JP3167714B2 - Nerve cell protective agent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a nerve cell protective agent, and more specifically, to protect nerve cells from disorders and the like by acting on the nerve cells as growth trophic factors, and The present invention relates to a medicament useful for preventing or treating dysfunction.

[Conventional technology]

Factors acting on the differentiation, growth and function maintenance of central nervous system cells have been found as nerve growth trophic factors from cultures and extracts of various tissues and cells. During growth of the nervous system, these factors elongate axons from nerve cells, properly induce axons to target tissues to be innervated, and only the necessary number of cells that have multiplied and proliferated. Is thought to play a role in surviving and eventually establishing innervation through synapse formation.

In other words, nerve growth factor (NGF) and brain-derived neurotrophic factor are produced in the brain, and work to maintain the functions of central cholinergic neurons (neural cells) and sensory neurons of the dorsal root ganglion, respectively. I think that the. When an antibody against NGF is administered to a neonatal animal or a fetus, remarkable atrophy of the sympathetic ganglion and disruption of the sensory nerve occur, respectively. NG
When F was administered to the brain of newborn rats, nerve fibers were found to extend from the exchange ganglia and enter the brain and spinal cord. In addition, it acts on cholinergic neurons in the septum of the brain to cause intracellular choline acetyltransferase (CTA)
It has been reported to increase the activity and acetylcholinesterase activity.

On the other hand, as a cause of Alzheimer's dementia, cholinergic neuropathy is assumed due to impairment of the basal ganglia of Meynert, and a remarkable decrease in CAT and acetylcholinesterase activity in the cerebral cortex has been reported. Therefore, it is expected that NGF is effective in treating Alzheimer's disease. In fact, it is known that cutting the septum-hippocampus projection pathway can reduce about half of the cholinergic neurons by administering NGF. Furthermore, there is a report that administration of NGF into the brain improved atrophy of cholinergic neurons and impaired memory in aged rats. In various ways, NGF has an action as a differentiation promoting factor in the development process of the nervous system and an action to maintain the function after maturation, and is promising as an agent for preventing or treating dementia.

[Problems to be solved by the invention]

However, despite such nerve growth trophic factors such as NGF, which have neuroprotective effects such as promotion of differentiation of the nervous system and maintenance of functions, they are present only in very small amounts in living organisms and are very isolated. Due to the difficulty, these factors could not be used directly as pharmaceuticals.

Accordingly, an object of the present invention is to provide a new medicine which can be obtained in large quantities and acts as a growth factor of the nervous system.

[Means for solving the problem]

Under such circumstances, the present inventors have conducted intensive studies to solve the above-mentioned problems, and found that calfobindin I, which is known as a human placenta-derived anticoagulant,
(Calphobindin I; J. Biochem. 106, 43-49 (1989), Pla
cental and Endometrial Proteins, p165-168 (198
8); hereinafter referred to as CPB-I), acting as a neuronal growth trophic factor and having an excellent neuroprotective effect, and completed the present invention.

That is, the present invention relates to human CPB-I or recombinant C
It is intended to provide a neuroprotective agent comprising PB-I as an active ingredient.

CPB-I, which is an active ingredient of the nerve cell protective agent of the present invention, is widely distributed in tissues and secretions in vivo, including the human placenta (Chem. Pharma. Bull, 38, 1957-1960, 1990). In a cell, it is a substance having a physiological activity such as an anticoagulant action present in the cytoplasm.

CPB-I can be extracted from human or animal organs (JP-A-62-174023). The CPB-I thus obtained has the following properties.

Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state) 34,000 ± 2,000 Isoelectric point (isoelectric point column electrophoresis using ampholite) 4.7 ± 0.1 Stability (a) Loss by heating at 50 ° C for 30 minutes Active (b) Stable at pH 4-10 (c) Stable at 37 ° C, 30 minutes in plasma Effect on blood coagulation system (a) Extend calcium recoagulation time (b) Extend prothrombin time (c) Activated partial thromboplastin Extend time Amino acid analysis Amino acid analysis shows aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, 1 /
2 The presence of cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine is observed.

Further, CPB-I can be expressed in Escherichia coli by a gene recombination technique using a gene fragment encoding human or animal CPB-I (Japanese Patent Laid-Open No. 64-20095).
CPB-I thus obtained by genetic recombination (hereinafter referred to as “recombinant CPB-I”) has the following amino acid sequence.

