CA1143682A - Biologically active fr-900156 substance, a process for the preparation thereof and use thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A new biologically active substance designated FR-900156 is provided which is prepared by cultivating under aerobic conditions a FR-900156 substance producing strain belonging to the genus Streptomyces in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salt; the new substance is useful in the therapeutic treatment of infectious diseases caused by pathogenic microorganisms and of cancer in humans and animals.

Description

~36~

BIOLOGICALLY ACTIVE
FR-900156 SUBSTANCE, A PROCESS FOR THE
PREPARATION THEREOF AND
USE THEREOF

This invention is concerned with a novel com-pound having biologically active properties, herein-after referred to as I;R-900156 substance and its pharmaceutically acceptable salts, l~ith a process for the preparation thereof and with a pharmaceutical composition containing the same. More particularly, it is concerned with a novel FR-900156 substance and its pharmaceutically acceptable salts which have enhancing activities of immune response and reticulo-endotherial system and accordingly are useful for,for example, the therapeutic creatment of infectous diseases caused by pathogenic microorganism and of cancer in human being and animals, with a process '~
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Eor the preparation thereof and with pharmaceutical compositions containing the same. -Accordingly, it ls one object of this invention to provide a novel compound, FR-900156 substance and its pharmaceutically acceptable salts which are use-ful or the therapeutic treatment of infectious disease caused by pathogenic microorganism, especial-ly gram-negative bacteria and gram-positive bacteria and fungi, and oE cancer in human being and animals.
Another object of this invention is to provide a process for preparation of the FR-900156 substance by fermentation of a FR-900156 substance-producing strain belonging to the genus Streptomyces in a nutrient medium.
A further object o~ this invention is to provide a parmaceutical composition containing, as an effective ingredient, FR-900156 substance or its pharmaceutically acceptable salts.
Still another object of this invention is to provide a method using FR-900156 substance or its pharmaceutically acceptable salts for therapeutic treatment of infectious diseases caused by bacteria and cancer in mammal.
The FR-900156 substance o-f this invention can .25 be produced by fermentation of a FR-900156 substance-producing strain belonging to the genus Streptomyces such as Streptomyces olivaceogriseus, Streptomyces violaceus and the like in a nutrient medium.

3Z ', THE MICROORGANISM

, The microor'ganism which can be used for the production of the FR-900156 substance is a strain belonging to the genus Streptomyces, among ~hich a strain of Streptomyces olivaceogriseus and Streptomyces violaceus have been newly isolated from a soil sample as a suitable strain of a FR-900156 substance-producing strain belonging to the genus Streptomyces.
It is to be understood that, for the production o~ the FR-900156 substance, this invention is not limited to the use of the particular organism as described herein, which is given for illustrative purpose only. This invention also includes the use of any mutants which are capable of producing the FR-900156 substance, including natural mutants which are produced by natural mutation of the organisms as well as artificial mutants which can be produced from the described organism by conventional means, such as X-rays, ultra-violet radiation, nitrogen mustard oils and the like. ' I. Re. Streptomyces olivaceogriseus nov. sp.C-353:
Streptomyces olivaceogriseus nov. sp.C-353 has been isolated from a soil sample collected at Kochi Prefecture, Japan and deposited with and added to a permanent stock culture collection' of the America Type Culture Collection under the number ATCC 31427.
Streptomyces olivaceogriseus nov. sp. C-353 (ATCC 31427) has the following morphological, cultural and physiological characteristics.
orphological characteristics:
Microscopic observations were made on cultures which were grown from 10 to 14 days on 36~Z

sucrose-nitrate agar, glycerin-asparagine agar, yeast-malt extract agar, oatmeal agar, starch-inorganic salts agar and Bennett agar. Sporophore morphology l~as observed Oll undisturbed plates cultures.
1. Type of branching of spore-Eorming hyphae:
~lonopodial branching

2. Form o spore-forming hyphae:
Retinaculiaperti ~closed spirals, loops)

3. Numbers of spores:
5 - 20 spores

4. Surface appearance and size of spores:
Smooth 0.6 - 1.2 ~ 1.0 - 1.9 micron

5. E~istence of zoospores:
Not observed

6. Existence of sporangiwn:
Not observed

7. Formation of spores:
At aerial mycelium . Fragmentation of substrate mycelium:
Not observed (2) Cultural characteristics:;
The following observations were made on slant cultures which were grown on various media at 30C -for . ,.
10 - 14 days.

T Aerial mass I Reverse side Soluble ~ledium ¦color ¦ of colony Pigment Sucrose-nitrate none or pale yellow, agar very thin, small colonies none powdery Glucose-aspara- light gray to pale yellow to pale gine agar greenish gray, yellowish brown, none powdery small colonies Glycerin-aspara- thin, powdery pale yellowish gine agar light gray brown, none or trac small colonies .
Starch-inorganic olive gray, grayish yellow salts agar powdery . brown, none small colonies light olive yellowish brown, Tyrosine agar gray, thinsmall colonies light brown powdery , _ __ Nutrient agar none flat none , .
Yeast-malt greenish gray, yellowish brown, extract agar powdery wrinkled none colonies .
light gray to colorless, Oatmeal agar n~ne greenish gray, small colonies powdery ~
"
Peptone-yeast none colorless to light brown iron agar pale yellow, .i slightly wrinkled Glucose-peptone white, colorless to plae gelatin stab thin powdery yellowa wrinkled brown colonies . , _, -white, colorless, Milk very thin sur~ace ring none powdery growth _ .

