JPH05271267A - Fr901459 substance, and its production and use - Google Patents

Abstract

PURPOSE:To provide the subject new compound exhibiting an excellent immunodepression effect and useful, e.g. as an inhibitor of a rejection of kidney transplantation, etc., and a medicine for preventing and improving autoimmune disease. CONSTITUTION:A compound of the formula, having following physical properties; Melting point: 156 to 158 deg.C; Specific rotatory power: [alpha]<23>D=-230 deg. (C=1.0, CHCl3); Elementary analysis (%): C 60.09, H 9.31 and N 12.34; Infrared absorption spectrum: 3350, 2960, 1635, 1520, 1485, 1470, 1410, 1390, 1370, 1340, 1310, 1290, 1270 and 1095cm<-1>. The compound of the formula can be obtained by culturing a cell strain belonging to Stachybotrys, e.g. Stachybotrys chartarum (FERM-BP-3364) in an aeration-agitation condition at 14 to 36 deg.C for 1 day to 1 week.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to FR901459 substance,
It relates to its manufacture and use. FR90145
Nine substances are novel substances that have been unknown and have been isolated and collected from cultures of Stachybotrys sp., Exhibit excellent immunosuppressive action, suppress agents for rejection due to organ transplantation, preventive and therapeutic agents for autoimmune diseases, etc. It is useful as various medicines.

[0002]

2. Description of the Related Art The living body has a mechanism for suppressing the immune response to self-components.
An immune response to self-components occurs, and various symptoms called autoimmune diseases based on pathological changes due to antigen-antibody reaction appear. Examples of this autoimmune disease include hemolytic anemia, uveitis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I purulent disease, rheumatoid arthritis, rheumatic fever, etc. However, it is well known that many of these exhibit relatively serious symptoms.

However, these autoimmune diseases are
As is generally said to be an incurable disease, there is currently no effective treatment.

On the other hand, biological organ transplants and implantable artificial organs are always accompanied by rejection reactions as a result of immune response (for example, organs or tissues such as heart, kidney, liver, bone marrow, and skin). Rejection reaction to transplantation, graft-versus-host reaction caused by bone marrow transplantation, etc.), if this rejection reaction is not suppressed, organ transplantation or implantation of artificial organs becomes impossible,
The precious human life will be lost.

[0005]

DISCLOSURE OF THE INVENTION The present invention is strongly desired to develop an excellent immunosuppressive agent in the fields of various medical treatments such as suppression of rejection reaction associated with living organ transplantation, prevention and treatment of autoimmune diseases. In view of the above, it has been made in order to further develop a novel immunosuppressive substance of a type different from the known ones.

[0006]

The present invention has been made to achieve the above object.

[0007] Therefore, the present inventors came to pay attention to the natural products from the viewpoint of safety and to pay attention to the fermentation products of the microorganisms. As a result of searching various microorganisms, they were newly separated from the soil collected in Ogasawara. Imperfect bacteria No. It was discovered that strain 19392 accumulated the target substance in the culture medium. Furthermore, when the physicochemical properties of this substance were studied in detail, it was confirmed that the substance was a novel substance that was previously unknown, and this substance was newly named FR901459 substance, and as a result of further research, its industrial production method was confirmed. Established and completed the present invention.

The FR901459 substance according to the present invention has the physicochemical properties shown in Table 2 below.

[0009]

[Table 2]

From the above physicochemical properties and other studies, it has been found that the chemical structural formula of FR901459 substance is as shown in the following chemical formula 1.

[0011]

[Chemical 1]

The FR901459 substance according to the present invention is, for example, an incomplete bacterium No. 1 newly isolated from a soil sample collected by the present inventors in Hahajima, Ogasawara Islands, Tokyo. 19392
Produced by stock.

Incomplete bacterium No. The 19392 strain spreads suppressively on various media, forming brown to olive colonies. No. The 19392 strain formed an anamorph consisting of a conidia stalk clearly distinguishable from vegetative hyphae, a conidia-forming cell covering the apex thereof, and a conidial mass on various media. The conidia formation method was the fiaro type. Also, no teleomorph was formed. From these morphological characteristics, N
o. The 19392 strain is a deficient fungus Stachybotrys sp.
e genus Stachybotrys Corda
1873). The mycological properties of this bacterium are shown below.

