JPH06343480A - Production of sf 2315a and its pharmacal application - Google Patents

Abstract

PURPOSE: To efficiently produce the subject compound capable of suppressing functions of T lymphocytes and useful as an immunosuppressant, an antitumor agent or the like by culturing a microorganism belonging to the genus Nocardia and producing SF2315A and collecting the SF2315A from the cultured product.

CONSTITUTION: A microorganism belonging to the genus Nocardia and producing SF2315A such as Nocardia mediterranei var. kanglensis 1747-64 strain (FERM BP-3718) is cultured under aerobic conditions and the SF2315A is then collected from the resultant cultured product to afford the objective compound represented by the formula. The daily dose of the SF2315A is preferably within the range of 5-500 mg/kg in the case of oral administration.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing SF2315A and its pharmaceutical use. More specifically, it relates to a method for producing SF2315A which is useful as an immunosuppressive substance applicable to rejection reaction due to organ transplantation, or as an antitumor agent, and its pharmaceutical use.

[0002]

Cyclosporin A selectively inhibits the proliferation of helper T cell clones and is used as an immunomodulator for organ transplant rejection (Agents).
and Actions, Vol. 6, pp. 468-4
75 (1976)). FK-506 is a recently discovered macrolide antibiotic (C ₄₄ H ₆₉ NO ₂₂ ) and is also used as an immunomodulator. However, these drugs are not free from toxicity and efficacy problems. Further, with the progress of medical technology, development of new drugs suitable for various uses is desired.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a more ideal immunosuppressive substance which can overcome various drawbacks of the immunosuppressive substances to date. Another object of the present invention is to provide a method for producing an immunosuppressive substance. Still another object of the present invention is to provide a new antitumor agent.

[0004]

Means for Solving the Problems The inventors of the present invention have extensively searched for metabolites of microorganisms and found that a strain belonging to the genus Nocardia produces a substance having strong immunosuppressive activity and antitumor activity. I have found The active substance was purified and isolated from the culture solution, and the physicochemical properties were examined. As a result, JP-A-62-296887 and Journa were found.
l of Antibiotics, Vol. 41, p
p. 835-848 (1988)) and was identified as SF2315A, which was previously reported. JP-A-62-29
According to the description in Japanese Patent No. 6887 and the like, specific uses of SF2315A are limited to medicines, veterinary drugs, and pesticides that focus on the antibacterial action of SF2315A against microorganisms. Therefore, the present invention relates to a method for producing SF2315A using a microorganism belonging to the genus Nocardia and its pharmaceutical use as an immunosuppressive agent and an antitumor agent.

The first gist of the present invention is the following formula

[Chemical 2] The method for producing SF2315A, wherein the SF2315A-producing bacterium belonging to the genus Nocardia is cultured, and SF2315A is collected from the culture. The second gist of the present invention is an immunosuppressive agent containing SF2315A or a pharmaceutically acceptable salt thereof as an active ingredient. The third gist of the present invention is an antitumor agent containing SF2315A or a pharmaceutically acceptable salt thereof as an active ingredient.

According to the present invention, SF2315A can be obtained by culturing SF2315A-producing bacteria belonging to the genus Nocardia in a nutrient medium to produce and accumulate SF2315A, and collecting SF2315A from the resulting culture. A typical example of the SF2315A-producing bacterium is Nocardia mediaterranei , a strain belonging to the genus Nocardia collected in the People's Republic of China.
var. Kanglensis 1747-64 strain. The mycological and physiological properties of this strain are shown below.

1. Morphological properties As a result of observing after 2 weeks at 28 ° C., the aerial hyphae are simply branched and their tips are wavy or looped. Fragmentation of basal hyphae is observed. No formation of sporangium or crusty silk is observed. Spore surface is smooth or rough, sometimes spiny, spores cylindrical, size 0.6-0.8x
It is 0.8 to 1.3 μm. In addition, spores are formed in 10 internal and external chains. 2. Growth in various media Table 1 below shows the growth conditions after 2 weeks at 28 ° C on various media.

[0008]

[Table 1]

3. Physiological properties Appropriate growth range: 27 to 37 ° C Nitrate reduction: Negative Gelatin liquefaction (on glucose / peptone / gelatin medium at 20 ° C): Negative Starch hydrolysis (starch / inorganic salt agar medium): Negative Nonfat milk Coagulation: Positive Peptonization of skim milk: Positive Formation of melanin-like pigment: Negative 4. Utilization of carbon source (Pridham-Godlieve agar
Top) L-arabinose + D-xylose + D-glucose + D-fructose + sucrose + inositol + L-rhamnose + raffinose-D-mannitol + 5. It is diaminopimelic acid meso-diaminopimelic acid in the cell wall .

