CS251077B2 - Method of new polypeptide antibiotic production with antitumor effect - Google Patents

Description

BACKGROUND OF THE INVENTION The present invention relates to a method for the manufacture of an antibiotic BBM-1644 having antitumor activity. It is a novel compound of the class of anticancer antibiotics of protein nature, such as neocarzinostatin, macromomycin and auromomycin.

Neocarzinostatin, also called zinostatin, is an acidic antibiotic of the nature of macromolecules! a protein having a molecular weight of 10,700 and consists of a single polypeptide chain containing 109 amino acid residues that are linked by two transverse disulfide bridges.

Neocarzinostatin production of Streptomyces carzinostaticus var. neocarzinostaticus is described in U.S. Patent 3,334,022 and in J. Antibiotics 18: 68-76 (1965). The amino acid sequence of neozarzinostatin has been described in Cancer Treatment Reviews 6: 239-249 (1979).

Macromomycin is a neutral or weakly acidic polypeptide antibiotic with an approximate molecular weight of 15,000. Macromomycin production by fermentation of Streptomyces macromomyceticus (NIHJ MC-42) is described in U.S. Patent 3,595,954 and in J. Antibiotics 21: 44-49 (1968). ). The purification of macromomycin and the characteristics of the purified compound are given in J. Antibiotics 29, 415-423 (1976).

Auromomycin is a weakly acidic polypeptide with a molecular weight of about 12,500 and an isoelectric point at pH 5.4. It consists of 16 different amino acids. Isolation of auromomycin from the culture medium after growth of Streptomyces macromomyceticus and the properties of the purified product are described in J. Antibiotics 330-339 (1979).

BBM-1644 can be distinguished from known antitumor polypeptide antibiotics, such as neocarzinostatin, macreomomycin and auromomycin, by physicochemical properties, such as molecular weight, amino acid content, and paper electrophoresis.

The subject of the invention is a process for the production of a novel polypeptide antibiotic having an antitumor effect of 22,000 molecular weight, water soluble, insoluble in methanol, ethanol, acetone, ethyl acetate and hexane, its infrared absorption spectrum in potassium bromide has peaks at 330-2 980 cm ⁴ and 1 650 and 1540 cm ⁴ , the ultraviolet spectrum has maximum at 275 and 310 nm in aqueous solution, in 0.01 N sodium hydroxide it has only one maximum at 285 nm, the optical rotation is [α] _D ²⁶ - 75.6 ° in a 0.25% aqueous solution, the antibiotic gradually decomposes at a temperature above 240%, characterized in that the Actinomadura sp. H 710 - 49 ATCC 39 144 producing this antibiotic in an aqueous nutrient medium containing useful carbon and nitrogen sources in submerged culture under aerobic conditions until the antibiotic accumulates in the nutrient medium.

The invention is illustrated in connection with the drawings, in which Fig. 1 shows the absorption spectrum of BBM-1 644 in infrared light in the pellets with potassium bromide (X-axis shows the wavenumber in cm ⁴ on the Y axis the absorption at%), Fig Figure 2 shows the absorption spectrum of the same antibiotic in ultraviolet light on curve 1 in water, curve 2 in 0.01 N hydrochloric acid and curve 3 in 0.01 N sodium hydroxide. The non-new length in nm on the X axis, the extinction on the Y axis.

The invention therefore relates to a process for the production of a novel antitumor protein protein antibiotic known as BBM-1644 by fermentation of a new strain of Actinomadura, designated Actinomadura sp. strain H710-49. This microorganism was isolated from a soil sample taken in the FRG. A biologically pure sample of the microorganism was deposited with the American Type Culture Collection, Washington, D. C., and designated ATCC 39,144.

The antibiotic BBM-1644 inhibits the growth of various Gram positive bacteria but also induces phage activity in certain bacteria and inhibits the growth of lymphatic and solid tumors, such as P388 leukemia in mice. Thus, the novel antibiotic can be used as an antibacterial agent or as an antitumor agent to inhibit tumors in mammals.