[0014]

[(1) Culture Properties] The culture properties on various media are shown in Table 3 below. Inoculate the center of corn-meal agar,
Growth was suppressed when cultured at 25 ° C for 14 days, and spread to a diameter of 1.5 to 2.0 cm. The surface of the settlement was flat, powdery, and yellowish brown. It also produced a red soluble pigment. The back of the village was brownish gray. Rich in anamorphs. When the same culture was carried out on potato dextrose agar, the growth was inhibitory (diameter 2.0-2.5 cm). The surface of the community was bulged, felt-like, and brownish gray. It also produced a red soluble pigment. The back of the village was brown. Anamorphs were abundant.

[0015]

[Table 3]

[0016]

[(2) Morphological characteristics] The morphological characteristics were observed based on the growth on corn-meal agar medium. Conidia structure is mono- or gregarious, erect, with septa. The conidia peduncle was clearly distinguished from vegetative hyphae, the base was colorless, the tip was dark olive-colored, and the upper part was often a fine rough surface. These had a length of 70 μm, a width of 2.5 to 3.5 μm, a slightly swollen tip, and 5 to 9 phialides were formed. The phialide is obovate, initially colorless and later dark olive-colored, smooth, length 7.5-11.5 μm, width 4.5-
It had a clear color at 5.5 μm and formed conidia. Conidia 1 cell, initially colorless, dark olive-gray when mature, smooth or zonal ridge, oval, 6.5-
Conidial mass was formed at the tip of the phialide at 8.5 × 3.0-4.0 μm. Vegetative mycelium has a septum and is colorless, smooth and branched. Mycelial cells are cylindrical, width 1.0-4.5
μm.

No. The 19392 strain can grow at 8-35 ° C, and the optimum growth temperature is 28-32 ° C (measured on potato dextrose agar medium).

No. The 19392 strain is Stachybotrys chaltalum (S
tachybotrys chartarum Hug
es1958). Furthermore, the above N
o. The characteristics of the 19392 strain are Jung and Divis (Jo
ng and Davis) (reference (1))
, Except for colony size and soluble pigment production. Therefore, this production strain was designated as Stachybotrys chaltalum. 19392 (Stachybotrys
chartarum No. 19392).

No. 19392 strain has been deposited in the Institute of Microbial Technology, Ministry of International Trade and Industry, Ministry of International Trade and Industry (accession number F
ERM BP-3364, Deposit Date: April 16, 1991
Day).

It should be understood that the production of FR901459 material is not limited to the use of the particular microorganisms described herein, merely for illustrative purposes. This invention includes artificial mutants and natural mutants obtainable from the described microorganisms by mutation treatment such as X-ray irradiation, ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, and 2-aminopurine. FR901459
It also includes the use of all mutants capable of producing the substance.

The FR901459 substance of the present invention is a substance-producing bacterium belonging to the genus Stachybotrys (eg Stac).
hybotrys chartarum No. 193
92) can be produced by inoculating a nutrient medium containing a carbon and nitrogen source capable of assimilation and culturing under aerobic conditions (eg, shaking culture, aeration and agitation culture).

As the carbon source, glucose, sucrose, starch, modified starch, fructose, glycerin and other carbohydrates are preferably used.

As the nitrogen source, oatmeal, yeast extract, peptone, gluten meal, cottonseed flour,
It is preferable to use cottonseed oil meal, soybean flour, corn steep liquor, dry yeast, wheat germ, peanut flour, chicken bone meat meal, etc., but ammonium salts (for example, ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.)
Inorganic and organic nitrogen compounds such as urea and amino acids can also be used advantageously.

It is advantageous to use these carbon sources and nitrogen sources in combination, but it is not necessary to use pure ones. This is because an impure substance may be advantageous in some cases because it may contain a growth factor or a trace element.

If necessary, the following inorganic salts may be added to the medium: sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, sodium iodide, iodine. Potassium chloride, magnesium salt, copper salt, cobalt salt, etc.

