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Title:
PRODUCTION OF AN IMMUNOSUPPRESSANT USING MICROORGANISMS
Document Type and Number:
WIPO Patent Application WO/1997/009298
Kind Code:
A1
Abstract:
This invention relates to a new bioactive compound, WF10917 substance or a salt thereof, having immunosuppressing activity, to a process for preparation thereof and to a pharmaceutical composition comprising the same, said compound having formula (I).

Inventors:
HINO MOTOHIRO (JP)
HATANAKA HIROSHI (JP)
TAKASE SHIGEHIRO (JP)
TSURUMI YASUHISA (JP)
KINA TORU (JP)
HASHIMOTO SEIJI (JP)
Application Number:
PCT/JP1996/002309
Publication Date:
March 13, 1997
Filing Date:
August 16, 1996
Export Citation:
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Assignee:
FUJISAWA PHARMACEUTICAL CO (JP)
HINO MOTOHIRO (JP)
HATANAKA HIROSHI (JP)
TAKASE SHIGEHIRO (JP)
TSURUMI YASUHISA (JP)
KINA TORU (JP)
HASHIMOTO SEIJI (JP)
International Classes:
A61K31/215; A61P17/00; A61P35/00; A61P37/06; C07C229/30; C12P13/00; (IPC1-7): C07C229/30; A61K31/20; C12P13/00; C12P13/04
Foreign References:
EP0410176A11991-01-30
Other References:
S. SASAKIM ET AL.: "Fungal metabolites. Novel potent immunosuppressants, mycestericins, produced by mycelia sterilia", JOURNAL OF ANTIBIOTICS, vol. 47, no. 4, 1994, TOKYO JP, pages 420 - 433, XP002018194
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Claims:
CLAIMS
1. A WF10917 substance of the formula: ( I ) » or a salt thereof.
2. A process for production of the WF10917 substance or a salt thereof, which comprises culturing Fungus No. 10917 (FERM BP 5223) in a nutrient medium and recovering the same.
3. Biological pure culture of Fungus No. 10917 (FERM BP5223).
4. A pharmaceutical composition containing WF10917 substance or a salt thereof.
5. A use of the WF10917 substance or a salt thereof as a medicament.
6. A method for treating or preventing rejection by transplantation, graftversushost diseases by medulla ossium transplantation, autoimmune diseases and psoriasis which comprises administrating the WF10917 substance or a salt thereof to human or animal.
7. Use of the WF10917 substance or a salt thereof for the manufacture of a medicament for therapeutic treatment or prevention of rejection by transplantation, graftversushost diseases by medulla ossium transplantation, autoimmune diseases and psoriasis in human or animal.
8. A compound of claim 1 for use as a medicament.
Description:
DESCRIPTION

PRODUCTION OF AN IMMUNOSUPPRESSANT USING MICROORGANISMS

TECHNICAL FIELD

This invention relates to a new bioactive compound, hereinafter entitled WF10917 substance or a salt thereof which are useful as a medicament.

DISCLOSURE OF INVENTION

The. present invention relates to a new bioactive compound, WF10917 substance.

More particularly, it relates to a novel compound, WF10917 substance or a salt thereof which has immunosupressing activity, to a process for preparation thereof, to a pharmaceutical composition comprising the same, which is useful as immunosupressing agents, and to a use thereof as a medicament.

Accordingly, one object of this invention is to provide the novel compound, WF10917 substance or a salt thereof which is of use for treating and preventing rejection by transplantation, graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases, psoriasis, and the like.

Another object of this invention is to provide a process for production of the WF10917 substance or a salt thereof by fermentation of a WF10917 substance-producing strain belonging to the fungi in a nutrient medium.

A further object of this invention is to provide a pharmaceutical composition containing, as an active ingredient, the WF10917 substance or a salt thereof.

Still further object of this invention is to provide a use of the WF10917 substance or a salt thereof for treating and preventing rejection by transplantation, graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases, psoriasis, and the like.

