JPH01108987A - New material nk86-0186 and production thereof - Google Patents

Abstract

NEW MATERIAL:NK86-0186 with a molecular weight of 186, having the following physico-chemical characteristics: (1) appearance: colorless powder, (2) molecular formula: C12H22N4O4, (3) specific optical rotation [alpha]D<20>+31.6 deg., (4) melting point: >=160 deg.C, (5) solubility: soluble to water and dimethylsulfoxide, sparingly soluble to methanol and insoluble to acetone and ethyl acetate, (6) color reaction; positive for ninhydrin reaction and Reidonsmith's reaction and magnetic for Pauly's reaction. USE:Useful for prevention or therapy of allergic diseases, prevention or suppression of the rejection in organ tranoplantation, prevention or therapy of autoimmune diseases, etc. PREPARATION:Streptomyces Sp-NK86-0186 (FERM P-9487) is subjected to culture on a medium and the resulting fungi are filtered to obtain a culture liquid, from which the objective new material can be isolated and purified.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention can be used for the prevention and suppression of the novel substance NKB6-0186, the prevention and treatment of autoimmune diseases, and the like.

[Conventional technology]

Conventionally known immunosuppressants include alkyl drugs, 1 antimetabolites, antibiotics, steroids, 1 folate antagonists, and plant alkaloids.

[Problems to be solved by the present invention]

Among conventional immunosuppressants, steroids have anti-inflammatory effects,
It is said that it exhibits immunosuppression rather than isobaric lymphocyte lytic action, and it is well known that it has a wide range of actions and is accompanied by various side effects.

Other immunosuppressants belong to so-called cytotoxic substances, and many of them act particularly on the nucleic acid 1 synthesis system, and are known to easily cause serious side effects in organs such as hematopoietic organs.

It selectively acts on immune cells such as lymphocytes.

There is a desire for a drug that has as few side effects as possible other than immunosuppressive effects.

[Means for solving problems]

Therefore, the present inventors conducted various searches for metabolites of microorganisms, and found that Streptomyces (stre-pt.
It has been discovered that a strain belonging to the genus P. omyces produces a new substance NKa6-0186 that has immunosuppressive effects.

The present invention was completed based on the above findings.

The new substance NKB6-0186 is produced by culturing NKB6-0186-producing bacteria belonging to the genus Streptomyces to produce and accumulate immunosuppressive substances.
Obtained by collecting a6-0186.

Streptomyces e.sp. (abbreviated as "stock").

The mycological properties of the NKB6-01B6 strain are shown below. Under a microscope, the NK86-0186 strain forms spiral aerial hyphae from branched basal hyphae, and no single whorled branch is observed. A mature spore chain consists of 20 or more spores, and the size of the spores is 0.8-0.9 x 1.2-1.
The size of the spores is about 7 microns.

It is smooth. Also, the spores cannot be detected.

2. Regarding the description of growth state colors in various media, the color standards of the Japan Color Research Institute were used.

(1) Sucrose/nitrate agar medium (cultured at 27°C) Aerial mycelium of a pale vinkle-reddish gray is grown on the colorless growth, and no soluble pigment is observed.

(2) Glucose-asparagine agar medium (cultured at 27° C.) White to pink gray aerial mycelia are grown on the growth of pale daisies to daisies, and the soluble pigment has a daisy flavor.

(3) Starch/inorganic salt agar medium (technical sp-medium 4゜2
(Cultivated at 7°C) Grayish red to reddish gray aerial mycelia are grown on the colorless to pale yellow growth, and the soluble pigment is only slightly tinged.

(4) Tyrosine agar medium (Engineering sp-medium 7, cultured at 27°C) Aerial mycelium of grayish white to light straw ash grows on the growth of light straw ash to brown ash, and the soluble pigment has a brownish tinge.

(5) Nutrient agar medium (cultured at 27°C) White to brown aerial mycelium grows on the pale yellow growth.

