JPH07258263A - Neuron differentiation promoter and its production - Google Patents

Abstract

PURPOSE:To obtain a neuron differentiation promoter, containing a specific pseurotin A (salt) as an active ingredient, having low toxicity and useful for an antidement, a neuron protecting agent, a therapeutic agent, etc., for peripheral neuropathy. CONSTITUTION:This neuron differentiation promoter contains pseurotin A expressed by the formula and its pharmacologically permissible salt as an active ingredient. Furthermore, the pseuroton A is obtained by culturing a microorganism, belonging to the genus Diheterospora and capable of producing the pseurotin A such as Diheterospora chlamydosporia NF01150 (FERM P-13984) strain and collecting the pseurotin A from the resultant culture. The daily dose of the pseurotin A is preferably within the range of 1-100mg/kg body weight in the case of parenteral administration.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel nerve cell differentiation-promoting agent containing chorotin A as an active ingredient,
For example, it is expected to be used as an anti-dementia agent, a nerve cell protective agent, or a therapeutic agent for peripheral neuropathy.

[0002]

2. Description of the Related Art Formula [I] used in the present invention

[0003]

[Chemical 2] Pseurotin A represented by Helvetica, Kimika,
It is a known compound described in Actor (Helvetica Chimica Acta) Vol. 59, p. 133, 1976. This compound is a monoamine oxidase inhibitor (Maikotoxin 22 33 (1985)), a chitin biosynthesis inhibitor (Biosci. Biotech, Biochem 57 961).
(1993)) and an activity as an apomorphine antagonist (EP Patent No. 0546475).

Nerve growth factor (hereinafter referred to as NGF) has an action to elongate neurites and an action to regulate the production of neurotransmitters, and it is shown in vitro that it has a regenerating action on nerve cells of old animals. Proven, [Age 8 Volume 19
Page (1985)] On the other hand, by adding NGF to PC-12 cells, a cell line cloned from rat adrenal medulla pheochromocytoma, the proliferation was stopped and the cells became sympathetic nerve cells with neurites. Known to differentiate. Due to such an action, it is one of the drugs that have been attracting attention as an anti-dementia drug in recent years.

Using these cells, it was investigated that, in addition to NGF, fibroblast growth factor and interleukin 6 also induce the elongation of protrusions. Recently, staurosporine, a low-molecular substance derived from microorganisms, has also been investigated. It has been shown to result in neurite outgrowth. (Neurochemistry, 26, 200-220 (198
7)].

[0006]

The above-mentioned staurosporine is extremely valuable in medicine because it is a low-molecular substance unlike NGF, but unfortunately it is highly toxic.
In actual use, there is still room for consideration. Under such circumstances, it is extremely important for medical treatment to provide a low molecular weight substance which is less toxic and exhibits a neurite outgrowth action at a low concentration.

[0007]

[Means for Solving the Problems] Therefore, the inventors of the present invention isolated a strain from soil and continued to study the product thereof, and as a result, the formula [I] was obtained.

[Chemical 3]

It has been found that the chlorotin A represented by the formula (1) has nerve cell differentiation promoting activity. The present invention has been completed based on the above findings.

The present invention will be described in more detail. First, it relates to an agent for promoting differentiation of nerve cells containing, as an active ingredient, chlorotin A and a pharmacologically acceptable salt thereof.

[0010] in the second, Jiheterosupora (Diheterospora)
PROBLEM TO BE SOLVED: To provide a method for producing chlorotin A and a pharmacologically acceptable salt thereof, which comprises culturing a chlorotin A (Pseurotin) A-producing bacterium belonging to the genus in a medium and collecting the chlorotin A from the obtained culture. It is a thing.

The first invention will be described in detail below.
The pharmacologically acceptable salt of the chorotin A used as an active ingredient in the present invention may be an acid addition salt, and the acid forming the acid addition salt may be, for example, hydrochloric acid,
Examples thereof include inorganic acids such as hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and methanesulfonic acid, and organic acids such as maleic acid, fumaric acid, oxalic acid, formic acid and acetic acid.

The neuronal differentiation promoting agent of the present invention containing chlorotin A as an active ingredient is expected to be used as, for example, an anti-dementia agent, a therapeutic agent for peripheral neuropathy caused by an anticancer agent, a neuronal cell protective agent, or the like. ing.