The WF10917 substance of the present invention can be represented by the following formula:

( I )

Suitable salt of the WF10917 substance (I) is conventional pharmaceutically acceptable salt and includes a metal salt such as an alkali metal salt (e. g. sodium salt, potassium salt, etc.) and an alkaline earth metal salt (e. g. calcium salt, magnesium salt, etc.), an ammonium salt, an organic base salt (e. g. trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N, N'- dibenzylethylenediamine salt, etc.), an organic acid salt (e. g. acetate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, formate, toluenesulfonate, etc.), an inorganic acid salt (e. g. hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, etc.), or a salt with an amino acid (e. g. arginine, aspartic acid, glutamic acid, etc.), and the like.

The WF10917 substance can be produced by fermentation of the WF10917 substance-producing strain belonging to the fungi such as Fungus No. 10917 in a nutrient medium.

It is to be understood that the production of the WF10917 substance

is not limited to the use of the particular organism described herein, which is given for the illustrative purpose only. This invention also includes the use of any mutants which are capable of producing the WF10917 substance including natural mutants as well as artificial mutants which can be produced from the described organism by conventional means such as irradiation of X-ray, ultra-violet radiation, treatment with N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine, and the like.

Particulars of Fungus No. 10917 is as follows:

Characteristics of producing strain No.10917

The fungus strain No.10917 was originally isolated from a soil sample, collected in Shingu-shi, Wakayama-ken, Japan. This organism grew very restrictedly on various culture media, and formed olivaceous colonies. Neither teleomorph nor anamorph of strain No.10917 was observed on various media or on an autoclaved leaf affixed to a corn meal agar plate. This strain did not also form differentiated hyphal structures: sclerotia, bulbils, chlamydospores and clamp connections. The lack of definite characteristics suggests that strain No.10917 might be classified as a member of the Mvcelia Sterilia (Hyphomycetes). Its mycological characteristics were as follows.

Cultural characteristics on various agar media are summarized in Table 1. Culture on corn meal agar grew very restrictedly, attaining 1.0-1.5 cm in diameter two weeks later at 25 °C. The colony surface was plane, felty and dark green. The reverse was the same color. Reproductive structures were not observed. Vegetative hyphae were smooth, septate, hyaline or sometimes dark brown, and branched. The hyphal cells were cylindrical and 1.0 - 4.0 μm in width. Chlamydospores were absent. Colonies on potato dextrose agar grew the similar rate as on corn meal agar. The surface at the center was raised, floccose and brownish gray, while that at the margin was plane, felty, producing colorless exudations and dark gray. The reverse was brownish gray and produced small amounts of orange soluble pigment. Both teleomorph and anamorph were not formed. On this medium, vegetative hyphae were rather darker than those on corn meal agar,

and sometimes funiculose. The hyphal cells were filamentous to cylindrical, sometimes ellipsoidal, and 1 - 5 μm in width.

Strain No.10917 was able to grow at the temperature range from 6 to 34°C, with the growth optimum at 28 to 30°C. These temperature data were determined on potato dextrose agar (made by NISSUI).

Mvcelia Sterilia, that is an asporogenic fungal group, is taxonomically heterogeneous. Thus, because of an uncertainty in the classification of this organism, it has been referred to simply as Fungus No.10917. This strain was deposited in National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) as FERM BP-5223 (deposited date: September 1, 1995).

Table 1. Cultural characteristics of strain No.10917.

Media Cultural characteristics

Malt extract agar' 1 G: Restrictedly, 2.0 - 2.5 cm S: Circular to irregular, raised, cottony, producing colorless exudations, brownish gray (6F2) at the center and greenish gray (30F2) at the margin R: Dark green (28F3) to greenish gray (28F2)

Potato dextrose G: Very restrictedly, 1.0-1.5 cm agar (Difco 0013) S: Irregular, raised, floccose and brownish gray

(5F2) at the center; plane, felty, producing colorless exudations and dark gray (1F1) at the margin R: Brownish gray (5D-E2) and producing small amounts of orange soluble pigment

Media Cultural characteristics

Czapek's solution G: Very restrictedly, 1.0-1.5 cm agar* S: Irregular, plane, thin and olive (2E-F3) R: Olive (1F3)

Sabouraud dextrose G: Very restrictedly, 1.0-2.0 cm agar (Difco 0190) S: Irregular, raised, felty, olive (2F4) at the center, brownish gray (1F2) at the middle, grayish brown (6F3) at the margin R: Brown (7E4), and grayish brown (6F3) at the margin, producing orange soluble pigment