The soluble pigment has a slight daisy taste.

(6) Yeast/malt agar medium (xsp-medium 2゜27
℃ culture) Light red daisies ~ white on the development of bright red daisies ~
It grows on aerial mycelia of straw ash and contains a large amount of soluble pigment.

(7) Oatmeal agar medium (Engineering sp-medium 3° 27°C
Culture) Pink-white to reddish-gray aerial mycelium grows on colorless to pale yellow growth, and no soluble pigments are observed.

(8) Starch agar medium (27°C culture) White to bright straw-gray aerial mycelium grows on the growing pale daikon, and the soluble pigment gives it a daisy flavor.

(9) Malate lime agar medium (cultured at 27°C) Pink gray aerial mycelium grows on the colorless growth, and the soluble pigment is reddish.

(10) Gelatin puncture culture In a simple gelatin medium (cultured at 24°C), white to pink gray aerial mycelia grow on pale yellow to yellow brown growth, and the soluble pigment has a brownish tinge. In glucose-peptone-gelatin medium (cultured at 24°C), aerial mycelia do not adhere to colorless to yellowish-brown growth, and no soluble pigment is observed.

(11) Skimmed milk (cultivated at 37°C) Colorless to pale yellow growth; Wake mycelia do not adhere, and soluble pigments are only slightly brownish.

5. Physiological properties (1) Growth temperature range Yeast starch agar medium (soluble starch 1.0%
, yeast extract 0.2 qb, powdered agar 2.0 qb, pH
7・0), 5.1G, 20°24.27,52,
As a result of testing at each temperature of 57.45℃, growth occurred at all temperatures except 5℃ and 45℃, but the optimum temperature was 24℃.
It seems to be around ~27℃.

(2) Liquefaction of gelatin (15% simple gelatin medium).

24°C culture, glucose fist peptone gelatin medium, 24
℃ culture) In simple gelatin medium, liquefaction begins around the 20th day.
Its effect is weak.

(5) Hydrolysis of starch (starch/inorganic salt agar medium and starch agar medium, both cultured at 27°C) In both media, ice-melting properties were observed from around the 5th day after culture, and the effect was weaker ~ Moderate.

(4) Coagulation and peptonization of skimmed milk (skimmed milk).

(Culture at 37°C) Peptonization started without coagulation around 20 days after culturing.
Its effect is weak.

(5) / Production of 5ene-like pigments (tryptone yeast broth, engineering SP-medium 1. peptone yeast iron agar medium, engineering SP-medium 6, tyrosine agar medium, engineering sp)
-Medium 7. Both were cultured at 27°C) Melanin-like pigments were observed in all the media.

(6) Utilization of carbon source (Pridham-Gotlieb agar medium, industrial sp-medium 9, 27°C culture) Utilizing glucose S L-arabis, sucrose, D-mannitol, '(/'/toll, Raffinose, rhamnose, D-fructose, and galactose are not used. Also, it seems that D-xylose is also used.

(7) Dissolution of malic acid lime (malic acid lime agar medium).

27℃ culture) Solubility was observed from around 14 days after culture.

Its effect is weak.

(8) Reduction reaction of nitrate (peptone water containing 0.1% potassium nitrate, cultured in SP-medium 8.27°C) is negative.

To summarize the above properties, 2,6-diaminopimelic acid contained in the cell wall of NK86-0186 strain is IIL-type. In addition, no spores were observed, and the aerial hyphae had a spiral shape.
Whorled branches are not allowed. The surface of the spores is smooth. Pink-gray to reddish-gray aerial mycelia were grown on pale yellow to large-scale growth in various media.

Soluble pigments have a strong taste. The melanin-like pigment is positive, the proteolytic ability is weak, and the ice-melting ability of starch is weak to moderate. In addition, dissolution of lime malate was observed,
Nitrate reduction reaction is negative.

This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology on July 17, 1985, and the accession number was No. 9.
No. 487 (FIRM P-9487).

