JPH05262663A - Nerve growth factor production accelerator - Google Patents

Abstract

PURPOSE:To obtain an accelerator for producing the nerve growth factor (NGF) containing malformins known as compounds having action to cause heteromorphism in plants. CONSTITUTION:The accelerator includes at least one from malformin A1 of formula I, malformin A2 of formula II, malformin A3 of formula III and malformin A4 of formula IV as an active ingredient. Said NGF production accelerator increases the capacity of NGF production in the target tissue, thus being efficacious in prophylaxis and treatment for nervous diseases caused by denaturation and deciduation of nerve cells such as Alzheimer's dementia and in restoration of damaged peripheral nerves. The compounds can be produced through fermentation, for example, by culturing Aspergillus sp. H143 (FERM-P-12673) and isolating it from the supernatant.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to malformin A1 and A.
The present invention relates to a nerve growth factor (hereinafter abbreviated as NGF) production promoter containing at least one of 2, A3 and A4 as an active ingredient. More specifically, the agent according to the present invention promotes the production and secretion of NGF, thereby preventing and treating Alzheimer's disease and central degenerative diseases such as dementia associated with cerebral blood vessel obstruction and general aging. Further, in the peripheral nervous system, it is useful as a therapeutic agent for functional recovery by repair of damaged site and a therapeutic agent against functional deterioration due to nerve disconnection or damage.

[0002]

2. Description of the Related Art NGF has a high synthetic ability in target tissues in both the exchange and sensory nervous systems of the peripheral nervous system, such as the ventricle, submandibular gland, and skin, which elongate nerve fibers and biosynthesize neurotransmitters. It is known that it has the effect of promoting the differentiation of nerve cells and differentiating nerve cells. In the central nervous system, it is revealed that it acts specifically on the cholinergic nervous system and is secreted synthetically in the hippocampal neocortical region which is the target cell like the peripheral system. It promotes differentiation and has a trophic factor effect of maintaining survival on nerve cells.

This cholinergic nervous system is important for memory and learning. For example, in Alzheimer-type dementia, degenerative loss of cholinergic neurons is observed (Annu. Rev.
Neurosci., Volume 3, 77 (1980)). In animal experiments, for example, NGF was introduced into the ventricles of aging rats with impaired learning ability.
It has been reported that continuous learning infusion improves memory learning ability (Nature, 329, 65-68 (1987)). Also,
Neuronal degeneration is prevented by NGF in Alzheimer's disease model rats in which the nervous system projecting from the septum to the hippocampus is damaged (Science, 235, 214-216 (198).
7)), and that ischemic neuronal damage in the hippocampus can also be prevented (Medical history, 145, 579-580).
(1988)).

Therefore, NGF is effective for central neurodegenerative diseases such as dementia and neuropathy and degenerative diseases, and it is expected that NGF improves the brain function lowered by these diseases. Further, it is also effective in repairing the damaged nervous system in the peripheral system, and is expected to improve and promote the functional decline due to the damage of the nervous system.

[0005]

Thus, NGF is expected to be an effective substance against various neurological diseases. However, NGF is a protein having a molecular weight of 10,000 or more, and it is considered that it is extremely difficult to directly administer NGF due to the difficulty of migration into the brain by peripheral administration and the problem of antigenicity. Therefore, in place of direct administration of NGF, development of a low molecular weight compound having an action of promoting NGF production in specific tissues is desired. As a substance having an NGF production promoting action, catechol derivatives such as catecholamine have been known so far (J. Biol. Che.
m., 261, 6039-6047 (1986), JP-A-63-83020), but the activity of these compounds cannot be said to be sufficient, and more effective new compounds are desired.

[0006]

Means for Solving the Problems As a result of earnest search for a new substance for promoting NGF production in order to solve the above problems, the present inventors have found that malformin (Handbook of Toxic Fungal Metabolite), which is known as a compound having a plant heteromorphism
, Academic Press, NY1981, 671, 673, 675, 678) have a strong NGF production promoting action, and have completed the present invention. That is, the present invention relates to malformin A1, A2,
The present invention relates to a nerve growth factor production promoter containing at least one of A3 and A4 as an active ingredient.

The above-mentioned malformins A1 and A used in the present invention
2, A3 and A4 are each represented by the following structural formula.

[0008]

[Chemical 1]

[0009]

[Chemical 2]

[0010]

[Chemical 3]

[0011]

[Chemical 4]

In the present invention, the above-mentioned malformin A1,
A2, A3 and A4 may be used alone or in appropriate combination of two or more kinds. This malformin is N
When used as a GF production promoter, it can be manufactured and administered in a wide variety of oral and parenteral dosage forms. When manufacturing a pharmaceutical composition from this malformin, the pharmaceutically acceptable carrier may be solid or liquid.

[0013] Orally administered drugs are usually in the form of powders, tablets, emulsions, capsules, granules, liquids and the like. Specific examples of excipients for internal use include powders and other powders for internal use. Examples of the excipient include lactose, starch, dextrin, calcium phosphate, calcium carbonate, synthetic and natural aluminum silicate, magnesium oxide, aluminum hydroxide, magnesium stearate, sodium bicarbonate, etc. Is
Examples include water, glycerin, and simple syrup.