Emerson Yp Ss agar G: Very restrictedly, 1.0-1.5 cm

(Difco 0739) S: Circular, somewhat raised, felty, dark green

(29F4), and gray (1D1) at the margin R: Dark green (30F2), and olive (3F3) at the margin

Corn meal agar G: Very restrictedly, 1.0-1.5 cm (Difco 0386) S: Circular, plane, felty and dark green (28F4)

R: Dark green (27F4)

MY20 agar* G: Very restrictedly, 1.0-1.5 cm

S: Circular, raised, felty to wet, producing large amount of colorless exudations, pale gray (IB 1) at the center, grayish orange (5B4) at the middle, and orange white (5A2) at the margin

R: Grayish orange (5B3)

Abbreviation G: growth, measuring colony size in diameter, S: colony surface, R: reverse.

*: The compositions of malt extract agar, Czapek's solution agar and MY20 agar were based on JCM Catalogue of Strains (Nakase, T., 5th ed., 503p., Japan Collection of Microorganisms and Life Science Research Information Section of the Institute of Physical and Chemical Research, Saitama, 1992).

These characteristics were observed after 14 days of incubation at 25°C. The color descriptions were based on Methuen Handbook of Colour (Kornerup, A. and J. H. Wanscher, 3rd ed., 525p., Methuen, London, 1978).

Production of the WF10917 substance

The WF10917 substance is produced when the WF10917 substance- producing strain belonging the fungi is grown in a nutrient medium containing sources of assimilable carbon and nitrogen under aerobic conditions (e. g. shaking culture, submerged culture, etc.).

The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, sucrose, starch, fructose or glycerin, or the like.

The preferred sources of nitrogen are yeast extract, peptone, gluten meal, cotton seed flour, soybean meal, corn steep liquor, dried yeast, wheat germ, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e. g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea or amino acid, or the like.

The carbon and nitrogen sources, though advantageously employed in combination, need not to be used in their pure form because less pure materials, which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use.

When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, zinc salts, iron salts, or cobalt salts, or the like.

If necessary, especially when the culture medium foams seriously a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone, or the like may be added.

Agitation and aeration of the culture mixture may be accomplished

in a variety of ways, such as agitation by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermenter, and the like.

The fermentation is usually conducted at a temperature between about 10°C and 40°C, preferably 20°C to 30°C, for a period of about 50 hours to 150 hours, which may be varied according to fermentation conditions and scales.

When the fermentation is completed, the culture broth is then subjected for recovery of the WF10917 substance to various procedures conventionally used for recovery and purification of biological active substance, for instance, solvent extraction with an appropriate solvent or a mixture of some solvents, chromatography or recrystallization from an appropriate solvent or a mixture thereof.

The salt of the WF10917 substance can be prepared by a conventional manner, during or after the recovery and purification of the WF10917 substance.

The WF10917 substance as obtained has the following physico- chemical properties:

Appearance:

Colorless powder Molecular formula :

C 23 H 41 N0 6 Elementary Analysis:

Calcd for C 23 H 41 N0 6 H 2 0

C 62.00, H 9.73, N 3.14 Found:

C 62.76, H 10.29, N 3.09 Molecular weight:

ESI-MS: m/z 428 (M + H) + Melting point:

148 - 153 °C (dec.) Specific rotation:

[α] D (23°C) +48 ° (c=0.1, in methanol)

Ultraviolet absoφtion spectrum: λ χ (methanol): 230 nm

Solubility:

Slightly soluble: methanol, chloroform

Insoluble: water, dimethyl sulfoxide Color reaction:

Positive: eerie sulfate reaction, iodine vapor reaction, ninhydrin reaction

Negative: Molish reaction, Ehrlich reaction, ferric chloride reaction Thin layer chromatography (TLC):

Stationary phase Developing solvent Rf value

Silica Gel 60 2-propanol: water: acetic acid 0.4

F 254 * (90:10:0.1, v/v)

* made by E. Merck

High Performance Liquid Chromatography (HPLC): Condition:

Mobile phase: acetonitrile:water:trifluoroacetic acid (65:35:0.1, v/v)

Column: YMC ODS AM-303** (S-5, 12θA, 4.6mmID x 250mm)

Flow rate: 1.5 ml/minute

Detection: UV at 230 nm

Retention time: 7.7 minutes

** trade name: made by Yamamura Chemical Institute Infrared Spectrum: v max (KBr): 3560 ' 3120 ' 2920 ' 2850 ' 1740 ' 1720 ' 1670 ' 1590 '