Microorganism

Actinomycetes strain H710-49 was isolated from a soil sample and processed in a conventional manner to a biologically pure culture to determine its properties. Strain H710-49 creates culture on both substrate and aerial mycelium. The culture produces long branched fibers that are not frequented by shorter fibers. Short spore chains are formed at the end of the mycelium. These chains contain 12 to 15, mostly 4 to 8 spores and are straight, curved or looped, spores have a matte surface and are ovoid to elliptical in size from 0.5 to 0.6 x 0.7 to 1.2 μΐη with oval or pointed end. Mature spores are also sometimes separated by empty hyphae, it is also possible to observe terminal swelling of hyphae on Czapka and Bennett agar. Moving spores, sporangia, and granules on sclerotis cannot be observed in any environment.

Unlike other Streptomyces species, the H710-49 strain grows slowly and produces a small amount of myceline in air in chemically defined environments as well as in natural organic environments. The culture mycelium color is colorless to reddish brown on oatmeal agar, starch and inorganic salt agar, and glycerol and asparagine agar. The culture itself is colorless, yellow, red-brown or dark gray-brown. Melanoid pigment does not form, but sometimes a lemon yellow pigment is formed on glycerol and asparagine, tyrosine a agar on Pridham-Gottlieb base agar supplemented with glycerol, L-arabinose, D-xylose, L-rhamnose, D-glucose , D-fructose, trehalose and D-mannitol. Strain H710-49 grows at temperatures of 20, 28 and 37, but not at

Table 1

Culture characteristics of H710-49 strain *

Broth with tryptone and yeast extract (ISP No. 1)

Sucrose and nitrate agar (Czapek 's agar j

Glucose and asparagine agar

Agar with glycerol and asparagine (ISP medium # 5)

Starch and inorganic salt agar (ISP No 4)

Tyrosine Agar (ISP # 7)

Nutrient agar

Yeast and malt extract agar (ISP No 2)

Oatmeal Agar (ISP No. 3)

Bennett agar

Iron, peptone and yeast extract agar (ISP No. 6)

077 and 41 ° C. It is susceptible to 10% sodium chloride, tolerates still 7% and is 0.001% resistant to iysozyme, does not use D-galactose, D-mannose, sucrose, raffinose and inositol. The properties of strain H710-49 are shown in Tables 1 and 2 below, the utilization of carbon sources is shown in Table 3 below.

G ”medium growth, flake culture, sedimentation, not pigmented

G very weak

R colorless or pale orange yellow ⁽ 73 ^j ***

A poorly developed white (263)

D 0

G poor

R yellow-white (92) to deep orange-yellow (69)

And very poor whites (263)

D 0

G poor to medium

R pale yellow (89) to dark orange-yellow (72)

And poor, white (263) to pale yellow-pink (31)

D bright yellow (83)

G poor to medium

R colorless to deep yellow (85)

And poor, pink-yellow (9) to pale yellow-pink (31)

D 0

G medium

R brown-orange (54) to slightly red-brown (43)

And poor, white (263) to pale yellow (89)

D strong yellow (84)

G poor to medium.

R yellow-white (92) to slightly yellow-brown (77)

A poor whites (263)

DD

Gmírný

R dark yellow (88) to dark brown (59)

And poor, white (263)

D light olive brown (94)

Gchudý

R colorless

And poor, white (263) to pink-white (9)

TO

G medium

R gray-yellow-brown (80) to dark yellow-brown (62)

A very poor) white (263)

D slightly olive brown. (95)

G poor

R gray-yellow (90) to dark gray-brown (62)

And poor, white (263)

D none to light yellow (77) * after incubation for 3 weeks at 28 ° C ** g = growth, R = back of culture, A = mycelium in air, D = diffusible pigment *** Colors and Numbers in brackets are given by Kelly, KL and DB Judd: ISCC-NBS color-name, illustrated by Centroid Colors. US Dept. of Comm. Circ. 553, Washington, D. C., November 1975.

