JP2001031572A - Medicine for central nervous neurocladism containing dictyosterol as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicine having excellent neurocladism action on central nervous cells, especially aged central nervous cells and useful as a therapeutic agent including an antidemential agent for neurodegenerative diseases by including a compound derived from a specific cellular mucosa. SOLUTION: This medicine comprises dictyosterol of the formula as an active ingredient. The compound of the formula can be obtained by culturing a cellular mucosa, extracting the compound of the formula which is intracellularly present with an organic solvent after finishing the culture, isolating the compound of the formula in the extract from the crude product by using a usually known purification method and washing the isolated compound with an organic solvent. The compound of the formula is preferably produced by culturing the cellular mucosa for 7-10 days by agar plate culturing method. The culturing temperature is suitably 20-25 deg.C. A form and a pathway which are pharmaceutically hitherto well-known are applied for administration of the medicine and the medicine is preferably administered at a daily dose of 0.001-100 mg divided into one to several portions.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for neurodegenerative diseases (such as Alzheimer's senile dementia), and more specifically, to dictyosterol (Dicty).
(Osterol) as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Chemical formula (1) which is a compound derived from cellular slime mold

[0003]

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Dictiosterol (Dict) represented by
yosterol has already been reported (Chemish Berchte, Chem. Ber., 108 , 1101 (197).
5), Proceeding of National Academic Science of USA (Proc. Natl. Acad. S)
ci. USA), Vol . 87 , No. 19, p. 7655 (1990)), but its pharmacological action is not known at all.

[0005] A central neurite regenerating agent is a drug that acts as a defense mechanism in the event of damage to a neural network, has a function of maintaining survival, and promoting germination and extension of neurites.

[0006] Mature central nervous cells do not divide or proliferate. Therefore, when nerve damage occurs due to stroke, trauma, or unexplained degenerative disease, the nerve cells will die and no new cells will replace them, and higher nerve function will be severely impaired.

Of particular importance for the regeneration of damaged neural networks is the sufficient extension of neuronal neurites toward target cells, and in recent years, the successful regeneration of severed neuronal processes even in mature cells. Has been reported to be possible by the action of projection extension factors such as neurotrophic factors (MD
Kawaja et al., Journal of Neuroscience (J.
Neurosci.), 12, 2849 (1992)). This indicates that it is possible to promote regeneration of the damaged neurons themselves or germination from surrounding healthy neurons to regenerate neurites.

Reconstructing a neural network by allowing damaged nerves to survive without dying and regenerating axons can lead to atrophy and loss of various nerve cells in a neurodegenerative disease that has been increasing in recent years. It is efficacious and suggests that central neurite regenerating agents are promising therapeutic agents.

Recently, various neurotrophic factors (BDN) have been attracting attention as therapeutic agents for neurodegenerative diseases including anti-dementia drugs.
F, GDNF).

[0010] Staurosporine is a substance having a neurite regenerating action (Neurology, 26, 299 (1987)).
And Lactacystin (J. Antibiotics, 44, 113 (1991)), P.
S-990 substance (Journal of Antibiotics, 47, 1175 (1994)), DQ
Substances such as 90C (JP-A-9-12468) are known. However, these substances are completely different in structure from the dictyosterols of the present invention.

[0011] In consideration of human medical use, substances having lower toxicity and exhibiting a process of regenerating the processes of central nervous cells at lower concentrations are expected to play a very important role.

[0012]

In view of the above background, there is a need for the development of a regenerating agent for central nervous cells, particularly mature central neurites.

[0013]

Means for Solving the Problems The present inventors have made intensive efforts to find a compound useful as a regenerating factor for central neurites, and as a result, dictyosterol represented by the following chemical formula (1). Found that this object was achieved, and completed the invention.

[0014]

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[0015] That is, for the first time, a pharmacologic effect of dictyosterol, which is a known compound but whose pharmacologic activity was unknown, to promote the process of extending central nervous cells was discovered, and a therapeutic drug for neurodegenerative diseases such as Alzheimer-type senile dementia ( (Including anti-dementia drugs).

The method for synthesizing dictyosterol represented by the above formula is known (for example, tetrahedron (Te
trahedron), 27 , 1261 (1971); Chem. Berch (Chem. Ber.), 108 , 1101 (1975)). In addition, Dictyostelium
Purpureum K1001) can be obtained by the following method.

(Manufacturing method) Cellular slime mold grows using bacteria such as Escherichia coli, Klebsiella pneumoniae and Bacillus subtilis as a nutrient source, and does not exist on natural or synthetic media like ordinary bacteria, actinomycetes and fungi. Cannot grow. Therefore, bacteria such as Escherichia coli are cultivated together with nutrients during culturing such as separation from the soil, subculture, production of bioactive substances, and the like.

As the culture method, there are an agar plate culture method and a liquid culture method, and the temperature suitable for the culture is 20 to 25 ° C., but the culture at about 22 ° C. is suitable. The production of dictyosterol is preferably performed by agar plate culture for 7 to 10 days.