1470, 1400, 1360, 1230, 1040, 1020, 990 cm "1 as shown in Fig. 1

H Nuclear magnetic resonance spectrum: (500 MHz, pyridine-dr) δ-rr

6.40 (IH dd, J=15.5 and 10.5Hz), 6.16 (IH, m), 6.06 (IH, dd, J=15.5 and 10.5Hz), 5.75 (IH, dd, J=15.5 and 7Hz), 5.68 (IH, m), 4.89 (IH, br d, J=llHz), 4.69 (IH, d, J=12Hz), 4.63 (IH, d, J=12Hz), 2.70 (IH, m), 2.35 (IH, m), 2.04 (2H, m), 1.80 (3H, s),

1.38 (2H, m), 1.32 - 1.20 (16H, m), 0.87 (3H, t, J=7Hz) as shown in Fig. 2

13 C Nuclear magnetic resonance spectrum:

(125 MHz, pyridine-d^) SQ

174.56 (s), 170.35 (s), 136.15 (d), 132.63 (d), 130.32 (d), 130.00 (d), 71.77 (d), 69.03 (s), 68.33 (d), 63.19 (t), 37.75 (t), 32.92 (t), 32.11 (t), 29.95 (t), 29.89 (t), 29.89 (t), 29.80 (t), 29.59 (t), 29.52 (t), 29.50 (t), 22.92 (t), 20.93 (q), 14.27 (q) as shown in Fig. 3. Nature:

Amphoteric substance

From the above physical and chemical properties, the WF10917 substance is inferred to have the following plane structural formula.

( I )

Biological properties of the WF10917 substance

The WF10917 substance (I) possesses pharmacological activities such as immunosuppressive activity, and the like, and therefore are useful for the treatment and prevention of immune-mediated diseases such as the resistance by transplantation of organs or tissue such as heart, kidney, liver, medulla ossium, skin, cornea, lung, pancreas, intestinum tenue, limb, muscle, nervus, etc.; graft-versus-host diseases by medulla ossium transplantation; autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, psoriasis, and the like.

Further, the WF10917 substance (I) is also useful for the treatment and the prophylaxis of inflammatory and hyperproliferative skin diseases and cutaneous manifestations of immunologically-mediated

illnesses, such as, psoriasis, atopical dermatitis, contact dermatitis and further eczematous dermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bϋllous Pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus, acne and Alopecia areata; various eye diseases such as autoimmune diseases and so on (e. g. keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's diseases, keratitis, heφetic keratitis, conical cornea, dystrophia epithelialis corneae, comeal leukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves' ophthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, etc.); reversible obstructive airways disease, which includes conditions such as asthma (e. g. bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma), particularly chronic or inveterate asthma (e. g. late asthma and airway hyper-responsiveness), bronchitis and the like; inflammation of mucosa and blood vessels such as gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel disease, inflammatory bowel disease, necrotizing enterocolitis, intestinal lesions associated with thermal burns, leukotriene B^-mediated diseases; intestinal inflammations/allergies such as Coeliac disease, proctitis, eosnophilic gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis; food related allergic diseases which have symptomatic manifestation remote from the gastro-intestinal tract, for example migraine, rhinitis and eczema; renal diseases such as interstitial nephritis, Goodpasture's syndrome, hemolytic-uremic syndrome and diabetic nephropathy; nervous diseases such as multiple myositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis, multiple neuritis, mononeuritis and radiculopathy; endocrine diseases such as hyperthyroidism and Basedow's disease; hematic diseases such as pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic puφura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia and anerythroplasia; bone diseases such as osteoporosis; respiratory diseases such as sarcoidosis, fibroid lung and idiopathic interstitial pneumonia; skin diseases such as dermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity and cutaneous T cell lymphoma; circulatory diseases such as arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa

