and cerebrovascular disorder, as compared with the conventional drugs for preventing nerve cell death caused by glutamate.
For example, it is useful as a prophylactic or therapeutic drug for dementia caused by cerebral ischemia associated with cerebral thrombosis, cerebral hemorrhage, hypoxia and the like.

[0016]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin organism name: Rat sequence Glu Ala Leu Glu Leu Ala Arg Gly Ala Ile Phe Gln Ala 1 5 10

[Brief description of drawings]

FIG. 1 is a drawing showing the inhibitory effect of rat peptide hippocampal neuronal cell death by the addition of this peptide. In the figure, the ordinate represents the survival rate (%) of the cells, and in the abscissa, A indicates that the medium did not contain glutamic acid and the peptide, and B indicates that the medium contained neither glutamic acid nor the peptide. In the case of C, C represents the case where glutamic acid and the present peptide were added to the medium. In addition, the addition time represents the time when the present peptide was added after the start of the treatment of the medium 4).

FIG. 2 is a drawing showing the inhibitory effect on rat hippocampal neuronal cell death by the addition of this peptide, varying the amount added. In the figure, the ordinate represents the survival rate (%) of the cells, and in the abscissa, A represents the case where glutamic acid and the present peptide were not added to the medium, and B represents that the glutamic acid was added to the medium,
The case where this peptide was not added is shown. Moreover, the addition amount represents the addition amount of the present peptide to the media 1) to 4).

Claims (1)

[Claims] 1. A nerve cell death inhibitor comprising a peptide represented by the amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing as an active ingredient.