Streptomyces sp. IKB6- used in the present invention
Strain 0116, like other Streptomyces strains,
NKB6 is a novel substance that is the object of the present invention, because its properties are easily changed. -0186C and below simply NK
Ql, -0186. ) can be used in the present invention.

To produce NKa6-0186 according to the present invention, the strain is first cultivated aerobically in a medium containing nutrients available to the microorganism. As the nutrient source, known ones conventionally used for culturing microorganisms can be used. For example, as the carbon source, glucose, fructose, glycerin, sucrose, dextrin, galactose, organic acids, etc. may be used alone or in combination. Can be used.

Inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, urea, ammonium nitrate.

Sodium sulfate, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soy flour.

Cottonseed oil cake, casamino acid, bactosoitone, nolupur vegetable, oatmeal, etc. can be used singly or in combination.

Other salt, calcium carbonate, magnesium sulfate, copper sulfate, iron sulfate, zinc sulfate, manganese chloride,
In addition to inorganic salts such as phosphates, organic substances such as amino acids, vitamins, nucleic acids, and inorganic substances can be appropriately added.

As a culture method, a liquid culture method, particularly a deep agitation culture method, is most suitable. The culture temperature was 20°C to 37°C, and the pH was
It is desirable to culture in neutral or slightly acidic conditions. In liquid culture, NK86-01 is usually cultured for 3 to 8 days.
86 is produced and accumulated in the culture medium.

When the production amount in the culture medium reaches the maximum, the culture is stopped.
The target substance is purified and isolated from the culture solution obtained by separating the bacterial cells from house to house.

The purification and isolation of this substance from the culture solution F generally involves the separation and isolation methods used to isolate microbial metabolic products from the culture solution.
Purification methods are utilized.

NKB6-0186 is soluble in water, methanol, dimethyl sulfoxide, and propanol. It is a substance that is insoluble or hardly soluble in general organic solvents such as acetone, and its purification is carried out by the method used for purifying so-called water-soluble basic substances.

Namely, adsorption/desorption method using activated carbon and ion exchange resin;
Methods such as Sephadex column chromatography can be used in appropriate combination.

For example, after adjusting the culture F solution to pH 7.0, it is adsorbed onto activated carbon powder, washed with water, eluted by linear concentration gradient elution with methanol, and the active fraction is concentrated to dryness.

The resulting yellow-brown coarse powder was mixed with a small amount of citrate buffer (
Sodium ion concentration, o, o5 mol, pH 2,6), packed into BP Sephadex oC-25 equilibrated with the same buffer solution, eluted by linear concentration gradient elution method of sodium chloride, and activated carbon powder. A pale yellow powder is obtained by desalting.

This powder was dissolved in a pyridine/formate buffer and equilibrated with the same buffer beforehand.
00 mesh) column.

Elution is carried out by raising the pH stepwise, and the active fraction is collected, neutralized, and then concentrated and freeze-dried. The obtained lyophilized product was dissolved in a small amount of 50% methanol water, filled into Sephadex 0LE2o equilibrated with the same solvent, eluted with the same solvent, and the active fraction was collected and concentrated.

A colorless powder of NK86-0186 was obtained by freeze-drying.

The titer of NK86-0186 in the culture and fractionation was determined by suppressing the uptake of H-thymidine in the 79 lymphocyte rejuvenation reaction induced by stimulation with popolysaccharide (LPS).

New substance NK96-0186 obtained as above
The physical and chemical properties of are shown below.