The parenteral administration forms include injections and the like. In the case of injections, sterile aqueous or non-aqueous solutions, suspensions, emulsions and the like are included. Examples of the aqueous solution and suspension include distilled water for injection and physiological saline. Examples of non-water-soluble solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and polysorbate 80 (trade name). Such compositions may further contain adjuvants such as preservatives, lubricants, emulsifiers and dispersants. These can be sterilized by, for example, filtration with a bacteria-retaining filter, blending of a bactericide, or irradiation. In addition, these can be used by producing a sterile solid composition and dissolving it in sterile water or a sterile solvent for injection before use.

Each of the above-mentioned preparations can be prepared according to a conventional method. When the compound represented by the structural formula of the present invention is used as a preparation, it is usually orally or parenterally administered to an adult at a rate of 0.1 to 100 mg / day.

The NGF production-promoting agent of the present invention improves the NGF production ability in target tissues to prevent or treat neurological diseases caused by degeneration or loss of nerve cells such as Alzheimer type dementia, and further damage. It is effective for promoting recovery of peripheral nerve tissue.

[0017]

EXAMPLES Examples of the present invention will be described in detail below, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded.

Reference Example Malformin A1, A2, A3, A4 can be produced by a fermentation method. For example, Aspergillus sp. H143 (FERM P-12673) is cultivated. It can be obtained by the operation shown in. That is, the above-mentioned strain is used for example with dextrin 2.0.
%, Glucose 1.0%, peptone 1.0%, corn steep liquor 0.5%, CaCO ₃ 0.2% (pH 7.0) in a medium containing 4%, and was obtained by culturing for 4 days. The culture supernatant is extracted with a solvent such as ethyl acetate. Malformin was prepared by adding this ethyl acetate extract to silica gel (developing solvent: 10%).
Methanol-containing chloroform), Toyopearl HW-40 (trade name) (developing solvent: methanol) and Sephadex LH-
It can be purified by chromatography using a carrier such as 20 (trade name) (developing solvent: methanol). It is preferable to use HPLC in order to obtain higher purity malformin. For example, Wako Pack 5C18 (trade name) (7.5 mm x
250 mm) and can be separated and eluted with 40% acetonitrile-water containing 0.5% TFA. The retention time was 19.9 minutes for Malformin A1, 14.7 minutes for A2, and A
3 is 23.8 minutes and A4 is 13.3 minutes.

Example 1 In order to examine the NGF production promoting effect of the present invention, the following experiment was carried out using LM cells. LM cells were cultured and maintained in 199 medium containing 0.5% bactopeptone, and when used in the test, they were seeded on a 96-well culture plate so that the cell density was 2 × 10 ⁴ cells / cm ^2, and CO 37 in ₂ incubators
When the cells were cultivated at ℃ for 5 days and became almost confluent,
100 μl of 199 medium containing 0.5% bovine serum albumin in which the test product was dissolved at a predetermined concentration was added. After 24 hours, the amount of NGF contained in the culture supernatant was measured. Enzyme-linked immunosorbent assay (Korsching and Thoenen, Proc. Natl. Acad. Sci. USA, 80
Vol. 3513-3516, 1983). Anti-mouse βNGF antibody was adsorbed, and 10 μl of the culture supernatant or standard βNGF was injected into each well of the 96-well microtiter plate after the blocking operation, and the mixture was left at 4 ° C for 15 to 18 hours. Remove the standard βNGF,
After washing each hole, β-galactosidase-labeled anti-mouse βN
Dispense 50 μl of GF antibody solution (0.05 U / ml) into each well and incubate at 4 ° C for 1
Let stand for 5-18 hours. After removing the β-galactosidase-labeled anti-mouse βNGF antibody solution, each hole was washed and
100 μl of -methylumbelliferyl-β-D-galactopyranoside solution was dispensed into each hole and left at 37 ° C for 2 hours, and then excited by an excitation light 365 by a microplate reader.
nm, fluorescence was measured at 450 nm. The measurement result was expressed as a magnification against the NGF concentration in the culture supernatant containing no test product. As a result, as a test product, malformin A1, A2,
As shown in FIG. 1 and FIG. 2, the fold increase in NGF concentration induced by production using A3 and A4 showed a remarkable NGF production promoting ability of 3 to 6 times at a concentration of 1 to 5 μM.

[0020]

INDUSTRIAL APPLICABILITY The nerve growth factor production-promoting agent of the present invention is used for the prevention and treatment of neurological diseases caused by degeneration and loss of nerve cells such as Alzheimer's dementia, and for promoting recovery of damaged peripheral nerve tissue. It is valid.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the concentrations of malformin A1, A2, A3, A4 and the increasing rate of NGF concentration.

FIG. 2 is a graph showing the relationship between the concentrations of malformin A1, A2, A3, A4 and the increasing rate of NGF concentration.

Continuation of front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI Technical display location (C12P 21/04 C12R 1:66) C07K 99:00 (72) Inventor Masanobu Munakata Okabata, Yaizu, Shizuoka Address: Sapporo Lobby Co., Ltd., Pharmaceutical Development Laboratory (72) Inventor, Tomiji Kobayashi, Okabata, No. 10, Okabata, Yaizu City, Shizuoka Prefecture

Claims (1)

[Claims] 1. Malformin A1, A2, A3 and A
A nerve growth factor production-promoting agent containing at least one of 4 as an active ingredient.