DD210075A5 - PROCESS FOR PREPARING ANTITUMORANTIBIOTICS BBM-1644 - Google Patents

Abstract

The invention relates to a process for the preparation of the anti-tumor antibiotic BBM-1644. This compound is a novel protein antitumour antibiotic prepared by adding a new Actinomadura strain called Actinomadura sp. Strain H710-49 (ATCC 39144) in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen, adjusting under aerobic submerged conditions, and optionally recovering the antibiotic from the culture medium. The antibiotic BBM-1644 is useful in medicine as an antibacterial agent and as an antitumor agent.

Description

The invention relates to a method for the preparation of the anti-tumor antibiotics BBM-1644. This antibiotic is useful in medicine as an antibacterial or antitumor agent.

Characteristic of the known technical solutions

The anti-tumor: antibiotic BBM-1644 prepared according to the invention is a new antibiotic from the group of protein-antitumor antibiotics, including, for example, neocarcinostatin, macromomycin and auromomycin

20 belong.

Neocarcinostatin (also referred to as Zinostatin) is an acidic protein macromolecule of molecular weight 10,700 consisting of a single polypeptide chain of 109 amino acids cross-linked by 2 disulfide bridges. The production of neocarzinostatin by fermentation of Streptomyces carcinostaticus var. Neocarzinostatikus strains is described in U.S. Patent 3,334,022 and in J. Antibiotics 18 , 68-76 (1965). The amino acid sequence of neocarcinostatin is described in Cancer Treatment Reviews _6_, 239-249 (1979).

Macromomycin is a neutral or weakly acidic polypeptide with a molecular weight of about 15,000. The production of macromomycin by

2 £ JUL1983 * iO623i

Fermentation of Streptomyces macromomyceticus (NIHJ NC-8-42) is disclosed in US Pat. No. 3,595,954 and in J. Antibiotics _2_1_, 44-49 (1968). The purification of macromomycin and the characteristic data of the purified compound are described in J. Chem Antibiotics 29 , 415-423 (1976).

10

Auromomycin is a weakly acidic polypeptide with a molecular weight of about 12500 and an isoelectric point at pH 5.4. It consists of 16 different amino acids. The isolation of

15 Auromomycin from the nutrient broth of Streptomyces

macromyceticus and the characteristic properties of the purified product are described in J. Antibiotics, 3_2_r 330-339 (1979).

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

Object of the invention

The aim of the invention was to provide a new antitumor antibiotic which is superior in its pharmacological properties to the antibiotics of the prior art.

Explanation of the essence of the invention

The object of the invention was therefore to provide a method for producing an anti-tumor antibiotic using bacterial cultures.

The invention therefore relates to a method for the production of the anti-tumor antibiotic BBM-1 644 , by a novel strain of Actinomadura, which is called Actinomadura sp. Strain H710-49 (ATCC 39144), in an aqueous nutrient medium, the contains assimilable sources of carbon and nitrogen under aerobic submerged conditions. until the mentioned organism has formed a certain amount of BBM-1644 in the culture medium, and then optionally adding BBM-1644 from the culture medium

20 wins. The invention includes the antibiotic

BBM-1644 in dilute solution, as a crude concentrate, as a crude solid and as a purified solid.

Figure 1 represents the infrared spectrum of BBM-1644 (KBr compact).

FIG. 2 shows the ultraviolet spectrum of FIG

BBM-1644 in water, 0.01 N HCl and 0.01 NaOH

The above-mentioned organism, Actinomadura sp. Strain H-710-4 9, was isolated from a soil sample collected in the Federal Republic of Germany. A biologically pure culture of the organism was obtained from American Type Culture Collection, Washington D.C. deposited under the name ATCC 39144.

BBM-16 44 inhibits the growth of various Gram-positive and acid-fast bacteria. The antibiotic also has phage-inducing properties in lysogenic bacteria and inhibits the growth of lymphoid and solid tumors, such as P388 leukemia, in mice. Therefore, the novel antibiotic prepared according to the invention can be used as an antibacterial agent or as an antitumor agent for inhibiting tumors in mammalian animals, including humans.

The Actinomyce fe strain, H-710-49 was isolated from a soil sample and prepared for characterization in the form of a pure biological culture by conventional methods. Strain H710-49 forms both a substrate and an aerial mycelium. The substrate mycelium is long, branched and not fragmented into short filaments. Short spore chains are located at the top or monopodial branches of the aerial mycelium. The spore chains contain 2 to 15 spores per chain (most often 4 to 8 spores) and have an elongated, hooked or looped shape. The spores have a warty surface and an oval to elliptical shape

(0.5 ^^ 0.6 χ 0.7 ^ s 1.2 μΐη) with a round or pointed end. Mature spores are often separated by empty hyphae. Swelling of the hyphae at the end is occasionally observed in the substrate mycelium in Czapek's and Bennett's agar. Agile spores, sporangia or skierotic granules were not observed in any of the media tested.