(3) Biologi.cal and physiological properties:
1. Temperature requirements (on Bennett agar slants) Z

growth from 15C to 40C optimum 2~C
2. Hydrolysis of startch (on starch-inorganic . salts agar) - hydrolyzed weakly 3. Liquefaction of gelatin ~on glucose-peptone gelatin stab) negative 4. Action on milk no coagulation no peptonization 5. Production of melanin (on tyrosine agar, peptone-yeast iron agar and tryptone-yeast broth) positive 6. Utilization of various carbon compounds (on Pridham-Gottlieb basal agar medium) L-Arabinose D-Xylose +
D-Glucose D-Fructose +
D-Galactose +
Sucrose +
Glycerin +
Inositol Lactose L-Rhamnose Maltose +
Raffinose D-Mannitol +
D-Mannose +
. Salicin Symbols : ~, good utilization; ~, doubtful utilization;
-, no utilization 7. Cell wall pattern I ~LL-diaminopimelic acid) - As a result of looking up the strain possessing the characte~istics mentioned abo~e by referring to the ~13~;~2 litera~ure, name]y "Bergey's ~lanual of Determinative Bacteriology" eighth edition (1975), and "The Inter-national Streptomyces Project Reports" written by E.B. Shirling and D. Gottlieb Cf. International Journal of Systematic Bacteriology Vol. 18, pages 69 and 279 (1968), Vol. 19, pages 391 (1969) and Vol. 22, pages 265 (1972), Streptomyces eurythermus and Streptomyces galbus ~Okami) have been detected as species having relatively analogous characteristics to those of the strain ATCC 31427.
The strain ATCC 31427, however, is different from these analogous species in the following:
Streptomyces eurythermus (Okami) :
A strain of the species can assimilate arabinose and can not assimilate inositol. Assimilation of rhamnose and raffinose by a strain of the species is indefinite. Loops are not formed. Straights or flexous mycelium are sometimes observed.
Streptomyces galbus:
Open-spirals are generally formed. A strain of the species produces a soluble yellow - yello~
green pigment, and can not assimilate sucrose.
In view of the result of the ob~servation, the strain ATCC 31427 can~be judged to be a new species belonging to the genus Streptomyces and this has been designated as Streptomyces olivaceogriseus nov. sp.
C-353.

II. Re. Streptomyces violaceus No.6724 Streptomyces violaceus No.6724 was isolated ~rom a soil sample collected at Ishigaki island~ at Okinawa Prefecture, Japan, and deposited with and added ~o a permanent stock culture collection of the American Type Culture Collection under the number ATCC 31481.
Streptomyces violaceus No.6724 has following

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morphological, cultural and physiological characteris-tics~
(1) ~lorphological characteristics ~licroscopic observa~ions were made on cultures which were grown on sucrose-nitrate agar, glycerin-asparagine agar, starch-inorganic salts agar, yeast-malt e~tract agar and oatmeal agar at 30C for 10 to 14 days.
1. Type o branching o~ spore-forming hyphae :
Monopodial branching 2. Form of spore-forming hyphae :
Spirales 3. Numbers of spores :
10 - so 4. Surface appearance and size of spore :
Spiny 0.3 - 0.7 x 0.6 - 1.1 ~
5. Existence of zoospore and sporangium :
Not observed ~ .
6. Formation of spores :
At aerial m~celium 7. Fragmen~ation of substrate mycelium :
Not observed ,2,5 ~368'~
g (2) Cultural characteristics The following observations were made on cultures which wer~ grown on various madia at 30C
for 10 to 14 days.
s Aerial mass Reverse side Soluble Medium color of colony Pigment Sucrose-nitrate purplish white, small colonies purple agar powdery . _ Glucose-aspara- purplish white, yellowish red, pink gine agar powdery small colonies _ Glycerin-aspara- pinkish white Yellowish red, gine agar -pink, powdery small colonies, plnrked slightly wrinkled _ Starch-inorganic whitish red, yellowish red, red salts agar short cottony slightly wrinkled Tyrosine agar none red, wrinkled none or colonies trace N none or very colorless-purple, reddish utrlent agar thin powdery flat ~ purple Yeast-malt pink-purplish reddish brown reddish extract agar pink, -purplish brown, purple short cottony wrikled colonies _ .
pink-purplish colorl~ss- pink-Oatmeal agar pink, powdery purplish pink, purple small colonies .,, Peptone-yeast colorless, iron agar none wrinkled colonies brown _ _ Glucose-peptone white, thin red, wrinkled faint gelatin stab powdery colonies brown M lk thin powdery red growth on reddish . surface yellow Reverse mycelium pigment is pH indicator, changing from red to violet (purple) with addition of 0.05N NaOH
or from violet to red ~pink) with addition of 0. 05N HCl.

.. . _ ., ., , . . .. . - . - - -36~'~

Soluble pigment is also pH sensitive, showing the same changes noted for reverse mycelium pigment.
(3) Biologicai and physiological properties 1. Tempe,rature reguirements (on Bennett agar slants) growth from 15C to 40C (optimum 28C) 2. Li~uefaction of gelatin ~on glucose-peptone gelatin stab) negative 3. Hydrolysis of starch (on starch-inorganic salts agar) pOsitive 4. Action on milk coagulation, no peptonization 5. Production of melanoid pigmen~
(on tyrosine agar~ peptone-yeast iron agar and tryptone-yeast e~tract broth) positive very weak or not on tyrosin,~e agar 6. Utilization of various carbon compounds (on Pridham-Gottlieb basal agar medium) L-Ara~bi'nose +
D-Xylose f 's L-Rhamnose +
,,25 D-Glucose +
D-Fructose +
- D-Mannose +
D-Galactose +
Sucrose +
Lactose +
Maltose +
Raff InOpSe +
Inulin Cellulose Chitin z Glycerin D~lannitol +
S.21icil1 +
I~ositol +
Na-Acetate Na-Citrate +
Na-Succinate Symbols : +, good utilization +, doubtful utilization -, no utilization q .,.25 The FR-900156 substance of this invention is produced when the FR-~00156 substance-producing strain belonging to the genus Streptomyces ~e.g. Streptomyces olivaceogriseus nov~ sp.C-353 and Strep~omyces violaceus, etc.) is grown in a nutrient medium con-taining 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, fructose, glycerin, starch and the like. Other sources which may be included are lactose, arabinose, xylose, dextrin, molasses and the like.
The preferred sources of nitrogen are yeast extract, peptone, gluten meal, cottonseed meal, 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 sulphate, ammonium phosphate, etc.), urea, amino acid and the like.
The carbon and nitrogen source~s, though advan-tageously employed in combination, need not 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 calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, magnesium salts, copper slats and the like. If neces-sary, especially when the culture medium foams seriously a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added.