Table 2

Physiological properties of strain H710-49

Test gelatin reaction temperature starch hydrolysis skim milk reaction melanoid pigment formation nitrate reduction NaCl resistance

Lysozyme pH

Result maximum growth at 28 ° C, peace at 20 to 37 ° C, does not grow at 10 and 41 ° C liquefaction hydrolysis is not coagulated, is completely peptoned, does not reduce resistance moderate, strain grows at 7%, but not at 10% the strain is resistant, growing at 0.001% growth at 8.0, not at 5.0

Environment

Bennet's agar medium with glucose, peptone and gelatin starch agar

Difco skimmed milk broth with trypton and yeast extract, agar with tyrosine and agar with iron, peptone and yeast extract Czapka's agar with glucose and nitrate and broth with glucose, nitrate and yeast extract agar with yeast extract and yeast extract tryptone and yeast extract agar with tryptone and yeast extract

Table 3

Utilization of carbon resources of strain ,, H710-49 ·

Glycerol +

D (- j-arabinose—

L (+ J-arabinose +)

D-xylose +

D-ribose +

L-rhamnose +

D-glucose +

D-galactose—

D-fructose +

D-mannose—

L (-j-sorbose - sucrose - lactose - cellobiose + melibiosis - trehalose + raffinose -

D (+) -melezitose-soluble starch + cellulose-dulcitol-inositol-

D-mannitol +

D-sorbltol-salicin * after incubation for 3 weeks at 28 ° C on a basic medium of Pridham-Gottlieb with inorganic salts.

The purified cell wall of strain H710-49 contains mesodiaminopimelic acid but does not contain glycine. The wall hydrolyzate contains madurose (3-O-methyl-D-galactose), glucose, ribose, and a small amount of mannose.The cell wall composition and sugar composition in this wall of strain H710-49 shows that the strain belongs to an organism with cell wall type III _B ·

The aforementioned properties of strain H710-49 are similar to the genus Actinomadura. A description of this strain and related strains of Actinomyceites was given in Goodfellow et al. Microbiol. 112, 95-111 years (1979), most species of this origin, isolated from soil, were classified into Cluster No. 7 of the 14 isolated groups. Strain number H710-49 is also closely related to the Cluster 7 family. In Nonomura and Ohara, J. Ferment. Technol. 49: 904-912 (1971) Actinomadura and Nonomura publications describe five saprophytic families of the genus [J. Enzyme. Technol. 52: 71-77 (1974)] and Preobrazhenskaya et al. [Actinomycetes j10 7 7 and Related Organisms 12: 30-38 (1977)] describe the identification and classification of the Actinomadura family. As a result of comparison with known strains, strain H710-49 has been assigned to a new family of Actinomadura, which is similar to A. roseola, A. salmonea, A. vinacea or A. sorallina according to Preobrazhenskaya et al. And Koka! 55/94391.

It will be appreciated that the production of BBM-1644 may be by other microorganisms, although the production of the antibiotic will be further described in connection with Actinomadura sp. strain H710-49 (ATCC 39144). It can also be a production using all natural and artificially induced variants and inutants of the H710-49 strain.

The BBM-1644 antibiotic may be prepared by culturing a strain of Actinomadura, preferably ATCIC 39144 or a mutant thereof, in a conventional aqueous medium. This environment is to contain the nutrient sources known in the cultivation of the family, namely useful carbon and nitrogen sources, inorganic salts and growth factors. The strain is usually grown in a submerged culture under aerobic conditions to produce larger quantities of antibiotic, while small quantities can also be used on agar or bottled cultures. The cultivation is carried out in a conventional manner.

The culture medium should contain a suitable carbon source, for example glycerol, L (+) -arabinose, D-xylose, D-ribose, D-glucose, D-fructose, soluble starch, D-mannitol or cellobiose. The nitrogen sources may be ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, optionally in admixture with organic nitrogen sources such as peptone, meat extract, yeast extract, corn extract, soy flour, cottonseed flour etc. If desired, inorganic salts may be added as sources of sodium, potassium, calcium, ammonium, phosphate, sulfate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt, ferrous, and the like.

In the production of BBM-1644, the strain can be grown at any temperature at which the strain grows satisfactorily, for example 20-37 ° C, usually 27-32 ° C. Optimal production in shake flasks can be achieved after incubation for 6 to 7 days. In tank fermentation, it is desirable to first obtain a vegetative inoculum material in a nutrient broth which is inoculated with a slant agar culture or a soil sample or a lyophilized culture of microorganisms. The active vaccine material obtained is aseptically transferred to the production tank. Antibiotic production was monitored by diffusion on agar using paper discs and Bacillus substilis M45 (Rec., Mutation Res. 16, 165-174 (1972)).