In summary, this strain has a cell wall of me
So-diaminopimelic acid, and according to the International Streptomyces Project (abbreviation ISP) method, the morphology of sporulating hyphae is Section Rectiflexibiles.
Or Reticina Culia Party (Recticuli)
aperti), the spore surface is smooth or rough,
The color of the mature hyphae is white (White color)
, no melanin-like pigment and no pigment is produced in the medium. The color of basal hyphae is light brown or light yellow, and fragmentation is observed. As carbon sources, L-arabinose, D-glucose, D-fructose, sucrose, inositol, L-rhamnose, D-xylose,
Utilizes D-mannose and D-mannitol but not raffinose.

[0011] Based on the above properties, Bergey's Manual of Determinating Bacteriology, edited by R. E. Buffanan & N. Gibbons.
erminative Bacteriology, 8th Edition, 1974 and Vergi's Manual of Systematic Bacteriology (Bergey's)
Manual of Systematic Bacte
As a result of conducting a search according to riology Volume 4, 1989), it was found that the above strain belongs to the genus Nocardia . Therefore, this strain was designated Nocardia m.
editori lane var. kanlensi
s 1774-64 strain (hereinafter referred to as 1747-64 strain)
I named it. The strain was deposited on February 20, 1991, with the Commission for Conservation and Management of Chinese Microbial Species in the People's Republic of China.
The consignment number CGMCC No: 0163 is given. The strain has been internationally deposited under the Butabest Convention on January 28, 1992, at the Institute for Microbial Technology of the Agency of Industrial Science and Technology, and the deposit number is assigned to Micro Works Research Article No. 3718 (FERM BP-3718). Has been done.

[0012] SF2315A can be obtained by culturing an SF2315A-producing bacterium belonging to the genus Nocardia represented by the above strains and collecting it from the culture. As a carbon source, glucose, starch syrup, dextrin, sucrose, starch, molasses, animal and vegetable oils and the like can be used as the carbon source in the medium used for culturing the production bacterium. As the nitrogen source, soybean flour, wheat flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, it is also effective to add inorganic salts such as sodium, potassium, magnesium and cobalt as needed. A suitable temperature for culturing is 15 to 37 ° C, but in most cases, culturing is performed at around 26 to 30 ° C. SF2315
Although the production of A varies depending on the medium and culture conditions, the maximum accumulation is usually reached in a period of 1 to 10 days in both shaking culture and tank culture. When the accumulation is highest, the culture is stopped and the target substance is isolated and purified from the culture medium.

When the SF2315A is collected from the culture solution, it can be extracted and purified by appropriately combining ordinary separation means utilizing its properties. The active substance is adsorbed and desorbed using a method of extracting the culture filtrate with a water-immiscible organic solvent such as ethyl acetate and an adsorption resin such as Diaion HP-20 and Amberlite XAD-2 to obtain a crude product.
Purification from the obtained crude extract is a known method used for collecting fat-soluble substances, for example, adsorption chromatography, gel filtration chromatography, solvent precipitation method, scraping of thin layer chromatography, high performance liquid chromatography, etc. Can be combined for pure isolation.

The SF2315A thus produced may be in the form of a pharmaceutically acceptable salt. Examples of these salts include alkali metal salts with sodium, potassium, lithium, etc., and alkaline earth metal salts with calcium, magnesium, etc. These salts can be obtained by a method known per se. SF23
When 15A is used as an immunosuppressant or an antitumor agent, it is administered alone, or mixed with an excipient or a carrier, and administered as an injection, an oral preparation, a suppository, or the like. As the excipient and the carrier, pharmaceutically acceptable and commonly used ones are selected, and their type and composition depend on the administration route and administration method. Although it varies depending on the kind of SF2315A content in the preparation, it is usually 0.1-100.
% By weight, preferably 1 to 98% by weight. For example, in the case of an injection, it is usually 0.1 to 30% by weight, preferably 1
It is advisable to contain from 10% by weight of the active ingredient. In the case of oral administration, it is used in the form of tablets, capsules, powders, granules, liquids, dry syrups, etc. together with a commonly used solid carrier or liquid carrier, and capsules, tablets, granules, powders, etc. Generally, it should contain 5 to 100% by weight, preferably 25 to 98% by weight of the active ingredient. When SF2315A is used as an immunosuppressive agent and an antitumor agent, the dose is as follows:
The therapeutically effective amount is generally 1 to 100 mg / kg · day for parenteral administration and 5 to 500 mg / kg · day for oral administration, depending on the symptoms, purpose of treatment and the like.