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As in the case of the preferred methods used for the production of other antibiotics in massive amounts, submerged aerobic cultural conditions are preferred for the production o~ the FR-900156 sub-S stance in massive amounts. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth is carried out in large tanks, it is preferable to use the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production of the FR-900156 substance. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism and culture them and then to transfer the cultured vegetative inoculum aseptically to large tanks. The medium, in which the vegerative inoculum is produced, is substantially the same as or different from the medium utilized for the production of the FR-900156 substance.
Agitation and aeration of the culture mixture may be accomplished in a variety of wa~s. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.
The fermentation is usually conducted at a tem-perature between about 20C a~d 40C, preferably 30C, for a period of about 50 hours to 100 hours.
The FR-900156 substance can be recovered from the culture medium by conventional means which are commonly used for the recovery of other known antibiotics.

In general, most of the FR-900156 substance produced are found in the cultured broth, and accordingly the F~-900156 substance can be separated from the filtrate, which is obtained by filtering of centrifuging ~he culture broth, by a conventional method such as concentration under reduced pressure, lyophilization, pH adjustment, treatment ~ith a resin (e.g. anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with an adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystal-lization and the like.
The FR-900156 substance thus produced in the culture broth can be isolated in the free form, i.e., FR-900156 substance per se and when the solution or its concentrate containing the FR-900156 substance is treated with a base, i.e. with an inorganic base such as an alkali metal compound (e.g. sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium ~0 hydroxide, etc.), an alkaline earth metal compound (e.g. calcium hydroxide, magnesium hydroxide, etc.), ammonia and the like~, with an organic base (e.g.
ethanolamine, triethylamine, dicycl~ohexylamine, etc.);
or with an acid i.e.~with an inorganic acid (e.g.
hydrochloric acid, sulfuric acid, phosphoric acid, etc.); or with an organic acid ~e.g. formic acid, acetic acid, p-toluenesulfonic acid~ citric acid, tartaric acid etc.) during operation of the processes, e.g. extraction, isolation or purification processes, the FR-900156 substance may be transformed into and isolated in the form of the corresponding salts thereof. Alternatively, thus prepared salts of the FR-900156 substance can easily be converted to the free form, i.e. FR-900156 substance per se in a conventional manner.

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, 15 - ' Further, the FR-900156 su~stance obtained in the free form may be converted t.o the corresponding salts thereof l,~i~h~a base or an acid as mentioned above in a conventional manner.
Accordingly, it is to be understood that this invention includes within the scope thereof the FR-~00156 substance as well as salts thereof as mentioned above.
The FR-900156 substance possesses the following physical and chemical properties ~The following data are ones of the product obtained by Example or fermentation (3)):
1) Form and color:
White powder 2) Nature of substance:
Amphoteric 3) Color reaction:
Positive; each reaction with ninhydrin, potassium permanganate and sulfuric acid Negative; ~ragendorff reaction and Ehrlich reaction 4) Solubility:
Soluble; water `
Sparingly.solubl.e; methanol ~
Insoluble; ethanol, acetone, ethyl acetate, .25 benzene, hexane, chloroform 5} MP;
143 - 148 ~dec.) 6) Specific rotation ~a]25 = -27.1 (c=0.4 in water) 7) Ultraviolet absorption spectrum:
end absorption 8) Infrared absorption spectrum (KBr):
1050, 1130, 1235, 1340, 1400, 1450, 1535, 368~ -1660, 1735, 2950, 2980> 3080, 3350 cm~

9) Elementary analysis:
Qualitativè analysis revealed that the FR-900156 substance comprises the following elements:
Carbon, Hydrogen, Nitrogen and Oxygen

10) Thin layer chromatography:

Stationary phase Solvent Rf Value *1 Eastman cellulose sheet ~uOH,Acetic acid0.35 l~ater(4:1:2) Silicagel sheet ~lerck *2 60% i-propanol-water 0.65 . . .
Note) *1: Trade name, made by Eastman Kodak Co.
*2: Trade name, made by ~vlerck ~ Co.