When this compound is used as a nerve cell differentiation promoting agent, it is administered alone or in admixture with an excipient or carrier as an injection, oral preparation, suppository, or the like.
As the excipient and the carrier, those which are pharmaceutically acceptable are selected, and the type and composition thereof are determined by the administration route and administration method. For example, water, alcohols or soybean oil, animal and vegetable oils such as peanut oil, sesame oil, and mineral oil as a liquid carrier, or sugars such as maltose and sucrose, amino acids, cellulose derivatives such as hydroxypropyl cellulose as a solid carrier in which synthetic oil is used. An organic acid salt such as magnesium stearate is used.

In the case of injections, physiological saline, various buffer solutions, saccharide solutions such as glucose, inositol and mannitol, ethylene glycol, propylene glycol, etc.
Glycols such as polyethylene glycol are desirable.
Inositol, mannitol, glucose, mannose, maltose, sugars such as sucrose, excipients such as amino acids such as phenylalanine, together with a suitable solvent for injection at the time of administration, such as sterile water, physiological saline,
It can also be administered by dissolving it in a liquid for intravenous administration such as glucose solution, electrolyte solution, amino acid solution.

The content of the compound in the preparation varies depending on the preparation, but is usually 0.1 to 100% by weight, preferably 1
Is about 98% by weight. For example, in the case of an injectable solution, it is preferable to contain the active ingredient in an amount of usually 0.1 to 30% by weight, preferably 1 to 10% by weight. In the case of oral administration, it is used in the form of tablets, capsules, powders, granules, liquids, dry syrups and the like together with the solid carrier or liquid carrier. Capsules, tablets, granules and powders generally contain 5-100% by weight, preferably 25-98% by weight, of the active ingredient.

The dose is determined depending on the age, body weight, symptoms, therapeutic purpose, etc. of the patient. Generally, the dose is 1 to 100 mg / kg for parenteral administration, 5 to 500 mg / k for oral administration.
g ・ It is a day.

The compounds are characterized by low toxicity, and all compounds are characterized by low toxicity accumulation after continuous administration. Even when the compound was intraperitoneally administered to mice once at a dose of 800 mg / kg, no sign of toxicity was observed.

Next, the second invention will be described in detail.
The serotin A-producing bacterium of the present invention belongs to the genus Diheterospora . For example, the NF01150 (deposition number FERM P-13984) strain isolated by the present inventors is an example of the strain most effectively used in the present invention. is there.
The mycological properties of this strain are as follows.

Bacteriological properties of NF01150

1. Potato dextrose agar (2
(5 ° C) grows very well and the diameter of the settlement is 5 after 14 days.
Reach 6-59 mm. On the surface of the village, aerial hyphae develop and become wool-like, and spread like concentric rings. Depending on the number of days of culture, the color is white to cream, and the back side is yellowish brown at the center and yellowish white at the periphery. 2. The malt extract agar medium (25 ° C) grows well,
Over the days, the diameter of the settlement reaches 53-54 mm. On the surface of the village, aerial hyphae develop and become wool-like, and spread like concentric rings. Depending on the number of days of culture, the color is white to cream, and the reverse side is yellow to tan. It also produces a yellow pigment in the medium. 3. The Tuapec agar medium (25 ° C) also grows well,
In days, the diameter of the settlement reaches 50-53 mm. The surface of the village spreads like wool and concentric rings. Depending on the number of days of culture, the color is white to pale yellow, and the back side is white.

4. Cornmeal agar medium (25 ° C) grows a little badly, and the diameter of the settlement is 47 to 50 mm after 14 days.
Reach The surface of the settlement is thin and flat with aerial hyphae.
Colorless to white depending on the number of culture days. 5. Oatmeal agar (25 ℃) grows well, 1
In 4 days, the diameter of the settlement reaches 54 to 56 mm. The surface of the colony is wool-like, and from the white color to the yellow color at the center after the cultivation days, and on the back surface is yellow to yellow-orange.