(Appearance; Colorless powder (2) Molecular weight; SI-M8 m/z 7286
(M”) (3) Molecular formula; 012H22N4°4 (4
) Specific rotation; (a), +s1. b°(a
t, H2o) (5) Melting point; 〉 160
°C (dec,) (6) Solubility; Soluble in water and dimethyl sulfoxide. Slightly soluble in methanol. Insoluble in organic solvents such as acetone and ethyl acetate (7)
Rf value by silica gel thin layer chromatography Silica gel thin layer (K i e e e 1 g e 1
60 F2540.25 m Merck),
n-butanol:acetic acid:water (12: 515v/v%)
and n-propanol S pyridine: acetic acid diwater (15:
10 s 5 + 12 v/v%) of each developing solvent system, the results show Rf == 0.10 and 0.57.

(8) Ultraviolet absorption spectrum Absorb at the end.

(9) Infrared absorption spectrum The infrared absorption spectrum measured with Calicum bromide tablets is
As shown in the figure. The absorption maximum value (wave number cm-') is shown below.

3425,! 5350. ! 5200. 294
0°2B70. 174G, 16! Sol 16
00°1535, 1465t 1445. 14
00°1130, 1115. 1060. 1010
゜1 000, 980. 960. 8
90°865, 820. 780. 7
30° (10) Hydrogen Nuclear Magnetic Resonance Spectrum The hydrogen nuclear magnetic resonance spectrum measured in heavy water is shown in Figure 2.

(11) Carbon nuclear magnetic resonance spectrum Carbon nuclear magnetic resonance spectrum measured in heavy water (Figure 3)
The chemical shift (δ-value) is 2 2 1.0'5. 1 8 1.7 7. 1 7
5.2 9゜55.19. 50.26.
49.27°47.91. 47+75. 3
7.96°3G, 60. 25.97. 25
．． 50°22+25°.

HKB6-0186 of the present invention is expected to be used as a pharmaceutical agent such as an immunosuppressant as described below.

The present compound forms a salt with an acid, but the acid for forming the salt may be 1, for example, any pharmacologically acceptable acid.
For example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc. are preferred.

If the compound is used as an immunosuppressant.

Injection alone or mixed with excipients or carriers.

It is administered orally or as a suppository. Pharmaceutically acceptable excipients and carriers are selected, and their types and compositions are determined by the route and method of administration. For example, water, alcohols or soybean oil as a liquid carrier;
peanut oil, sesame oil.

Animal and vegetable oils such as mineral oil, or synthetic oils are used as solid carriers, sugars such as maltose and sucrose, amino acids, cellulose such as hydroxypropyl cellulose,
Stearic acid, a derivative of stearic acid! Organic acid salts such as gnesium are used. In the case of injections, generally physiological saline, various buffer solutions, saccharide solutions such as glucose, inositol, mannitol, ethyl glycol, phlopylene glycol, etc.

Glycols such as polyethylene glycol are preferred.

In addition, it is made into a lyophilized preparation together with excipients such as sugars such as inositol, mannitol, glucose, mannose, maltose, and sucrose, and amino acids such as phenylalanine. water.

It can also be administered by dissolving it in a liquid for intravenous administration, such as a glucose solution, an electrolyte solution, or an amino acid solution.

The content of this compound in the preparation varies depending on the preparation, but is usually 0.1 to 100% by weight, preferably 1 to 98% by weight.
i%. For example, in the case of injection solutions, 1 usually 0.1 to S
The active ingredient may be contained in an amount of 0% by weight, preferably 1 to 10% by weight. When administered orally, tablets, capsules, powders, granules, and liquid preparations may be used together with the solid carrier or liquid carrier.

It is used in the form of dry syrup, etc. capsule,
Tablets, granules and powders generally contain 5 to 100% by weight of active ingredient, preferably 25 to 98% by weight.

The dose is determined depending on the patient's age, weight, symptoms, therapeutic purpose, etc., but the therapeutic dose is generally 1 to 10 mg for parenteral administration.
0η/Kp・day, 5-50G+1+1i1/ by oral administration
It is KP day.

The present compounds have low toxicity, and each compound is characterized by low toxicity accumulation upon continuous administration.

This compound was administered intraperitoneally to mice at a dose of 800η/KP.
No signs of toxicity were observed after multiple administrations.