11) ~vlolecular weight:
Mass spectrometory (Field desorption method):
M=519 (base peak~ 1=520)

12) Nuclear magnetic resonance absorption spectrum:
As shown in the Figure of accompanying drawing.
(Solvent: D2O, Reference: TMSP)

13) Amino acid analysis:
~ lar ratio Glycine ~ 1.00 ,25 Glutamic acid 1.06 Alanine 1.04 ~, ~-Diaminopimelic acid 1.03 ~Molar ratio is expressed as assuming Glycine = 1.00)

14) ~lolecular formula:

Further, physical and chemical properties of more purified sample of FR-900156 substance (i.e. a product obtained by Example for fermenta~ion (4)) was measured, ;8;~

as a result of ~lhich ~IP of said sample is 147 - 153C
~dec.) and others properties thereof were the same as the above.
From analysis of the above physicochemical properties and the further investigation for eluci-dation of chemical structure, the chemical structure of the FR-900156 substance has been elucidated as follows:
OH CH

~D) ~L) lH2 I ~L) (D) ~D-Lactyl-L-alanyl-~-D-glutamyl-(L)-meso-diaminopimelyl(L)-glycine) . ,~, BIOLOGICAL PROPERTIES OF

The FR-900156 substance and its pharmaceutically acceptable salts of this invention have been found to possess enhancing activities of immune response (i.e. enhancing activities of cellular immunity and humoral antibody production) and reticuloendotherial system, enhancing activity o blood stream of carbon, mitogenic activity, inducing activity of interferon, ~36~Z

protective ef-ficacy in e~perimental infection and anticancer activity.
Accordingly, the FR-900156 substance and its pharmaceutically acceptable salts are useful ~or the therapeutic treatment o~ infectious diseases caused by pathogenic microorganism~ especially gram-negative bacteria and gram-positive bacteria and fungi, and of cancer in human being and animals.
For the purpose of showing pharmaceutical utility of the FR-900156 substance, pharmacological test data are illustrated in the follo~ing.
1. EN~ANCING ACTIVITIES OF CELLULAR IM~UNITY AND
HU~IORAL ANTIBODY PRODUCTION
Guinea pigs ~groups of five) were given 0.1 ml of FIA ~Freund's Incomplete Adjuvant) emulsion containing 500 ~g of ovalbumin in both posterior footpads. Control groups received antigen in FIA
only, whereas the test groups received the antigen with FR-900156 substance in FIA. The ani~mals were skin-tested on day 14 and bled on day 16.
The results are as the following table 1.

_ Dose Cellular Humoral immunity ,-g, immunity ste) skin -hemagglutination hemolysin titer reactions *l titer ~+S.E.) ~S.E.) (mm dia- ~log2) *2 ~log2) *2 meter, ~S.E.) 0 0 9.1 + 0.19 4.5 + 0.~5 0.1 8.2 + 2.8 *3 9.9 + 0.10 *3 6.4 ~ 0.76 l 14.5 + 2.1 *3 11.5 + 0.61 *3 7.7 + 0.64 *3 11.0 + 1.~ *3 12.2 + 0.56 *3 7.1 + 0.58 *3 100 4.2 + 1.7 *3 10.0 + 1.01 - 5.9 + 0.89 - 19 ~
Note: *l : The skin test was performed on the back by intradermal injection of 5 ~g of antigen - dissolv,ed in 0.1 ml of saline.
Skin reaction of the test site was measured at 48 hours.
*2 : Antibody es-timation ~ras carried out as follows:
O~albumin-coated sheep red blood cells were prepared by chromium chloride.
Antibody titer was expressed as the reciprocal of the highest dilution of serum evoking threshold hemogglutination and hemolysin.
The results were converted to log2 unit.
*3 : Significance was calculated by Student's t-test; P<0.05 2. MITOGENIC ACTIVITIES FOR MOUSE SPLEEN CELLS
[Materials and Methods]
~1) Animal :
Mice used for this experiment were male BALB/C
strain, aged 13 weeks (Test 1) or female BALB/C strain, aged 9 weeks (Test 2).
(2) Tissure Culture Medium : ~
The tissure culture medium employed was a complete medium designat~ed Roswell Park Memorial Institute (RPMI)-1640. All media employed contained 100 units/ml of penicillin G and 100 ~g/ml of streptomycin sulfate and 10% fetal calf serum.
(3~ Cell Preparation :
Spleens were removed under sterile conditions, and washed with Hanks solution and then teased in the tissue culture medium. The cells were suspended in the tissue culture medium to con-tain 8 x 106 cells/ml.

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~4) Culture Conditions :
Into each hole of l~;icrotest II tissue culture plate (8 x 12 hole)~maker: Falcon Plastics Co.) were poured 0.1 ml of the above cells suspen-sion and 0.1 ml of the prescribed concentrate of the test compound as described below and then the cultures were incubated intriplicate at 37C in a humidified atmosphere (95% air, 5% CO2) for 48 hours.
The control culture contained 0.1 ml of the culture medium instead of the medium containing the test compound.
(5) ~3H] Thymidine uptake :
In all tests, 20 ~Q of 10 micro-curine (~ Ci)/
ml of tritiated thymidine ~3H-thymidine) was added to each hole for the final 24 hours of culture. After the culture was completed, the resultant cells were filtered with a filter paper, Whatman GF83 and washed successively with saline and with 5% trichloroacetic acid.
The filter paper was dried and placed in a scintillator~(~oluene 1~ containing 0.1 g of p-bis[5-phenyloxazoyl]benzen;e and 4 g of 2,5-diphenylo~xazoyl), and 3H-thymidine incorpo-rated into DNA was measured.
(6) Stimulation Index :
3H-thymidine uptake(net cpm) Stimulation Index= at treatmen-t at control (7) Test Compound :
FR-900156 substance 3~

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[Results]

Concent- - 3H-thymidine Stimulation ratlon' ' uptake net Inde~

100 2,02~ + 89 4.9 1~486 + 120 3.6 Test 11 835 + 64 2.0 0 417 + 13 1.0 .
100 3,666 + 42 4.1 3,223 + 402 3.6 Test 21 2,741 + 319 3.0 0 901 + 105 1.0 3. PROTECTIVE EFFICACY IN EXP~RIMENTAL INFECTION
IN ~IICE
In determing the protective efficacy against experimental infections in mice, the FR-900156 sub-stance was dissolved and diluted in sterile water to provide three-fold concentrations of drug for'testing.
Male DDY-strain mice, aged 6 ~eeks and averaging 24 - 26 g in weight; were used in groups of four mice ~25 each.
Overnight cultures of Escherichia coli No.22 in ~ifco Nutrient Broth was diluted to 1/100 in fresh medium and incubated at 30C with'shaking. When the cell density of 1 x 108/ml was obtained, 0.2 ml. of the culture was injected intraperitoneally. All the animals receiving the challenge and not treated with the drug died within 48 hours of the infection.
One-fift'h ml. o-f the FR-900156 substance solution was injected subcutaneously, 1~4,5, and 6 days before the infection.