6. This strain grows rapidly on Tuapeck agar and forms abundantly with conidia on the entire surface of the colony. The hyphae are 1.6 to 2.6 μm in diameter, are colorless and have long and narrow partition walls. The formation of conidia consists of two types, Aleuro-type conidia and Fiaro-type conidia. Aleuro-type conidia formation structure: Conidia peduncle has a length of 2.5 to 30 μm and a diameter of 1.6 to 2.6 μm, and swells conidia toward the settling part of the conidia.
Conidia are Aleu-type conidia, colorless to yellowish-brown, spherical to oval, and irregular in shape. When the cell wall matures, it becomes a thick wall. The diameter is 16 to 29 μm. Fiaro-type conidia formation structure: Conidia peduncle has a long and narrow septum, single generation (1) to serrata (5)
Produces a fear ride. The phialide is elongated and has a slightly bulged base, 17 to 30 μm × 1.3 to 2.0 μm,
colorless. Conidia are filoro-conidia, varying in size, oval to short cylindrical, rounded at the tip, cut out at the base, colorless on the smooth surface, 1.9-4.5 × 1.3 ~ 2.2
μm. Conidia gather at the tip of the phialide to form a drop shape (spherical shape). The optimum growth temperature of this strain is around 25 ° C, and it does not grow at 37 ° C. Growth pH range is 3-10
And the optimum pH is around 5.5.

7. Due to the above mycological properties, this strain is
“Ainsworth and Bisby's Dictionaryof the Fungi"
(By DL Hawksworth BCSutton and GGAinsworth.
According to 7th ed., CMI, Kew, 1983), it was identified as a genus of Diheterospora with fungal phyla, incomplete subphyla, and incomplete filamentous fungi. After further searching for species, Diheterospora ch
Dihete almost matched because this strain with the description of lamydosporia
It was identified as rospora chlamydosporia, Diheterospora chla
It was named mydosporia NF01150.

In order to produce chlorotin A according to the present invention, a microorganism belonging to the genus Diheterospora and capable of producing chlorotin A is cultured in a medium and Pseu is added to the culture.
Generate and accumulate rotin A, and then collect this.

The culturing method is basically similar to the culturing method of filamentous fungi, but the deep culturing method by liquid culturing is usually advantageous. The medium used for the culture is strain NF-0115.
Any medium may be used as long as it contains a nutrient source used by No. 0. As the nutrient source, known ones conventionally used for culturing filamentous fungi can be used. For example, as a carbon source, glucose, galactose, mannitol, dextrin, starch, starch syrup (starch malt saccharified product), soybean oil alone or It can be used in combination.

As the inorganic and organic nitrogen sources, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean oil residue, oat mill, casamino acid, bactosoyton. , Soluble soluble protein, etc. can be used alone or in combination.

Other inorganic salts such as sodium chloride, magnesium sulfate, copper sulfate, zinc sulfate, manganese chloride, calcium carbonate, and phosphate can be added, if necessary, and the growth of the bacterium and the production of chorotin A can be promoted. Organic matter,
For example, nucleic acids, amino acids, vitamins and inorganic substances can be added appropriately.

Culturing is usually carried out under aerobic conditions such as shaking or aeration-agitation culture. From the practical point of view, deep aeration stirring culture is preferred. The pH of the medium is, for example, 5.0 to
Although it is 8.0, it is preferably around neutral. Culture temperature is 25
~ 28 ° C is preferred. As for the culture time, in the case of liquid culture, the culture is usually performed for 2 to 4 days, and the culture is terminated when the accumulated amount of choriotin A in the culture reaches the maximum.

The culture conditions such as medium composition, liquid culture of the medium, culture temperature, agitation speed, and aeration rate are appropriately adjusted and selected so as to obtain preferable results depending on the kind of strain to be used and external conditions. It goes without saying that it will be done. When foaming occurs in liquid culture, a defoaming agent such as silicone oil, vegetable oil, or surfactant is used as appropriate.

The chlorotin A accumulated in the culture thus obtained is usually produced in the culture filtrate.

To collect Shurotin A from the culture filtrate, the means usually used for collecting metabolites from the culture liquid of a microorganism may be used alone or in any combination.
Or it is used repeatedly. That is, for example, filtration,
Centrifugation, dialysis, concentration, drying, freezing, adsorption, desorption, methods utilizing the difference in solubility in various solvents (eg, precipitation, crystallization, recrystallization, phase transfer, countercurrent distribution, etc.), chromatography, etc. Is used.