In particular, if the medium strongly foams, liquid paraffin, animal oil, vegetable oil, mineral oil, silicone or the like may be added when necessary.

For large-scale industrial production of the target substance, it is preferable to carry out aeration-agitation culture as in the case of other fermentation products. For small-volume production, shake culture using a flask is suitable.

When the culture is carried out in a large tank, F
In order to prevent the growth delay of the bacteria in the production process of the R901459 substance, it is preferable to first inoculate and culture the produced bacteria in a relatively small amount of medium, then transfer the culture to a large production tank and carry out the production culture there. In this case, the composition of the medium used for the pre-culture and the composition of the medium used for the production culture may be the same or may be different if necessary.

The culture is preferably carried out under aeration and stirring conditions,
For example, known methods such as stirring with a propeller or other machine, rotation or shaking of a fermenter, pumping, blowing of air are appropriately used. Use sterilized air for ventilation.

The culturing temperature may be appropriately changed within the range in which the present FR901459 substance-producing bacterium produces this substance, but it is usually 1 to 40 ° C., preferably 14 to 36 ° C. The culturing time varies depending on the culturing conditions and the culturing amount, but is usually about 1 day to 1 week.

After completion of fermentation, the desired FR9 was obtained from the culture.
01459 material is recovered. That is, bacterial cells are directly extracted with water and / or an organic solvent, or disrupted mechanically or by a known means such as ultrasonic waves, and then extracted with water and / or an organic solvent, followed by a conventional method. Recover and purify according to. In the case of a culture solution, it may be directly recovered and purified by a conventional method.

As a method for recovering and purifying, for example, water extraction, organic solvent, solvent extraction with a mixed solvent thereof; chromatography; recrystallization from a single solvent or a mixed solvent, or a conventional method is appropriately used alone or in combination. it can.

The FR901459 substance is recovered and purified by appropriately utilizing the known method as described above, but it may be carried out as follows, for example. First, the acetone extract of the culture,
Extract with ethyl acetate, hexane, or a mixed solvent thereof, and evaporate or distill the extract to concentrate it. The concentrated residue may be chromatographed or recrystallized and further lyophilized if necessary.

The pharmaceutical composition according to the present invention is FR9014.
In addition to oral administration agents in the form of solids, semi-solids or liquids, parenteral administration agents such as external preparations are prepared by adding 59 substances and / or salts thereof as an active ingredient to an inorganic or organic carrier that is commonly used. Formulate.

The preparations for oral administration include tablets, pills, granules, soft and hard capsules, powders, fine granules, powders,
Examples thereof include emulsions, suspensions, syrups, pellets and elixirs. Formulations for parenteral administration include
Injection, drip, infusion, ointment, lotion, tonic,
Sprays, suspensions, oils, emulsions, suppositories and the like can be mentioned.
To formulate the active ingredient of the present invention, a conventional method may be followed, including a surfactant, an excipient, a coloring agent, a flavoring agent, a preservative,
Stabilizers, buffers, suspensions, isotonic agents and other commonly used adjuvants are used as appropriate.

The dose of the pharmaceutical composition according to the present invention varies depending on the type, the type of disease to be treated or prevented, the administration method, the age of the patient, the symptoms of the patient, the treatment time, etc.
In the case of intravenous administration, the active ingredient (FR
901459 substance) 0.01 to 1000 mg / kg,
Preferably, 0.1 to 100 mg / kg is administered, the same is 0.01 to 1000 mg / kg in the case of intramuscular administration, preferably 0.1 to 100 mg / kg, and the same is 0.5 to 2000 mg / kg in the case of oral administration. kg, preferably 1
It is administered within the range of 1000 mg / kg.

Hereinafter, the present invention will be described in more detail with reference to Examples.

[0038]

Example 1

[0039]

[(1) Fermentative production of FR901459 substances] Sucrose 4%, cottonseed oil cake 2%, dry yeast 1%, peptone 1
%, KH ₂ PO ₄ 0.2%, CaCO ₃ 0.2%, Tween 80 0.1%, Adekanol LG-109 (manufactured by Asahi Denka) 0.2
Pre-culture medium consisting of 5% was dispensed into each of 10 500 ml Erlenmeyer flasks, 120 ml each.
Sterilized at 25 ° C for 30 minutes. NO.
19392 strain (Stachybotrys chart
arum No. 19392 strain (FERM BP-33
64)) slant culture was inoculated with 1 platinum loop each, and shake-cultured at 25 ° C. for 3 days.