~3~2 Two days after infection, the test was consldered complete and survival records o:~ that day were made.
The test results are shown in Table 3.

Dose (mg/mouse/day)Survival/infected .
O . O . 1 10/10 0.003 10/10 O . 001 10/10 0.0003 8/10 0.0001 4/10 Control 0/10 4. ANTICANCER ACTIVITY
Fema~e rats o-f Donru Strain, aged 6 weeks, were used in groups of three rats each.
Suspension of ascites hepatoma A~I 66 in 0.5 ml.
of Hank's solution was transplanted intrap~eritoneally (5 x 104 cells/rat). About 13 days later, all the control animals died of ascites. Prolongation of survival time in comparison with the controls is the criterion of effectiveness. ~
Therapy was given 5, 6, 7, 8, 9, days before 2,5 tumor transplantation. The PR-900156 substance was dissolved and diluted in sterile saline water to provide threefold dilutions or testing and the said sterile saline solution of the FR-900156 substance was given intraperitoneally to the animals. The test results are shown in Table 4.

_ Dose (mg/rat/day) Life span ~days) -1.0 ~ 30, >30 0.3 13,>30, ~30 0.1 13,'30, '30 0.03 13,13, 13 Control ~saline) 13, 13, 13 -5. BLOOD STRE~ CLEARANCE OF CARBON
Reagents:
1. Carbon suspension. Rotring drawing ink ~170 mg carbon/ml) was diluted to 1/5 of the original concentration in saline containing 1% gelatin.
2. 0.1% aqueous sodium carbonate solution.
Procedure:
Mice ~DDY male 5 - 6 W) were injected via the tail - vein with a dose of 0.01 ml/g body we~ght of carbon.
Blood samples were taken by means of a pointed capillary pippet calibrated,to,hold a 50 ~1 and - previously washed in heparin. This was plunged into the retroorbital venous s.inus at the nasal angle of the eye. The samples were removed at 2j,5 3 and 6 min. The blood was immediately discharged into 3.0 ml of the sodium carbonate solution.
This hemolyzed the blood and allowed the quantita-tion of carbon. The samples were then read in a spectrophotometer at 660 nm, the log concentration being obtained from a previously determined standard curve. The clearance value K may be determined by plotting log carbon concentration against time according to the following relation-ship;

~3~BZ
~ ~a (log Cl - log C2) I~ =
- : . T2 Tl in which Tl and T2 represent the time in min when the sample were withdrawn and Cl and C2 represent the concentrations of carbon in the blood at the time Tl and T2, respectively.
EXA~IINATION OF EFFECT OF THE FR-900156 SUBSTANCE ON
CARBON CLEARANCE
The aqueous soIution of the drug as given was administered subcutaneously to mice. Twenty-four hours later, blood stream clearance of carbon was measured. K value obtained with treated mice was compared with that of control mice. The test results are shown in Table 5.
EFFECT OF VARIOUS DRUGS ON CARBON CLEARANCE

Drug Dose K /K
(mg/mouse) treated control Krestin 10 1.1 1 . 0 Levamisol 10 - (death) 2,5 1 0 5 Tuftsin 400 1.4 125 1.0 ~FR-900156 substance 0.25 2.9 0.06 1.5 Control (saline) 1.0 6. INDUCING ACTIVITY OF INTERFERON
Male ICR strain mice aged 4 to 6 weeks were used m these experiments. Spleens of these mice were ... .. . . .

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removed and the capacity of spleen cells to produce interferon in vitro by the treatment of FR-900156 substance t~as tested.
Single spleen cell suspensions were prepared in medium RP~ 1640 supp'ement with streptomycin (100 y/ml), penicillin G (100 ~(/ml), 1% glutamine and 2-mercaptoethanol (5 x 10 5 ~l).
These were cultured at 37C at a concentration of 4 x 107 cells/ml with or without FR-900156 substance dissolved with culture medium (dose: 100, 10, 1 ~g/ml).
After 24 hours, supernatants of these cultures were collected and those interferon activities were titrated by cytopathic effect (CPE) inhibition test in L cell-VSV system. Antiviral titer was expressed in IU/ml based on standard interferon.
The results are as the follol~ing table 6.