Pseurotin A is mainly produced and accumulated in the culture filtrate. Therefore, in order to separate and collect this compound, it is sufficient to collect it from the culture filtrate from which the bacterial cells have been removed. For example, from the culture filtrate obtained by removing cells and other cells from the culture,
Pseurotin A was extracted with ethyl acetate and concentrated, and then silica gel column chromatography, Sephadex LH-
20 Chlorotin A can be isolated by chromatography or the like.

[0033]

Next, the neurite outgrowth effect of choriotin A on PC12 cells will be described. Green
Et al. [Ann, Rev. Neurosi., 3, p. 353 (1980
Year)], and judged by morphological change.

That is, PC12 cells were planted at 1 × 10 ⁴ / ml in a modified Dulcobeco's modified Eagle medium supplemented with 10% fetal bovine serum and 10% horse serum, and overnight at 37 ° C. in a collagen-coated 96-well multiplate. 5% CO ₂
Cultured below. A sample is added here and the morphological change after 1 day is observed with a microscope.

As a result, the effective concentration of pseurotin A for causing neurite outgrowth on PC12 cells was 25.
The range was from μg / ml to 0.4 μg / ml.

As a control, the activity of staurosporine showing the same action was measured in the same manner as the above-mentioned method.
Protrusion extension was observed at 02 μg / ml.

Further, the concentration of Pseurotin A which kills PC12 cells by cytotoxicity after 1 day by the above method was 30 μm / ml, while that of staurosporine was 0.05 μm / ml. Therefore, it was found that Scheurotin A has low toxicity.

[0038]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited thereto.

Example 1 In a 500 ml Erlenmeyer flask, glucose 1%, sucrose 2%, soybean flour (trade name; adipron E-3) 2
%, KH ₂ PO ₄ 0.1%, MgSO ₄ .7H ₂ O
Dispense 100 ml of liquid medium containing 0.05%,
Diheterospora chlemydosporia NF sterilized by steam sterilization at 20 ℃ for 20 minutes and then cultivated at 26 ℃ on potato dextrose magar slant medium
-01150 (Deposit No. FERM P-13984)
One platinum loop was inoculated from each of the slant cultures of No. 1 and cultured by rotary shaking at 26 ° C. for 3 days to obtain a seed mother.

30 liter jar fermenter 1
Add 15 liters of the above liquid medium to the base,
Steam sterilized for 0 minutes. The above-mentioned three seeds were transplanted to this, and the mixture was aerated and agitated at 26 ° C. for 72 hours under a stirring rate of 25 rpm and an aeration rate of 10 l / min.

The supernatant obtained by filtering the culture broth was extracted with 15 liters of ethyl acetate to obtain 10.2 g of a concentrated dry solid. This is subjected to silica gel column chromatography (φ3 × 20c
m) and eluted with CHCl ₃ : MeOH = 50: 1. Each fraction was traced by a bioassay method using PC12 cells, and active fractions were collected and concentrated under reduced pressure to obtain 380 mg of dried solid matter. This was subjected to LH-20 column chromatography (φ3 × 180 cm), eluted with MeOH, and active fractions were collected to isolate 100 mg of chorotin A.

[0042]

[Effect of the Invention] As described above, Scheurotin A is PC12.
It is useful as a nerve cell differentiation promoting agent because it has a cell projection-promoting activity and is less toxic than microbial-derived staurosporine, and is useful as, for example, an anti-dementia agent or a peripheral nerve disorder in the treatment of an anticancer agent. It is considered to be applied as a therapeutic agent and a neuronal cell protective agent.

Claims (4)

[Claims] 1. The following formula [1]: A neuronal differentiation promoting agent containing Pseurotin A represented by the formula (Pseurotin A) and a pharmacologically acceptable salt thereof as an active ingredient 2. A culturing Jiheterosupora (Diheterospora) Shurochin A (Pse-urotin A) producing bacteria belonging to the genus
A method for producing chlorotin A, which comprises collecting chlorotin A from the obtained culture. 3. Pseurotin A belonging to the genus Diheterospora
The producing strain is Diheterospora chlamydosporia.
spora Chlamydosporia ). 4. Pseurotin A belonging to the genus Diheterospora
The producing bacterium is Diheterospora chlamydosporia NF01
The manufacturing method according to claim 3, which is 150.