Insulation and cleaning

After the fermentation is complete, the antibiotic is predominantly in the liquid portion of the fermentation broth after filtration or centrifugation of the mycelium. For this reason, the fermentation material is separated into a mycellous cake and a liquid fraction, and the filtrate is then concentrated and dialyzed using a semipermeable membrane, such as a cellophane tube, to remove impurities. The remaining antibiotic-containing solution is then saturated with salts, such as ammonium sulfate, to precipitate BBM-1644 as a crude solid. This solid dissolves in water and can be freed from salt by water-proofing.

The antibiotic can be further purified in a conventional manner as well as other acidic polypeptides. An aqueous solution containing the antibiotic can be absorbed on an ion exchanger, for example DEAE Sephadex, DEAE-cellulose, CM-Sephadex or CM-cellulose and eluted with a neutral salt solution. Usually, several chromatographic steps are required with increasing salt concentration. The aqueous fractions containing the antibiotic are then concentrated, for example by lyophilization.

Physico-chemical properties of antibiotics

BBM-1644

The antibiotic is isolated by lyophilization as an amorphous white powder. Electrophoresis at 4,500 V in 0.05 M barbital buffer at pH

8.6 the antibiotic moves as an acid

8.7 cm towards the anode per hour. It does not have an exact melting point and decomposes progressively above 240 ° C.

It is soluble in water, practically insoluble in organic solvents such as methanol, ethanol, acetone, ethyl acetate and n-hexane. Optical rotation is [α] _D ² -? 75.6 ° in 0.25% aqueous solution. As shown in Figure 2, the absorption spectrum of the antibiotic in ultraviolet light has peaks at 275 nm (Elem ¹ % 8.2) and 310 nm (Elcm ¹ % 4.6, hump) in aqueous solution. Spectrum in water and 0.01M hydrochloric acid is almost identical, in 0.01 N sodium hydroxide, however, is observed only a single peak at 285 nm (- - - ¹ - 8.9). The infrared spectrum in potassium bromide is shown in Figure 1. The spectrum shows the presence of NH and OH groups (330-2) of 980 cm ^-1 and amide groups (1,650 and 1,540 cm ⁴ ). The antibiotic provides a positive reaction with Folin-Lowry reagent, xanthorpotein, biuret, and ninhydrin reagent and decolorizes potassium permanganate solution. It is negative in anthrone and Sakaguchi reactions. Simultaneous chromatography on Sephadex G-75 with egg albumin with a molecular weight of 43,000, chymotrypsinogen with a molecular weight of 25,000 and ribonuclease A 13,700 results in an antibiotic just after chymotrypsinogen and its molecular weight.

The carbon content is 46.60%, hydrogen 6.45%, nitrogen 13.34% and sulfur 0.20%. Amino acid analysis revealed 13 types of amino acids as shown in Table 4. The antibiotic does not contain basic amino acids such as lysine, hystidine, and arginine.

Table 4

The amino acid composition of BBM-1644

Aminoacid

Relative amino acid composition * *

alanine 8.8 aspartic acid 6.0 half of the Cystin molecule 1.0 glutamic acid 5.8 glycine 8.8 isoleucine 2.3 leucine 1.0 phenylalanine 1.4 proline 4.1 serine 1.6 threonine 8.2 tyrosln 0.6 valine 11.1

* leucine content was determined to be · 1.0.

The antibiotic is sufficiently stable at a pH of 2 to 9, and the stability decreases rapidly beyond these limits. The aqueous antibiotic solution is stable for 2 hours at 50 ° C at neutral pH. When exposed to ultraviolet light, antibiotic activity is lost within 20 minutes.

The above physicochemical properties of BBM-1644 show that they are other compounds of the class of protein antibiotics with antitumor activity, including neocarzinostatin, macromomycin and auromomycin. The new antibiotic can be distinguished from the previously known antibiotics by molecular weight, individual amino acid content and paper electrophoresis. The mobility of BBM-1644, not ocarzinostatin and macromomycin, is shown in Table 5 below.