Dose IF Titer ~IU/ml) 0 ` ~6.5 ~5 - _ 7. ACUTE TOXICITY IN MICE
1 g/kg ~i.v.) : no toxicity 8. CELL TO~ICITY ~mouse L cell) 500 ~g/ml : no toxic effect For therapeutical administration, the FR-900156 substance and its pharmaceutically acceptable salts of this invention can be formulated for administration in any convenient way, for example, in the form of 3~8~ -pharmaceutical composition which comprises an active substance of this invention in admi~ture with a non-toxic pharmaceut;cally acceptable carrier.
A pharmaceutically acceptable salt of the FR-900156 substance may include a salt with an inorganic or organic base such as a sodium salt, potassium salt, calcium salt, ammonium salt, ethanolamine salt~
triethylamine salt, dicyclohexylamine salt and the like, and an acid addition salt with organic or inorganic acid such as formic acid salt, acetic acid salt, p-toluenesulfuric acid salt, citric acid salt, tartaric acid salt, hydrochloric acid salt, sulfuric acid salt, phosphoric acid salt and the like.
The pharmaceutical composition of this invention can be used in the form of a pharmaceutical preparation, for example,in solid, semisolid or liquid form, which contains an active substance of this invention in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or p~renteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsuies, suppositories, solutions, emulsions;~ suspensions, and any other form suitab~le for use. The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn 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 and coloring agents and perfumes may be used. The pharmaceutical compositions can also contain preservative or bacteriostatic agents to keep the active ingredient in the desired preparations stable in activity. The active object compound is included in ~3~ Z
- ~7 -the pharmace~tical composition in an amount sufficient to produce the desired therapeutic effect upon the process or conditi~n of diseases.
For applying this composition to humans, it is preferably to apply it by intravenous, intramuscular or oral administration. l~hile the dosage or therapeutically effective amount of the object compound of this invention varies from and also depends ~upon the age and condition of each individual patient to be treated, a daily dose of about 2-100 mg of the active ingredient/kg of a human being or an animal is generally given fortreating diseases, and an average single dose of about 50 mg, 100 mg, 250 mg and 500 mg is generally administered.

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~3~32 The following examples are given for the purpose o illustrating the present invention :- _ . .
E~ample : For fer,mentation :
5 (1) A vegetative medium 1 (pH 7.0) was prepared from the following ingredients:
Vegetative medium 1 Soluble starch 2% by wt.
Gluten meal 1% by wt.
Dried yeast 1% by wt.
Corn Steep Liquor 1% by wt.
Tap water q.s.
100 ml. of the medium 1 in each of ten 500 ml.
flasks were sterilised in conventional manner and then inoculated with a loopEul of culture from a stock slant of Streptomyces olivaceogriceus ATCC 31427. The organism was grown in the medium at 30C for 48 hours on a shaker.
Into a 30-litre Jar-fermentor, there ~ere placed 20 litres of the vegetative medium 2 prepared from the following ingredients:
Vegetative~medium 2 Soluble starch 2% by~ wt.
Cottonseed meal 0.5% by wt.
Gluten meal 0.5% by wt.
Dried yeast 0.5% by wt.
Corn Steep Liquor 0.5% by wt.
Tap water q.s.
~ The vegetative medium 2 (pH 7.0) was sterilised in a conventional manner and then inoculated aseptically with the whole volume of the vegetative inoculum culture prepared above. The organism was grown in the medium 2 at 30C for 2~ hours.
The whole volume of the vegetative inoculum thus prepared was aseptically inoculated into a 2000-litre fermentor, con~aining 1600 litres of the fermentation medium prepared from the following ingredients : :
Fermentation medium Soluble starch 2% by wt.
Cottonseed meal 0.5% by wt.
~heat germ 0.5% by wt.
Dried yeast 0.25% by wt.
Corn Steep Liquor 0.25% by w~.
KH2PO4 0.5% by wt.
a2HPO4 12 H2O 0.5% by l~t.
CoC12.6 H20 1.25 mg/l Tap water q.s.
The organism was cultured in the fermentation medium for 72 hours at 30C. During the growth period, the broth was stirred with a propeller operating at 170 r.p.m. and sterile air was passed through the broth.at a rate of 1600 litres per minute.
After the fermentation was comple-ted,~20 kg "Radiolite" (trade ~ffl~, a filter aid material sold by Showa Chemical Company, Japan) was added to the culture broth and the mixturè was filtered to remove mycelia.
1600 litres of the filtrate was passed through a column of activated charcoal ~800 litres) and then was washed .~5 with 1600 litres of water. Elution was carried out with 3000 litres of 50% aqueous acetone and then the eluate was concentrated to a volume of about 600 litres.
The concentrate was passed through a column of DEAE-Sephadex (trade ~fffl~r, made by Pharmacia A.B.) ~200 litres) which has previously been buffered with phosphate buffer (pH 6. ? . Th/e column was successively washed with 200 litres of water and 200 litres of 0.1 M
sodium chloride solution and then eluted with 400 litres of 0.3 M sodium chloride solution. The aqueous eluate was passed through a column of an activated charcoal 1~3~3%