and myocardosis (e. g. autoimmune myocarditis, virus myocarditis); collagen diseases such as scleroderma, Wegener's granuloma and Sjogren's syndrome; adiposis; eosinophilic fasciitis; periodontal disease such as lesion of gingiva, periodontium, alveolar bone, substantia ossea dentis; nephrotic syndrome such as glomerulonephritis; male pattern alopecia or alopecia senilis; muscular dystrophy; Pyoderma and Sezary's syndrome; Addison's disease; active oxygen-mediated diseases, for example, organ injury such as ischemia-reperfusion injury of organs (e. g. heart, liver, kidney, digestive tract) which occurs on preservation, transplantation or ischemic diseases (e. g. thrombosis, cardiac infarction): intestinal diseases such as endotoxin- shock, pseudomembranous colitis, colitis caused by drug or radiation: renal diseases such as ischemic acute renal insufficiency, chronic renal insufficiency: pulmonary diseases such as toxinosis caused by lung- oxygen or drug (e. g. paracort, bleomycins), lung cancer, pulmonary emphysema: ocular diseases such as cataracta, siderosis, retinitis, pigmentosa, senile macular degeneration, vitreal scarring, corneal alkali burn: dermatitis such as erythema multiforme, linear IgA ballous dermatitis, cement dermatitis: and others such as gingvatis, periodontitis, sepsis, pancreatitis, diseases caused by environmental pollution (e. g. air pollution), aging, carcinogenis, metastasis of carcinoma, hypobaropathy; diseases caused by histamine or leukotriene C4 release; and Behcet's disease such as intestinal-, vasculo-, or neuro-

Behcet's disease, and also the one which affects oral cavity, skin, eye, vulva, articulation, epididymis, lung, kidney and so on; and so on.

And further, the WF10917 substance (I) may have liver regenerating activity and/or activities of stimulating hypertrophy and hypeφlasia of hepatocytes. Therefore, they are useful for treatment and prevention of hepatic diseases such as immunogenic diseases (e. g. chronic autoimmune liver diseases such as the group consisting of autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis (e. g. necrosis caused by toxins, viral hepatitis, shock or anoxia), B -virus hepatitis, non-A/non-B hepatitis, cirrhosis and hepatic failure such as fulminant hepatic failure, late-onset hepatic failure and "acute-on-chronic" liver failure (acute liver failure on chronic liver diseases).

And further, the WF10917 substance (I) may be useful for various

diseases because of its useful pharmaceutical activity such as augmenting activity of chemotherapeutic effect, preventing of treating activity of cytomegalovirus infection, anti-inflammatory activity, and so on.

As examples for showing biological activities of the WF10917 substance, some biological data are explained in the following.

Test 1. Suppression of blastogenic responce of murine spleen cells by mitogen stimulation

The lymphocyte blastogenesis test was perfoπned in microtiter plates with each well containing 1 x 10 splenic cells of Balb/c mice in 0.1 ml RPMI1640 medium supplemented with 10% fetal bovine serum, 50mM 2-mercaptoethanol, penicilln (100 units/ml) and streptomycin (lOOμg/ml), to which lipopolysaccharide of E. coli (LPS) or anti CD3 antibody(2Cll) was added. The cells were incubated at 37°C in a humidified atmosphere of 5% CO for 96 hours. After the culture period, suppressive activities of the test samples in lymphocyte blastogenesis were quantitated by a MTT [3-(4,5-dimethylthiazolyl-2- yl)-2,5-diphenyltetrazolium bromide] dye reduction' assay.

The WF10917 was dissolved in methanol and further diluted in RPMI 1640 medium and added to the culture to give final concentrations of lOOOng/ml or less.

The results are shown in Table 2.

The WF10917 substance suppressed murine lymphocyte blastogenesis induced by anti CD3 antibody but not by LPS.

Table 2 Effect of the WF10917 substance on murine lymphocyte blastogenesis induced by anti CD3 antibody and LPS

Concentration Suppression of blastogenesis (%) (ng/ml) anti-CD3 LPS

0 0 0

10 25.2 5.9

20 46.5 -4.2

40 48.7 2.6

80 57.9 7.5

160 55.8 14.8

315 59.0 9.2

630 55.8 25.3

1250 64.5 43.3

2500 63.9 67.5

5000 48.2 91.5

Test 2. Effect of WF10917 substance on antibody production in mice

Female Balb/c mice were immunized with 2,4-dinitrophenylated Ascaris Extract (DNP- ascaris) and alum by intraperitoneal injection.