The human CPB-I or the recombinant CPB-I used in the present invention does not necessarily have to have the same amino acid sequence as described above. That is, CPB-I has a structure in which one or more amino acids are added or truncated from the N-terminus or C-terminus, and one or more amino acids in the structure of natural CPB-I are different from those of other CPB-I. Those having a structure substituted by an amino acid can also be used as long as they have the activity of CPB-I.

Such CPB-I and recombinant CPB-I act as growth trophic factors for nerve cells as shown in Examples below, and various diseases based on dysfunction of nerve cells, such as senile dementia such as Alzheimer's dementia and the like. It is useful for prevention and treatment of cerebrovascular disorders and the like. CPB-I is a protein derived from a living body and has an LD ₅₀ value of 200 mg / kg when administered intravenously to mice.
As described above, it is a highly safe drug showing almost no toxicity to the human body.

Examples of the method for administering the neuroprotective agent of the present invention include intravascular administration to arteries and veins, intramuscular administration, and direct administration to dysfunctional sites. In addition, dosage, age, weight,
Although it depends on the symptoms and administration method, it is usually 0.0001 to 10 as CPB-I or recombinant CPB-I per adult per day.
0 mg is preferred.

When administering the neuroprotective agent of the present invention, CPB-I
Or recombinant CPB-I as it is, or serum albumin, gelatin, artificial cerebrospinal fluid, mannitol,
It may be used in the form of a mixture with a phosphate buffer, saline or the like, and the form may be a lyophilized product or a liquid.

〔The invention's effect〕

The nerve cell protective agent of the present invention comprises the active ingredient CPB-I.
In addition, recombinant CPB-I acts on nerve cells as a growth trophic factor, is easily available in large quantities, and is used for various diseases based on dysfunction of nerve cells, such as senile dementia such as Alzheimer-type dementia and cerebrovascular disease. It is useful for prevention and treatment of disorders and the like.

〔Example〕

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

In this example, CPB-I was disclosed in
No. 1 obtained in Example 1 above, and the recombinant CP
For BI, the one obtained in Example 4 of JP-A-64-20095 was used.

Example 1 (1) Primary culture of fetal rat cerebral cortex A cerebral hemisphere was removed from a rat fetus on the 16th day, and after minced,
Digest with trypsin (0.1%) and DNase (0.01%) and pass the tissue through a stainless mesh (250 μm). Cells were seeded at a concentration of 5 × 10 ² cells / mm ² on 24-well plates coated with poly-L-lysine. The medium was 1% fetal bovine serum, 0.5 mg / ml sodium bicarbonate, 5 mg / ml glucose, 50
U / ml penicillin, 100 μg / ml streptomycin, 1
0 μg / ml insulin, 10 μg / ml transferrin, 10 μg / ml selenium (ITS premix.Colab), and 10 μg / ml
Minimum Bssentia containing 0 μM putrescine (Miles)
l Medium (MEM) was used. Twenty-four hours after seeding, CPB-I was added, the culture was continued for another four days, and the surviving nerve cells were measured under a microscope.

(2) Neurotrophic factor activity of human CPB-I 10 ^-8 to 10 ^-6 g / ml of CPB-I was added to the above measurement system, and the number of surviving neurons was measured. As shown in FIG. , 3
A nerve cell survival effect was observed by adding CPB-I at a concentration of × 10 ⁻⁸ g / ml or more. In particular, the effect was remarkably recognized at 10 ^{−7 to} 3 × 10 ⁻⁷ g / ml.

When a rabbit polyclonal antibody against human CPB-I was added to this system, this neuronal trophic factor activity was lost.

The measurement of neurotrophic factor activity was measured using primary culture of rat cerebral cortex during the fetal period.

Example 2 Formulation for topical injection: CPB-I 1 to 100 mg physiological saline, appropriate amount, total volume 10 ml Example 3 Formulation for intravenous injection: CPB-I 1 to 100 mg Ringer's solution, appropriate volume, total volume 10 ml

[Brief description of the drawings]

FIG. 1 shows the amount of CPB-I added and the number of viable neurons (cells / m
It is a drawing showing the relationship with m ² ).

Continued on the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/00 A61P 25/00 A61P 25/28 C07K 14/47 ZNA C12N 15/09 CAPPLUS (STN) MEDLINE (STN) BIOSIS (STN) EMBASE (STN)

Claims (2)

(57) [Claims] A neuroprotective agent comprising human calfobindin I or recombinant calphobindin I as an active ingredient. 2. The method according to claim 1, which is used as an anti-dementia agent.
The nerve cell protective agent according to the above.