[For production]

This compound exerts a suppressive effect on the function of mouse lymphocytes, which are immunocompetent cells. That is, the method by Waithe et al.
Of Experimental engineering mmunolog
y p. 26, 1, 1978) Same as K.

When we investigated its effect on the lymphocyte rejuvenation response, we found that this compound inhibited the rejuvenation of T lymphocytes stimulated with Con A (concanavalin A) and the rejuvenation of 3977 lymphocytes stimulated with LPS (lipopolysaccharide). Rejuvenation reaction was significantly suppressed.

Next, the pharmacological action of the present compound will be specifically explained using test examples.

Test Example I Inhibition of T 1777 blastogenesis reaction by Con A BA
Pound the tile cells of LB10 mice into microplates 2X.
Dispense the test compound to 105 cells/0.2 ml/well, add each concentration of the test compound to each well except for the control group, and add 5 μg/t of Con A to all wells.
After adding the cell suspension to 37°C,
The cells were cultured for 72 hours in a 5% carbon dioxide incubator. For the lymphocyte regeneration reaction, 1 μC1/well of 3H-thymidine was added 6 hours before the end of the culture, and the amount taken up into the cultured cells was measured using a liquid scintillation counter. The uptake count when only Con A was added was Adpm, Co
n When A and the drug are added f) tJ'17 to Bdp
As m, (1-Bdpm/Aapm) x
The value of loo was taken as the inhibition rate of each drug against juvenile rejuvenation. The results are shown in Table 1.

Table 1 Inhibition temperature of the T17 lymphocyte blastogenesis reaction by con A K of this compound (pttaka) Inhibition rate (chi) 0.8 404.0
9020.0
100 Control (no drug added) 0 As is clear from the above table, this compound strongly suppressed the T lymphocyte blastogenesis reaction.

Test Example 2 Inhibition of B-lymphocyte blastogenesis by LPS (lipopolysaccharide) According to the method of Test Example 2 (however, E. coli IIPB was added to 100 μf/d instead of conA), young The amount of 3H-thymidine incorporated into the cultured B cells was measured. The inhibition rate by the test compound was determined in the same manner.

As shown in Table 2, one compound significantly inhibited LPS-induced 3977 sphere juvenile development.

Table 2 Inhibition temperature (μf/d') of B17 lymphocyte blastogenesis by LP13 of this compound Inhibition rate (chi) 0.16
210.8
644・0
90 controls (no drug added)
0 [Effects of the Invention] From the above results, 1 NKB6-0186 of the present invention has extremely low acute toxicity and exhibits excellent immunosuppressive effects such as suppressing the function of T lymphocytes and 3977 cells, and is superior to conventional immunosuppressants. It is thought that the mechanism of action is different from that of 1, and it is considered to be a new immunosuppressant with fewer side effects.

An example of the present invention will be shown below, but this is merely an illustration and is not intended to limit the present invention in any way, and various modifications may be made.

Example 1 2% soluble starch in a 500ml Erlenmeyer flask.

Glucose 0.5%, peptone 0.5%, yeast extract 0
．． 5%, potassium phosphate 9m 0. Os%, Sulfur fR Mac4
Medium containing 0.05 tons of Si and 0.5% soybean flour (pH 7.2
) 10 Q mg was dispensed and sterilized in an autoclave at 120°C for 20 minutes. This was inoculated with one loopful of strain NK86-01136 (Feikoken Bacteria No. 9487).

In a rotary shaker, culture was carried out at 190 revolutions/min at 27° C. for 2 days. Separately, add glycerin A%, polypeptone O, and SL to a 500 d Erlenmeyer flask.
Dispense 100R1 of a medium (pH 7.0) containing 0.3% powdered yeast extract, 0.5% meat extract, 0.3% sodium chloride, and 0.05% magnesium sulfate.