Next, modified starch (Modified st
arch, MS 3600 (manufactured by Japan Food Processing)) 6%,
2% wheat germ, 2% corn steep liquor, 2 soybean flour
%, (NH ₄ ) ₂ SO ₄ 1%, NaNO ₃ 0.2%, C
aCO ₃ 0.2%, Adecanol (Adekano)
l) LG-109 0.025%, Silicon (Sili
Cone) pH of medium consisting of 0.025% KM70
Was adjusted to 6.1 to prepare a main culture medium, and 150 l of this main culture medium was injected into a 200 l jar fermenter. After sterilizing this at 125 ° C. for 30 minutes, the whole amount of the preculture obtained above was inoculated and cultured at 25 ° C. for 4 days.
The stirring was performed at 200 rpm and the aeration rate was 100 l / min.

The amount of FR901459 substance in the culture is
HPLC [column: YMC AM-303 (S-5, 1
20A, 4.6mm ID x 250mm) YMC C
o. , LTD (Kyoto, Japan); Solvent: 9
0% methanol, 2.5 mM KH ₂ PO ₄ buffer, pH
7.0; Detection: UV 210 nm; Flow rate 1 ml / m
in]. As a test sample, an equal amount of acetone was added to all broths, filtered, and then concentrated to an appropriate amount.

[0042]

[(2) Extraction and purification of FR901459 substance] Table 4 below
The FR901459 substance was extracted and purified according to the flow of 1.

[0043]

[Table 4]

50 l of the fermentation broth obtained above was mixed with 6 N
The pH was adjusted to 8.9 with NaOH, and an equal amount of acetone (40 l) was added thereto, followed by thorough stirring. After leaving this at room temperature for 24 hours, radio light (manufactured by Seiwa Chemical Industry Co., Ltd.)
2 Kg was added and filtered with a filter press. The obtained filtrate (80 l) was applied to a column of DIAION HP-20 (manufactured by Mitsubishi Kasei) 5 l (SV = 5), washed with 15 l of 50% acetone water, and then eluted with 15 l of 90% acetone water. 5 l of deionized water was added to this elution zone to dilute it. This was applied to a 4 l activated carbon (manufactured by Wako Pure Chemical Industries) column filled with 50% acetone water, and 50% acetone water 20
1, washed with 20 l of deionized water, and then eluted with 40 l of ethyl acetate. The elution zone was concentrated under reduced pressure, dehydrated with sodium sulfate, and then dried. The residue was dissolved in 1.2 l of a mixture of hexane and acetone (11: 2), and applied to 1.4 l of a silica gel column filled with a mixture of hexane and acetone (5: 1). After developing with hexane: acetone = 3: 1, 4.5 l, elution was performed with hexane: acetone = 2: 1, 10 l.
The active area was concentrated to dryness, dissolved in ethyl acetate, and hexane was added to powderize the active area. As a result, 34 g of a white powder of the target substance was obtained.

[0045]

[(3) Physicochemical Properties of FR901459 Substance] The physicochemical properties of the FR901459 substance thus obtained are as shown in Table 2. The amino acid analysis was performed as follows. First, 1 mg of FR901459 substance was sealed in a sealed tube using 6N HCl (1 ml).
It was hydrolyzed at 10 ° C. for 20 hours. After completion of the hydrolysis, the reaction mixture was evaporated to dryness, which was then analyzed by an amino acid analyzer (Hita).
chi 835 Automatic Amino-A
Cid Analyzer). The results are Thr (1), Sar (1), Ala (2), Le.
It was u (2). It was found that its chemical structure was chemical formula 1.