Table 5

Electrophoresis on paper *

Antibiotic

Mobility in mm from application site

BBM-1644 +887 neocarzinostatin +11 macromomycin —20 * 4500 V, 1 hour, barbital buffer pH 8.6

The neocarzinostatin family of antibiotics has two common maxima in ultraviolet light at 285 and 350 nm, while the new antibiotic has a maximum absorption at 285 and 310 nm. In Bíochem. Res. Commun. 95: 1351-1356 [1980] it is reported that the maximum at 350 nm may be due to the presence of non-protein chromophores that are essential for biological activity. The antibiotic BBM-1644 appears to carry a different chromophore group from other antibiotics.

Biological properties of BBM-1644

The antibiotic activity of BBM-1644 was determined by conventional agar dilution. Nutrient agar was used for both gram positive and gram negative bacteria. Nutrient agar containing 4% glycerol and Sabouraud medium was used for fungi and bacteria growing in acidic media. Efficacy was expressed as minimum inhibitory concentration (MIC) on agar, results are shown in Table 6 along with results for neocarzinostatin. The antibiotic BBM-1644 is very effective against Gram positive bacteria and bacteria growing in an acid environment, it does not affect Gram-negative bacteria and fungi. The antibacterial spectrum is similar to that of neocarzinostatin, with some microorganisms being more effective.

Antimicrobial activity in vitro

Microorganism

BBM-1644

MIC in µg / ml neocarzinostatin

Staphylococcus aureus

FDA 209P

Staphylococcus aureus

Smith

Streptococcus pyogenes

A20201

Micrococcus luteus

PCI 1001

Micrococcus flavus

D12

Bacillus subtilis

PCI 219

Escherichia coli

NIHJ

Klebsiella pneumoniae

D-II

Proteus vulgaris

A9436

Pseudomonas aeruginosa

A9930 ’

Mycobacterium smegmatis 607 D87

Mycobacterium phlei

D88

Candida albicans

IAM 4888

0.4

0.2

6.3

1.6

1.6

0.8> 100> 100> 100> 100

12.5

3.1> 100

1.6

0.8

3.1

1.6

1.6

3.1> 100> 100> 100> 100

12.5> 100

The ability of the antibiotic to induce a prophage in the case of lysogenic bacteria (ILB) was determined according to the method of Lein et al. Plaques were counted on agar plates containing test substance (T i and control substance (C). A T / C ratio of plaques greater than 3 was considered statistically significant, ILB efficacy was expressed as the minimum concentration inducing prophage. of Table 7, the ILB value for the novel antibiotic is 1 µg / ml, comparable to neocarzinostatin.

Effect of BBM-1644 on lysogenic bacteria

ILB activity (T / C) * in µg / ml

100 10 1

BBM-1644 neocarzinostatin

10.4 10.3 6.0

28.6 20.4 10.8

* statistically significant T / C efficiency is greater than 3.0

Antitumor activity of the antibiotic was determined in BBFi mice against P388 lymphocytic leukemia. Each mouse was inoculated intraperitoneally with 3 x 10 5 tumor cells. The antibiotic was also administered intraperitoneally 24 hours after tumor implantation. The antibiotic was administered once daily on days 1, 4, and 7 after infection (schema 1 to 9). The comparator was neocarzinostatin, the results are shown in Table 8. The antibiotic BBM-1644 was highly effective against leukemia in mice at a dose of 0.03 to 1.0 mg / kg / day in both cases. Efficacy was approximately the same for 9 days, in the latter case approximately three times greater than that of neocarzinostatin under the same conditions, expressed as the minimum effective dose.

Anti-tumor efficacy against P388 leukemia

T / C% MST *

Dose in mg / kg / day, ip, q3d x 3

Table 8

1 0.3 0.1 0.03 0.01 BBM-1644 188 188 138 125 neocarzinostatin 163 150 125 113 113 Girl in mg / kg / day, ip, qd 1 to 9 0.3 0.1 0.03 0.01 BBM-1644 125 175 138 113 neocarzinostatin 163 138 125 113

efficacy when the T / C value is * mean survival has a statistically significant equal to or greater than 125%.

The anti-tumor efficacy of the novel antibiotic was also demonstrated by another efficacy trial against P388 leukemia in mice, the results of which are shown in the following table.