(200 Q), washed with 200 litres of water and then eluted with 400 litres of 50% aqueous acetone. The eluate was concentrated and then reeze-dried to give 800 g of a white powder. The powder was dissolved into 25 litres of water and the solution was passed through a column of C~l-Sephadex ~l -form)(25 Q). The column was eluted with 25 litres o~ water and the el~late was concentrated and then freeze-dried to give 33 g. of yellowish white powder. The powder was placed on the top of a column of cellulose ~1.2 Q). The column was washed with 1000 ml. of 70% aqueous propanol and then eluted with 1000 ml. of 60% aqueous propanol. The eluate was concentrated and freeze-dried to give 4 g. of white powder. The powder was dissolved into 150 ml. of water and then the solution was subjected to column chromatography on Sephadex G-15 (2.8 Q).
The column was developed and eluted with water.
The active fractions were collected and concentrated and then freeze-dried to give 4 g. of a whi~te powder.
The powder dissolved into 25 ml. of water and the solution was subjected to column chromatography on CM-Sephadex ~H form~(400 ml). The column was developed and eluted with water. ~he active fractions were collected, concentrated and then freeze-dried to give 40 mg. of the FR-900156 substance in the form white powders (purity: about 70%).
(2) Fermentation was carried out in the same manner as described in Example (1). After the fermentation was completed, 20 kg. "Radiolite" (trade ~e, a filter aid material sold by Showa Chemical Company) was added to the culture broth and the mixture was filtered to remove mycelia. 1600 Litres of the filtrate was passed through a column of activated charcoal (800 litres) and then was washed with 1600 litres of water.
Elution was carried out with 3000 litres of 50% aqueous 3~;~2 acetone and then the eluate was concentrated to a volume of about 600 litres. The concentrate was passed through a coiumn of DEAE-Sephadex (trade made by Pharmacia A.B.)(200 litres) which has pre-viously been buffered with phosphate buffer (p~l 6.0).The column was successively washed with 200 litres o-F
water and 200 litres of 0.1 M sodium chloride solution and then eluted with 400 litres of 0.3 M sodium chloride solution. The aqueous eluate was passed through a column of an activated charcoal (200 litres), washed with 200 litres of water and then eluted with 400 litres of 50% aqueous acetone. The eluate was concentrated and then freeze-dried to give 800 g. of white powder. The powder was dissolved into 25 litres of water and the solution was passed through a column of G~-Sephadex (H+ form)(25 litres). The column was eluted with 25 litres of water and the eluate was concentrated and then freeze-dried to give 33 g. of yellowish white powders. The powders were~placed on top of a column of cellulose (1.2 litres). The column was washed with 1000 ml. of 70% aqueous propanol and then eluted with 1000 ml. of 60% aqueous propanol.
The eluate was concentrated and fre~ze-dried to give 4 g. of white powder.~ The powder was dissolved into 300 ml. of water and then passed through a column of DEAE-Sephadex (trade ~, made by Pharmacia A.~.) (1.4 litres) which has previously been buffered with phosphate buffer ~pH 6.0). The column was washed with 1.5 litres of 0.1 M sodium chloride solution and then eluted with 3 litres of 0.2 M sodium chloride solution The active fractions were collected and then passed through a column of activated charcoal ~300 ml).
The column was washed with water and then eluated wit 800 ml. of 50% aqueous acetone. The eluate was 3~ concentrated and then freeze-dried to give 700 mg. of white pol-~ders. The po~ders were dissolved into 20 ml.
of l~ater and then passed throughia column of C~-Sephadex ~H form)(500 ml). The column was eluted with water and the active fractions were collected, and then concentrat~d to give 10 ml. of concentrate. The concentrate was subjected to a column chromatography on Sephade~ G lS ~500 ml) and developed with water.
The active fractions were collected and concentrated and then freeze-dried to give 70 mg. of white powders.
The powders were dis,solved into 25 ml. of water and subjected to preparative thin layer chromatography on cellulose (made by Eastman Kodak Co.): a developing solvent was mixture o butanol, acetic acid and water (4:1:2). Elution was carried out with 50 ml. of water and the eluate was concentrated and then freeze-dried to give 20 mg of the FR-900156 substance in the form of a white powder.
(3) Fermentation was carried out in the same manner as described in Example (1). After the ferm,entation was completed, 20 kg. "Radiolite" (trade ~*ffle, a filter aid material sold by Showa Chemical Company) was added to the culture broth~and the mixture was filtered to remove mycelia. 1600 Litres of the~ filtrate was passed through a column o activated charcoal (800 litres) and 25, then was washed with 1600 litres of water. Elution was carried out with 3000 litres of 50% aqueous acetone and then the eluate was concentrated to a volume- of about 600 litres. The concentrate was passed through a column of DEAE-Sephadex (trade ~e, made by Pharmacia A.B.)(200 litres) which has previously been buffered with phosphate buffer (pH 6.0)., The column was successively washed with 200 litres of water and 200 litres of 0.1 M sodium chloride solution and then eluted with 400 litres of 0.3 M sodium chloride solution.
The aqueous eluate was passed through a column of an 1~3~2 activated charcoal (200 litres~, washed with 200 litres of water and then eluted with 400 litres of 50% aqueous acetone. The elu~e was concentrated and then freeze-dried to give 800 g. of white powder. The powder was dissolved into 25 litres of water and the solution was passed through a column of CM-Sephadex ~H form) ~25 litres). The column was eluted with 25 litres of water and the eluate was concentrated and then Ereez-dried to give 33 g. of yellowish white pol~der. The powder was placed on top of a column of cellulose (1.2 litres). The column was washed with 1000 ml. of 70% aqueous propanol and then eluted with 1000 ml. of 60% aqueous propanol. The eluate was concentrated and freeze-dried to give 4 g. of white powder. The powder was dissolved into 300 ml. of water and then passed through a column of DEAE-Sephadex ~trade ~
made by Pharmacia A.B.)(1.4 litres) which has previously been buffered with phosphate buffer (pH 6.0). The column was washed with 1.5 litres of 0.1 M ~sodium chloride solution and then eluted with 300 litres of 0.2 M sodium chloride solution. The active fractions were collected and then passed through a column of activated charcoal (300 ml). The Golumn was washed with water and then e~luated with 800 ml. of 50% aqueous ~5 acetone. The eluate was concentrated and then freeze-dried to give 700 mg. of white powders. The powders were dissolved into 20 ml. of water and then passed through a column of CM-Sephadex (H form)(500 ml).
The column was eluted with water and the active fractions were collected, and then concentrated to give 10 ml. of concentrate. The concentrate was freeze-dried to give 400 mg of a powder. The powder was placed on the top of a column of cellulose (500 ml).
Elution was carried out with a mixture of n-butanol, acetic acid and water (4:1:2). The active fractions ~43~

were collec~ed and freeze-dried to give 200 mg of a then placed On Se h diSsolved intO 7 substance in the form of a white powder (4~ A whi-te powder of FR-900156 substance obtained by the Example (3) was further purified by conducting repeatedly the above purification means to give a more p~rified product or FR-900156 substance.