2 ml of the culture solution was transferred to a flask that had been sterilized in an autoclave at 120°C for 20 minutes, and cultured with shaking in a rotary shaker at T90 revolutions/minute at 27°C for 5 days. The culture in this method was treated with Celite as an auxiliary agent to obtain a culture p solution 201. Add this F solution to activated carbon bundle 0 (for Wako Tsumugi KK i chromatography) 2000aj
HKB6-0186 was adsorbed through a column packed with
After washing with water.

Elution was performed using a methanol linear concentration gradient elution method consisting of 3000 mg each of water and 80 timethanol water.

The active fractions were collected and concentrated to dryness under reduced pressure to obtain 15.71 f of a yellow-brown crude powder.

Next, this powder was dissolved in a small amount of citric acid buffer (sodium ion concentration 0.05 mol, pH 2.6).

BP-Sephadec JC-2 equilibrated with the same buffer in advance
51500d column, the buffer and 1.0
The sample was eluted with a linear concentration gradient elution method consisting of 3000 molar salt in the same buffer solution each containing 3,000 ml of the same buffer solution to obtain a salt sample with a concentration of 0.
An active fraction of 6 to 0.7 mol was obtained. This was collected, neutralized with a 1 mol aqueous sodium hydroxide solution, adsorbed on a column with a 50R/cm bundle of activated carbon, and washed with an aqueous sodium chloride solution and water.

The active fraction was collected by desalting operation using 80% methanol water and concentrated to dryness under reduced pressure. The resulting pale yellow powder 313.4η was dissolved in a small amount of pyridine:formate buffer (pyridine concentration 0.2 mol, pH 3.02) and equilibrated with the buffer beforehand.
500d of the above buffer solution, then the same buffer solution (pyridine concentration 0.2M, pH 3,60) SOOsj
It was eluted. The obtained active fractions were collected, neutralized with 0.1M aqueous ammonia under water cooling, concentrated under reduced pressure, and then freeze-dried.

The obtained white powder was dissolved in a small amount of 50% methanol water and Sephadec pLH2050 equilibrated with the same solvent in advance.
The active fraction was collected by desalting by passing through a column of 0jIj and eluting with the same solvent, concentrated under reduced pressure, and lyophilized.
NK86-ota6 colorless powder 76, IT! 1g was obtained.

[Brief explanation of the drawing]

Figure 1 shows NK86-01 measured as a potassium bromide tablet.
86 infrared absorption spectrum. FIG. 2 is a hydrogen nuclear magnetic resonance spectrum of NK86-0186 measured in heavy water. FIG. 3 is a carbon nuclear magnetic resonance spectrum of NK86-0186 measured in heavy water. Patent applicant Nippon Kayaku Co., Ltd.

Claims (1)

[Claims] New substance NK86-0186 characterized by having the following physical and chemical properties (1) Appearance; colorless powder (2) Molecular weight; SI-MSm/z; 286 (M^+) (
3) Molecular formula; C_1_2H_2_2N_4O_4(4)
Specific optical rotation; [α]^2^0_D+31.6° (CI, H
_2O) (5) Melting point: >160°C (dec.) (6) Solubility: Soluble in water, slightly soluble in methanol. (7) Rf value determined by silica gel thin layer chromatography n-butanol:acetic acid:water (12:3:5v/v%) and n-propanol:pyridine:acetic acid:water (15:1
0:3:12v/v%) developing solvent system shows Rf=0.10 and 0.57. (8) Ultraviolet absorption spectrum: Absorbs at the end. (9) Infrared absorption spectrum: The spectrum measured with potassium bromide tablets is as shown in FIG. (10) Hydrogen nuclear magnetic resonance spectrum: The spectrum measured in heavy water is as shown in Figure 2. (11) Carbon nuclear magnetic resonance spectrum; the spectrum measured in heavy water is as shown in FIG. (12) Color reaction; positive for ninhydrin reaction and Lydon-Smith reaction, negative for Pauli reaction.