[0015]

Action: SF2315A exerts a strong inhibitory effect on the function of mouse lymphocytes, which are immunocompetent cells. That is, U
method by Umezawa et al. (Umezawa et.a.
t. , "Supression of Tissue
Graft Rejection by Sperqu
alin ”, The Journal of Anti
biotics, Vol. 38, pp. 283-28
4, 1985), the effect on the lymphocyte blastogenesis reaction was examined, and SF2315A remarkably suppressed the blast transformation reaction of T lymphocytes stimulated by ConA (concanavalin A). The above results indicate that SF2315A significantly suppresses the function of T lymphocytes. Since this suppressive action means suppression of humoral immunity and cellular immunity,
SF2315A is extremely useful for suppressing the rejection reaction in organ transplantation or skin transplantation, which is considered to be caused by the abnormal increase. Further, SF2315A is also extremely useful for the treatment of autoimmune diseases thought to be mainly caused by various autoimmunity, for example, lupus nephritis. Also, SF231
5A strongly suppresses the growth of K562 leukemia cell line, which is one of the tumor cells. Therefore, SF2315A is also extremely useful as a therapeutic agent for tumors such as leukemia.

[0016]

EXAMPLES The present invention will be described in more detail below with reference to examples and test examples. Example 1 As an isolated and purified seed medium of SF2315A , glucose 2.5%, sunrich (soybean flour) 0.5%, yeast extract 0.5%, ammonium sulfate 0.5%, potassium chloride 0.25%, second 0.02% potassium phosphate, 0.02% magnesium sulfate, 0.5% calcium carbonate, silicon KM-700 as a defoaming agent.
100 ml of a medium containing 01% was poured into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 30 minutes. Nocardia mediaterra was added to this medium.
nei var. kanglesis 1747-
Inoculation of 1 platinum loop of 64 strains of agar slant culture at 27 ° C, 1
The cells were cultured on a rotor shaker at 80 rpm for 2 days to give a primary culture. Then, add 500 m of seed medium having the same composition as above.
Dispense 100 ml into an Erlenmeyer flask and
Sterilized at 20 ° C. for 30 minutes. 2 ml of the first-type culture solution was transplanted into this incubator and cultured at 27 ° C. for 2 days, which was designated as the second-type culture. Then, as a production medium, glucose 5.0
%, Yeast extract 1.0%, ammonium chloride 0.5%,
0.5% calcium carbonate, and silicon K as an antifoaming agent
M-70 0.05%, Pronal ST-1 0.03
20 liter of a medium consisting of 30% was placed in a 30 liter jar fermenter and sterilized at 120 ° C. for 30 minutes. This culture tank was inoculated with 400 ml of the above-mentioned second-type culture solution, aerated at 27 ° C., aeration (6 liters / minute), and stirred (220
Cultivation was performed for 5 days at a rotation speed of 5 minutes.

After the completion of the culturing, the filter aid perlite was added to the culture solution and filtered to obtain 74 liters of the culture filtrate. 74 liters of the obtained filtrate was passed through a column filled with 2.7 liters of Diaion HP-20 (manufactured by Mitsubishi Kasei),
The target substance was adsorbed. Then, the column was washed with distilled water and 50% aqueous methanol solution. Further, methanol was passed through the column to collect the target substance. The recovered liquid was concentrated under reduced pressure to obtain 300 ml of a concentrated liquid. This concentrated solution was extracted twice with 250 ml of ethyl acetate to collect the desired product in the ethyl acetate layer. The ethyl acetate layer was concentrated under reduced pressure to give a brown oil (9.6 g).
Got This crude material was dissolved in 70 ml of methanol, 25 g of silica gel (Art. 7734 manufactured by Merck & Co., Inc.) was added, and the mixture was concentrated to dryness under reduced pressure. The dried solid was suspended in chloroform, applied to a column of 1100 ml of silica gel 60 (Art 7734, manufactured by Merck & Co.) preliminarily filled with chloroform, and washed with chloroform. Then, the target substance was eluted by passing a solvent of chloroform: methanol (50: 1). The eluate was concentrated to dryness to give a brown oil 7
90 mg was obtained. 15 ml of this crude material in methanol
Dissolved in silica gel 60 (Merck's Art773
4) 5 g was added and concentrated to dryness under reduced pressure. Then, this was suspended in n-hexane and pre-filled with n-hexane on silica gel (Art 7734 manufactured by Merck), 1
After applying to a 60 ml column and washing with n-hexane, the target component was eluted with a solvent of n-hexane: acetone (5: 1). The eluted fraction was concentrated to dryness under reduced pressure to obtain 210 mg of a reddish brown paste-like substance. This was suspended in n-hexane, the insoluble matter was filtered off, and the insoluble matter was dried to obtain 29 mg of a reddish brown powder. After dissolving this in a small amount of methanol, Sephadex LH- filled with methanol
20 (manufactured by Pharmacia), pass through a 60 ml column,
It was developed with methanol. The effective fractions were collected and concentrated to dryness under reduced pressure to obtain 15 mg of reddish brown powder. This was dissolved in a small amount of ethanol with heating and cooled to precipitate orange crystals. The filtered crystals are dried to give pure SF2315A.
10.5 mg was obtained.