[0046]

[Example 2: Inhibitory effect of FR901459 substance on in vitro mixed lymphocyte reaction (MLR)] C5 in each well
5 × 10 ⁵ 7BL / 6 responsive cells (H-2b) and BALB / C stimulated cells (H- treated with mitomycin C (treated with 258 μg / ml of mitomycin) at 37 ° C. for 30 minutes and washed 3 times with RPMI1640 medium) 2d) 5x1
0 ⁵ to 0.2 ml of 10% fetal calf serum-added PRMI16
40 medium [sodium hydrogen carbonate 2 mM, penicillin (5
0 unit / ml) and streptomycin (50 μg / ml)
ml) is added] to the microtiter plate. The cells were cultured at 37 ° C. for 68 hours in an incubator kept at 100% humidity, 5% carbon dioxide and 95% air, and pulsed with ³ H-thymidine (0.5 μCi) for 4 hours to incubate the cells. Collect. The target compound of the present invention is dissolved in ethanol, diluted in RPMI1640 medium and added to the culture so that the final concentration is 0.1 μg / ml or less.

The results are shown in Table 5 below. FR of this invention
The substance 901459 was carried out to suppress mouse MLR.

[0048]

[Table 5]

[0049]

Example 3: FR901459 substance for survival of allogeneic skin grafts in rats Example 3: Effect of FR901459 substance for survival of allogeneic skin grafts in rats Donor (Fisher) rat abdominal skin graft recipient (WKA)
Implant in the lateral chest of rats. Remove bandages on day 5. Examine the grafts daily until there is a rejection reaction in which more than 90% of the graft epithelium is necrotic.

The FR901459 substance is dissolved in olive oil and administered intramuscularly for 14 consecutive days starting from the day of transplantation.

As shown in Table 6 below, 1 part of olive oil was added.
Skin allografts are all rejected within 8 days in rats that have been intramuscularly administered for 4 consecutive days.
Daily treatment with 9 substances clearly prolonged skin allograft survival.

[0052]

[Table 6]

From the above results, it was revealed that the FR901459 substance has an immunosuppressive action, and FR901459
It has been found that the substance is useful, for example, in suppressing rejection associated with transplantation and in the preventive treatment of autoimmune diseases.

When a test was conducted on the acute toxicity of the FR901459 substance by intraperitoneal administration in ddy mice, no death was observed at a dose of 100 mg / kg, confirming that it is highly safe.

[0055]

[Example 4: Production of injection] (1) Substance produced in Example 1 5g (2) Food salt 9g (3) Chlorobutanol 5g (4) Sodium hydrogencarbonate 1g

All the components (1) to (4) were added to 100 m of distilled water.
After dissolving in 1 l, 1 ml each was dispensed into ampoules to produce 1000 injections.

[0057]

INDUSTRIAL APPLICABILITY The present invention provides a FR901459 substance, which is a previously unknown novel pharmacologically active substance, exhibits excellent immunosuppressive action, and has a rejection reaction associated with medicines such as organ transplantation. It is very useful as a suppressive agent, a preventive and / or therapeutic agent for autoimmune diseases and the like.

Since the structure of the active ingredient according to the present invention has also been clarified, in addition to industrial production by microorganisms, production by organic synthesis is possible, and as a result, various derivatives can be produced. The production of compounds and the development of new applications can also be expected.

[Brief description of drawings]

FIG. 1 is a drawing showing a 13C nuclear magnetic resonance spectrum of FR901459 substance.

FIG. 2 is a drawing showing 1H nuclear magnetic resonance spectrum of FR901459 substance.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI technical display location C12R 1: 645) (72) Inventor Shigehiro Takase 1-12-10 Ishikawa City Soja, Ibaraki 72) Inventor Michio Yamashita 3-11-11 Namiki, Tsukuba City, Ibaraki Prefecture (72) Inventor Masakuni Okuhara 2-14-10 Umezono, Tsukuba City, Ibaraki Prefecture

Claims (3)

[Claims] 1. A FR901459 substance having the physical properties shown in Table 1 below. [Table 1] 2. Stachybotr (Stachybotr)
A method for producing a FR901459 substance, which comprises culturing a FR901459 substance-producing bacterium belonging to the genus ys) to produce the FR901459 substance, and collecting the FR901459 substance. 3. An immunosuppressive agent comprising the FR901459 substance as an active ingredient.