Effect of BBM-1644 on P388 leukemia

Material Administration Dose IP mg / kg / inj.

neocarzinostatin D. 1,4 and 7 1.6 BBM-1644 D. 1 0.8 0.4 0.2 0.1 0.05 2.56 D. 1,4 and 7 1,28 0.64 0.32 0.16 0.08 0.04 0.02 1,28 QD 1 to 9 0.64 0.32 0.16 0.08 0.04 0.02 0.01 0.64 Checks 107 0.32 0.16 0.08 0.04 0.02 0.01 0.005 10% NaCl 10 ^B 10% NaCl 10 ⁵ 10% NaCl 104 10% NaCl

9. Details of this procedure have been reported in Cancer Chemother. Rep. 3: 1-87, Part 3, 1972. MST MST AWG g Survival days % T / C d.6 Day 5 (30) 8.0 89 6/6 10.5 117 -4.3 6/6 15.5 172 -3.2 6/6 15.0 167 —2.5 6/6 13.5 150 -1.2 6/6 12.0 133 -1.7 6/6 9.0 100 ALIGN! 4/6 14.0 156 —4.1 6/6 14.5 16 1 -4.9 6/6 16.5 133 —4.8 6/6 14.0 156 —4.1 6/6 12.0 133 _2.8 6/6 10.5 117 -2.7 6/6 11.0 122 -2.5 6/6 23.5 261 - 3,3 6/6 19.0 211 -3, b 6/6 18.5 206 —4.3 6/6 14.5 161 -3.8 6/6 12.0 133 —3.3 6/6 12.0 133 -2.9 6/6 10.0 111 -2.5 6/6 10.0 111 - 1,9 6/6 8.0 89 -5.2 6/6 10.0 111 -4.2 6/6 19.0 211 -4.3 6/6 18.0 200 -4.2 6/6 15.5 172 —3.5 6/6 13.0 144 —3.3 6/6 12.0 133 -2.2 6/6 11.0 122 -1.1 6/6 8.0 ___, 10/10 9.0 - —0.3 20/20 11.0 - - 10/10 14.0 - - 10/10

1S

Vaccination materials:

10 ⁶ ascitic cells ip [4- titration]

Host:

CDFi female mice

Toxicity:

fewer than 4/6 mice alive on day 5

Evaluation:

MST ~ median survival

Effect:

% T / C = MST in treated animals / MST in control animals x 100

Evaluation method:

% T / C higher than or equal to 125% is considered to be a statistically significant antitumor effect.

The acute toxicity of BBM-1644 was determined in dd Y mice by a single intraperitoneal injection, the LD 50 calculated as 5.8 mg / kg.

As has been shown, the novel antibiotic has antibacterial activity against some bacteria and can be used against infectious diseases in mammals and other animals. In addition, the antibiotic can be used as a disinfectant for medical or dental instruments.

Prophage induction in lysogenic bacteria and antitumor activity against leukemia. P388 in mice shows that the novel antibiotic can also be used to inhibit tumor growth in mammals.

For this purpose, an effective dose of the antibiotic or pharmaceutical composition containing the antibiotic is administered to a mammal having a bacterial infection or cancer.

A pharmaceutical composition containing an effective antibiotic is produced using a conventional inert carrier or diluent in a conventional manner. The compositions are usually administered parenterally.

Formulations for parenteral administration are sterile aqueous or non-aqueous solutions, suspensions or emulsions, and may also be sterile solid form preparations which are dissolved, before administration, in sterile water, saline or other sterile injectable media.

The amount of antibiotic administered in a single dose will vary with the route of administration, the disease and the condition of the patient.

A number of factors affect the effect of a substance such as age, body weight, sex, food, time of administration, route of administration, elimination rate, administration of other drugs, drug sensitivity and disease severity. The medicament may be administered continuously or intermittently, the optimum route of administration will be readily determined by one skilled in the art.

The invention will be illustrated by the following examples. DEAE cellulose is diethylaminoethyl cellulose, Sephadex G-5'0 is a filter gel, supplied by Pharmacia Fine Chemicals, Inc. DEAE Sephadex A-50 is a diethylaminoethylanion exchange gel, supplied by Pharmacia Fine Chemicals, Inc. Sephadex® is a trademark of Pharmacia Fine Chemicals, Inc.