With respect to the thus obtained crystal (10.5 mg), the physicochemical constant was measured, and the analytical value was SF23.
15A Reference value (Journal of Antib)
iotics Vol. 41, pp. 835-848,
1988). As a result, the isolated active substance in the culture solution was identified as SF2315A substance. The main physical properties are described below. Molecular weight (FAB-MS): m / z 325 [M + H] ⁺ ultraviolet absorption spectrum: The spectrum measured with a methanol solution is shown in FIG. Red external absorption spectrum: The spectrum measured with a potassium bromide tablet is shown in FIG. Hydrogen nuclear magnetic resonance spectrum: The spectrum measured in deuterated dimethyl sulfoxide is shown in FIG.

Test Example 1 Effect of SF2315A on lymphocyte blastogenic reaction Spleen cells (2 × 10 ⁵ cells / well) of C5713L / 6 mice were subjected to S in the presence of concanavalin A (5 μg / ml).
Incubated with F2315A for 72 hours. Proliferation of lymphocytes was evaluated by adding 37 KBq of [ ³ H] -thymidine 6 hours before the end of culture and measuring the amount of incorporation into the cells. The result is that the concentration of SF2315A is 1 μg / ml.
The inhibition rate was 97.7%. Test Example 2 SF2315A against proliferation of K562 leukemia cell line
Action K562 cells (1 × 10 ⁵ cells) were cultured with SF2315A for 72 hours. After culturing, the number of cells was measured and evaluated using a Coulter counter. The result is SF231
The inhibition rate was 89.8% at a concentration of 5 A of 1 μg / ml.

Example 2 Production of Tablets SF2315A 30 parts by weight, crystalline lactose 120 parts by weight,
147 parts by weight of crystalline cellulose and 3 parts by weight of magnesium stearate were tabletted with a V-type mixer to give tablets of 300 mg each.

[0021]

As is clear from the results shown in Test Examples 1 and 2, SF2315A showed an inhibition rate of 97.7% against the lymphocyte blastogenic reaction at a concentration of 1 μg / ml,
Inhibition rate against proliferation of K562 leukemia cell line 89.8%
showed that. Therefore, SF2315A is useful for suppressing the rejection reaction in organ transplantation and for treating autoimmune diseases because it significantly suppresses the function of T lymphocytes, and also for treating tumors.

[Brief description of drawings]

FIG. 1 Nocardia mediaterranei
var. 1 shows an ultraviolet absorption spectrum of SF2315A isolated and purified from a culture of strain Kanglensis 1747-64.

FIG. 2 Nocardia mediaterranei
var. 1 shows an infrared absorption spectrum of SF2315A isolated and purified from a culture of strain Kanglensis 1747-64.

FIG. 3 Nocardia mediaterranei
var. 1 shows a hydrogen nuclear magnetic resonance spectrum of SF2315A isolated and purified from a culture of strain Kanglensis 1747-64.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Seiichi Saito 4-7-2-407 Matsuba-cho, Kashiwa-shi, Chiba Prefecture (72) Inventor Wang Nankin No. 1 Nishizato, Tendan, Beijing Xuanwu District, People's Republic of China

Claims (4)

[Claims] 1. The following formula: The method for producing SF2315A, which comprises culturing an SF2315A-producing bacterium belonging to the genus Nocardia and collecting SF2315A from the culture. 2. The SF2315A producing bacterium is Nocardi.
a Mediterranei var. kangl
ensis 1747-64 strain (FERM BP first 3715
No .: FERM BP-3718). 3. An immunosuppressive agent containing SF2315A or a pharmaceutically acceptable salt thereof as an active ingredient. 4. An antitumor agent containing SF2315A or a pharmaceutically acceptable salt thereof as an active ingredient.