After the completion of the culture, dictyosterol present in the cells is extracted with an organic solvent such as methanol. Dictiosterol present in the extract is isolated from the crude product using a commonly known purification method such as liquid-liquid partitioning or adsorption chromatography, and washed with an organic solvent such as methanol to obtain a single dictyosterol. Sterols can be obtained.

When the regenerating agent of the present invention is administered to a human, conventional pharmaceutically well-known forms and routes are applied. For example, powders, tablets, capsules, fine granules, granules, injections, solutions, ointments, patches, etc., are used orally or parenterally, and the dosage is
It depends on age, weight, symptoms, route of administration, etc.
It is administered in a dose of 0.001 mg to 100 mg per day in one or several divided doses.

[0021]

The production method of the compound of the present invention and its activity will be described in detail with reference to examples.

Production method 1 E. coli solution (Escherichia coli NIHJ JC)
-2) in an A medium (glucose (Kanto Chemical) 0.5%, polypeptone (Nippon Pharmaceutical) 0.5%, yeast extract (Kyokuto Pharmaceutical) 0.05%, KH ₂ PO ₄ (Nippon Physical Chemical) 0.2
_{25%, Na 2 HPO 4 ·} 12H 2 O ( Japan physics and chemistry chemicals)
_{0.137%, MgSO 4 · 7H 2} O ( Wako Pure Chemical) 0.0
5%) at 37 ° C. for 18 hours, and a suspension of the cellular slime mold Dictyostelium purpureum K1001 (cellular slime mold suspended in sterile distilled water).
m A agar (A medium supplemented with 1.5% agar (Nippon Pharmaceutical)). 22 ° C, under light irradiation, 1
For 0 days, 700 agar plates (φ9 cm) were cultured.

The fruiting body is suspended in distilled water, freeze-dried,
Extracted with 90% methanol. The extract was concentrated, water-saturated saline (1: 1) was added, and the mixture was extracted with ethyl acetate. Thereafter, the ethyl acetate extract was concentrated, dissolved in acetonitrile, added with 0.2 M acetic acid, and extracted with hexane. Further, acetonitrile-0.1% ammonia water (4:
1) Then, it was washed with saturated saline and concentrated. The residue was subjected to silica gel column chromatography (Wako gel).
C-300, developing solvent: ethyl acetate / hexane = 1:
When subjected to 4), a crude product of dictyosterol was obtained.
The crude was washed with methanol and dried to give 38 mg of a single dictyosterol as a white powder.

The ¹³ C-NMR spectrum data of the acetic ester of dictyosterol was in agreement with the data described in the literature (Steroids, 53 , 625 (1989)).

EXAMPLES Biological activity of dictyosterol Test method: Brain Research, 651
Volume , pp. 101-107 (1994).

That is, a slice of the hypothalamus was prepared from the brain of a 3-week-old rat, and then pronase (0.02%, Calbiochem) and thermolysin (0.02%, Sigma) at 37 ° C. for 20 minutes. Enzyme treatment was performed. The suprachiasmatic nucleus was punched out of this slice, and pipetting was performed with a glass capillary to obtain a cell dispersion. This cell dispersion was placed on a plastic plate (Nunc) previously coated with 0.1% polyethyleneimine.
Microliters / well were added. L-glutamine (0.29) was added to 10% fetal bovine serum and MEM (Nissui Pharmaceutical) medium.
g / liter, Wako Pure Chemical), sodium pyruvate (0.11 g / liter, Wako Pure Chemical), glucose (9.0 g / liter, Wako Pure Chemical), sodium hydrogen carbonate (2.0 g / liter, Wako Pure Chemical) ) And a medium prepared with sodium selenite (30 nM, Wako Pure Chemical Industries) at 37 ° C. in a 5% carbon dioxide gas incubator.

The sample was dissolved and added to the medium at the start of the culture.

On the 4th to 5th days of the culture, while observing the morphological change of the cells with a microscope, the number of nerve cells extending the projection more than twice the cell body was counted, and compared with the medium control without the sample.

The results are shown in Table 1.

[0030]

[Table 1]

Dictiosterol has a neurite regenerating activity on mature central nervous cells of 0.01 to 1.0 μg / day.
ml.

[0032]

As shown in Table 1, dictyosterol, a compound of the present invention, has an excellent process of regenerating projections on central nervous cells, especially on mature central nervous cells, and has neurodegenerative diseases including anti-dementia drugs. Useful as a therapeutic.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B064 AH07 BG01 BG09 CA05 DA01 4B065 AA58X BB28 BC48 CA05 CA44 4C086 AA01 AA02 DA11 ZA02 ZA16 ZC52 4C091 AA01 BB01 CC03 DD01 EE04 FF01 GG01 MM01

Claims (2)

[Claims] [Claim 1] Chemical formula (1) A regenerating agent for central nervous cell processes containing dictyosterol as an active ingredient. 2. The regenerating agent for central neurites according to claim 1, wherein the central nervous cells are mature central nervous cells.