'-25 (5) 100 ml of a medium containing corn starch 2%
~by ~t.), gluten meal 1%, dried yeast 1% and corn steep liquor 1% ~ere poured into each oE ten 500 ml flasks and sterilised in a cenventional manner and then inoculated ~ith a loopful of culture from a stock slant of Streptomyces violaceus ATCC 31481.
The organism was grown in the medium at 30C for 48 hours on a shaker.
, Into a 30-litres Jar-fermentor, there were placed 20-litres of the same medium as above. The medium was sterilised in a conventional manner and then inoculated aseptically with the whole volume of the inoculum culture prepared above. The organism was grown in the medium at 30C for 30 hours.
The whole volume of the inoculum thus prepared was aseptically inoculated into a 400-litres fermentor, containing 320 litres of the medium (pH 6.5) containing sol~lble starch 2% ~by wt.), gluten meal 1%,lcottonseed meal 1% and sodium sulfate (10 hydrates) 2%.
The organism was cultured in the medium a,t 30C
for 72 hours. During the growth period, the broth was stirred with a propeller operating at 170 r.p.m.
and sterile air was pàssed through the broth at a rate ~- of 320 litres per minute. After the fermentation was complete, 4 kg of "Radiolite" was added to the cultured broth and the mixture was filtered t,o remove mycelia.
300 litres of the filtrate was passed through a column of activated charcoal (150 litres) and then washed with 300 litres of water. Elution was carried out with - , , ,~

368~

600 1itres of 50% aqueous acetone and then the eluate was concentrated to a volume of about 120 litres.
The concentrate was passed through a column of DEAE-Sephadex (30 litres) which has previously been buffered with phosphate buffer (pH 6.0). The column was successively washed with 30 litres oE water and 30 litres of 0.1 ~I sodium chloride and then eluted with 0.3 M sodium chloride. The eluate (60 litres) was passed through a column of an activated charcoal (30 litres), washed with 60 litres of water and then eluted with 60 litres of 50% aqueous acetone. The eluat`e was freeze-dried to give 120 g of a white powder. The powder was dissolved in 4 litres of water and the solution was passed through a column of CM-Sephadex (H form)(14 litres). The column was eluted with water and the eluate was concentrated and then freeze-dried to give 20 g of a powder. The powder was placed on the top of the column of cellulose. Elution was carried out with an aqueous propanol and th~e ,active fractions were collected and freeze-dried to give 5 g of a powder. The powder was dissolved in 500 ml of water and the solution was passed through a column of DEAE-Sephadex (1.3 litres) which has previously been buffered with phosphate buffer (pH 6). The column was washed with 0.1 M sodium chloride and eluted with 0.2 ~I sodium chloride. The active fractions were collected and passed through a column of an activated charcoal (900 ml). The column was washed with water and eluted with 50% aqueous acetone (200 ml). The eluate was concentrated and free~e-dried to give 1 g of a powder.
The powder was dissolved iIl 300 ml of water and the solution was passed through a column of C~l-Sephadex (H form)(250 ml). The column was developed and eluted with water. The active fractions were collected, concentrated and then freeze-dried to give 800 mg of a powder. The powder was dissolved in 20 ml of water and the solution was mixed with a small amount of cellulose. The mixture was subjected to column chromatograph on cellulose. The column was washed successively t~ith 100 ml of acetone and 300 ml of mixture of n-butanol: acetic acid: water (4:1:1) and then developed and eluted with a mixture of n-butanol:
acetic acid: water (4:1:2)~1 litre). The active fractions were collected and freeze-dried to give 30 mg of a powder. The powder was dissolved in 20 ml of water and the solution was passed through a column of Sephadex G 15 ~250 ml). The column was developed and eluted with water and the active fractions were collected and then freeze-dried to give 5 mg of FR-900156 substance.

Example : for the pharmaceutical composition:
~1) Preparation for injection The required quantities of the FR-900156 sub-s~allce were distributed into vials, each containing 500 mg of the active ingredient. The vials were sealed hermetically to exclude bacte4ria. Whenever the vial is required`for use, 2 ml of sterile distilled water for injection is added to the vial and then the aqueous solution is administered by injection.
~2) Preparation of tablet A suitable formulation for a tablet consists of the following mixture.
FR-900156 substance 200 mg Mannitol ' 400 mg Starch 50 mg Magnesium stearate 10 mg ~3) Preparation of capsule FR-900156 substance 300 mg ~lagnesium stearate 15 mg The above ingredients ~ere mixed and then inserted into a hard gelatin capsule in a conventional manner .

.15 .

Claims (7)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A process for the production of FR-900156 sub-stance of the formula:

which comprises cultivating under aerobic conditions a FR-900156 substance-producing strain selected from the group consisting of Streptomyces olivaceogriseus and Streptomyces violaceus in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salt and recovering said FR-900156 substance from said culture medium.

2. A process according to claim 1, wherein the FR-900156 substance-producing strain is a strain of Streptomyces olivaceogriseus.

3. A process according to claim 1, wherein the FR-900156 substance-producing strain is a strain of Streptomyces violaceus.

4. A process according to claim 2, wherein the FR-900156 substance-producing strain is Streptomyces olivaceogriseus ATCC 31427.

5. A process according to claim 3, wherein the FR-900156 substance-producing strain is Streptomyces violaceus ATCC 31481.

6. The substance FR-900156, as defined in claim 1, whenever produced by the process of claim 1, 2 or 3, or by an obvious equivalent thereof.

7. The substance FR-900156, as defined in claim 1, whenever produced by the process of claim 4 or 5, or by an obvious equivalent thereof.