Example 1

Fermentation of BBM-1644

Actinomadura sp. H710-49 was used to inoculate 100 ml medium in a 500 ml Erlenmeyer flask, medium containing 1% mannitol, 2% peptone and 1% yeast extract, pH before sterilization was 7.2. The culture was incubated for 72 hours at 32 ° C on a rotary shaker at 250 rpm, 5 ml of culture was transferred to another medium, 100 ml of the same medium. This medium was incubated under the same conditions as the first culture. 5 ml medium was used to inoculate 100 ml medium in 500 ml Erlenmeyer flasks, medium containing 2.5% mannitol, 0.5% glucose, 1% soy flour, 0.5% / o peptone, 1% / o extract of meat, 0.3% calcium carbonate and 0.2% sodium chloride. The fermentation was carried out at 28 ° C on a rotary shaker at 250 rpm. Antibiotic production was monitored by diffusion on agar using paper discs and Bacillus subtilis M45 (Rec ~ mutant) as the test organism. The antibiotic accumulates gradually in the nutrient medium and, after 6 to 7 days, is approximately 300 µg / ml.

Example 2 liters of the broth obtained from Example 1 are separated into a mycelial cake and the supernatant by centrifugation (Kokusan No. 4A). The filtrate is concentrated to 1/10 the original volume at less than 40 ° C and the concentrate is dialyzed in a Union Carbide tube against tap water. The solution inside the tube is concentrated to 1.5 liters and centrifuged at 8,000 g to remove insoluble matter. The clear supernatant was saturated with ammonium sulfate and allowed to stand at 5 ° C for 5 hours. The resulting precipitate is centrifuged, dissolved in 300 ml of water and freed from salts by dialysis against water. 700 ml of dialysis solution contained 22 g of crude solid BBM-1644 as shown by lyophilization of part of the solution. The remainder of the solution was used for further purification without concentration. The antibiotic solution was applied to a DEAE cellulose column (Cl, 400 mL) and the column was washed with 1 liter of water and then developed with 1/15 M phosphate buffer pH 7.0 containing 0.3 M sodium chloride. The 300 ml of active fractions are decanted, dialyzed against tap water for 18 hours and chromatographed on a column containing 400 ml of DEAE cellulose in a straight line.

18 in a 1/15 M phosphate buffer pH 7.5. The column is developed with the same buffer containing increasing amounts of sodium chloride up to 0.2 M. The active eluate is freed from salts by dialysis and applied to a column containing 17 ml of Sephadex A-50. The column is developed with 1/15 M phosphate buffer pH 7.5 containing increasing sodium chloride concentrations up to 0.3 M. Active fractions are determined by B. subtilis M45 stanov determination, pooled and dialyzed against running water for 18 hours. The salt-free solution is chromatographed on a column containing 18 ml of DEAE Sephadex

A-50 using 1/15 M phosphate buffer pH 7.0 with increasing amounts of sodium chloride up to 0.3 M as eluent. Antibiotic fractions were collected, pooled, evaporated to a volume of 10 mL and applied to a Sephadex G-50 column to remove salts. The column is eluted with deionized water and the active fractions are combined and lyophilized to give 120 mg of a white powder. The BBM-1644 antibiotic sample thus obtained is homogeneous as evidenced by polyacrylamide gel electrophoresis.

Claims (1)

OBJECT OF THE INVENTION A method for the production of a novel antitumor polypeptide antibiotic having a molecular weight of 22,000, the antibiotic is water soluble, insoluble in methanol, ethanol, acetone, ethyl acetate and hexane, its infrared absorption spectrum in potassium bromide has a maximum at 330 to 2980 cm ⁴ and 1650 and 1540 centimeters per l, the ultraviolet spectrum has maxima at 275 and 310 nm in aqueous solution, in 0.01 N sodium hydroxide it has only one maximum at 285 nm, the optical rotation is [a] D ² B - 75.6 ° in a 0.25% aqueous solution, the antibiotic gradually decomposes at a temperature above 240 ° C, characterized in that the Actinomeadura sp. H 710-49 ATCC 39 144, producing this antibiotic in an aqueous nutrient medium containing useful carbon and nitrogen sources in a submerged culture under aerobic conditions until the antibiotic accumulates in the nutrient medium.