The WF10917 substance was dissolved in 10% (HCO-60 R )/saline and administered subcutaneously for 7 consecutive days, beginning at the day of immunization. Seven days after of immunization, serum were collected and antibody titer against DNP was determined by the ELISA. Briefly, 50ml of DNP conjugated bovine serum albumin was added to each well of microtiter plates and incubated for 16 hours at 4°C. The microtiter plates were washed with PBS containing 0.1% of Tween 80(T-PBS) and blocked with 0.5 % of bovine serum albumin- PBS. After washing with T-PBS, 50 ml of diluted mice sera were added and incubated for a hour at room temperature. One hour later, anti murine IgGl monoclonal antibody horse raddish peroxidase conjugate was added and incubated for one hour. After one hour incubation, plates were washed with T-PBS and added orthophenylenediamine dissolved in citrate buffer (pH4.5). Antibody titer were measured using a microplate absoφtion

spectrophotometer at 492 nm.

As shown in Table 3, the antibody production was markedly suppressed by the administration of the WF10917 substance.

Table 3 Effect of the WF10917 substance on antibody production in mice

: p< 0.05 ** : P<0.01 *** : P<0.005

The pharmaceutical composition of this invention can be used in the form of pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the WF10917 substance or its pharmaceutically acceptable salt, as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral administrations. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, injections, ointments, liniments, eye drops, lotion, gel, creme, and any other form suitable for use.

The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, com starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening, solubilizing and coloring agents and perfumes may be used.

For applying the composition to human, it is preferable to apply it

by intravenous, intramuscular, topical or oral administration. While the dosage of therapeutically effective amount of the WF10917 substance varies from and also depends upon the age and condition of each individual patient to be treated, in the case of individual patient to be treated, in the case of intravenous administration, a daily dose of 0.01 - 10 mg of the WF10917 substance per kg weight of human being, in the case of intramuscular administration, a daily dose of 0.1 - 10 mg of the WF10917 substance per kg weight of human being, in case of oral administration, a daily dose of 0.5 - 50 mg of the WF10917 substance of human being is generally given for treating.

Following examples are given for the puφose of illustrating the present invention in more detail.

Example 1

(1) Fermentation

An aqueous seed medium (60 ml) containing sucrose (3%), dried yeast (3%), cotton seed flour (1%), CaC0 (0.2%), Adecanol LG-109

(deforming agent, Asahi Denka Co., Ltd.) (0.05%), and Silicone KM- 70 (deforming agent, Shin-Etsu Chemical Co., Ltd.) (0.05%) (pH 6.3 adjusted with IN NaOH) was poured into a 250-ml Erlenmeyer flask and sterilized at 120°C for 30 minutes. A loopful of Fungus No. 10917 was inoculated from a slant culture into the flask. The flask was cultured on a rotary shaker (220 φm, 5.1 cm-throw) at 30°C for 5 days and then transferred at the rate of 2% to 160 ml of the same sterile seed medium in the 500-ml Erlenmeyer flasks. The flasks were shaken on a rotary shaker (220 φm, 5.1 cm-throw) at 30°C for 4 days. The resultant seed culture was inoculated to 20 liters of sterile production medium consisting of sucrose 4%, dried yeast 2%, CaCO? 0.5%,

Adecanol LG-109 0.05%, and Silicone KM-700.05% (pH 6.3) in a 30- liter jar fermenter. Fermentation was carried out at 25 °C for 4 days under aeration of 20 liters/minute and agitation of 250 φm.

(2) Isolation

The cultured broth (140 liters) was extracted with 140 liters of acetone-methanol (1:1) mixture by intermittent mixing. The acetone- methanol extract was filtered with an aid of diatomaceous earth and an equal volume of water was added. The resultant cake was reextracted with 70 liters of 80% aqueous methanol. The methanol extract was filtered with an aid of diatomaceous earth and 70 liters of water was added. These extract was combined and passed through a column (10.5 liters) of Diaion HP-20 (Mitsubishi Chemical Co., Ltd.). The column was washed with 50% aqueous methanol, 70% aqueous methanol, water, and ethyl acetate and then eluted with methanol. Twenty liters of water was added to the eluate (30 liters), and applied on a column (4 liters) of YMC gel (ODS-AM 120-S50, YMC Co,. Ltd.). The column was developed with 90% aqueous methanol. An active fraction (2 liters) was concentrated under reduced pressure to give the WF10917 substance in the form of colorless crystals (681 mg).