In contrast to common species of the genus Streptomyces, strain H710-49 grows slowly and forms a faint aerial mycelium in chemically defined and natural organic media. The aerial mycelium is white and assumes a pink shade after sporulation in oatmeal agar, inorganic salt-starch agar and glycerol-asparagine agar. The substrate mycelium is colorless, yellow, reddish-brown or dark-gray brown. A melanoid pigment is not produced, however, a lemon yellow diffuse pigment is present in glycerol asparagine agar, tyrosine agar and Pridham-Gottlieb's basic agar supplemented with glycerol, L-arabinose, D-xylose, L-rhamnose, D-glucose , D-fructose, trehalose and D-mannitol.

The strain H710-49 grows at 20 ° C, 28 ⁰ C and 37 ⁰ C, but not at 10 ⁰ C or 41 ⁰ C. It is sensitive to NaCl at 10%, but not at 7%, and resistant to lysozyme at 0.001%. D-galactose, D-mannose, sucrose, raffinose and inositol are not utilized by the strain. The physiological properties and the characteristics of the cultures of the strain H710-49 are summarized in Tables 1 and 2. The utilization of carbon sources by the strain is summarized in Table 3.

table

Characteristic features of the culture of strain H710-49

10 15 20 25 30 35

Tryptone Yeast Extract Broth (ISP No. 1)

Sucrose nitrate agar G (Czapek's Agar). R A D Glucose-asparagine agar G R A D Glycerin-asparagine agar G (ISP No. 5) R

inorganic salts-starch agar (ISP No. 4)

D G R A

Tyrosine Agar (ISP No. 7) G

good to moderate; flaky, sedimented and unpigmented

 sparse

colorless to pale orange yellow (73) *** sparse; white. {263) no bad

yellowish-white (92) to dark orange-yellow (69) very sparse; white (263) none

poor to moderately pale yellow (89) to dark orange-yellow (72) poor; white (263) to pale yellow-pink (31) bright yellow (83) poor to moderate, colorless to deep yellow (85) poor; White rose (9) to pale yellow rose (31) not moderately

brown-orange (54) to moderately reddish brown (43) poor; white (263) to slightly yellow (89) strong yellow (84)

Nutrient agar

Yeast Extract Malζ Extract Agar (ISP No. 2)

Oatmeal Agar (ISP No. 3)

Bennett's agar

Peptone Yeast Extract Iron Agar (ISP No. 6) G poor to moderate R yellowish white (92) to moderate

yellow-brown (77) A bad; white (263) D no tolerant R dark yellow (88) to dark brown (59) A sparse; white (263) D slightly olive brown (94) G poor R colorless A poor; white (263) to white

pink (9) D no tolerances R gray-yellow brown (80) to dark gray-brown (62)

A very sparse; white (263) D moderately olive brown (95) G poor R greyish yellow (90) to dark

gray-brown (6 2) A bad; white (263) D no to moderately yellowish-brown (77)

observed after 3 weeks of incubation at 28 ° C, ** Abbreviations: G - growth; R - color of the back side; A - aerial mycelium; D - diffusible pigment

*** The numbers in parentheses correspond to the color standard in "Kelly, K.L. & D.B. Judd: ISCC-NBS color name charts illustrated with Centroid Colors." US Dept. of Comm., Circ 553, Washington, D.C.

 Nov. 1975 "

Table 2 Physiological properties of strain H710-49

Turn wax temperature range

Gelatineverflussigung

starch hydrolysis

Reactions in skimmed

Melanoidbildung

nitrate reduction

Speak to

hydrolyzed "liquefied maximum growth at 28 ° C, moderate growth at 2O ⁰ C and 37 ° C. No growth at 10 ⁰ C and 41 ⁰ C.

not coagulated and completely peptonized not observed

Reduced

Resistance to NaCl Moderately resistant. growth

at 7%, but not at

10%

 Lysozyme resistant. Growth at

0.001%

 pH growth at 6.0 though

not at 5.0.

Method and Medium Bennet 's Agar

Glucose Peptone Gelatin Medium Agar Plate Difco Skimmed Milk

Tryptone Yeast Extract Broth, Tyrosine Agar and Peptone Yeast Extract Iron Agar Czapek ¹ S Glucose Nitrate Broth and Glucose Yeast Extract Nitrate Broth Tryptone Yeast Extract Agar

Tryptone-yeast extract agar

Tryptone-yeast extract agar

Table 3 Utilization of carbon sources by strain H710-49

Glycerol + D (-) - arabinose

L (+) - arabinose +

D-xylose +

D-ribose +

L-rhamnose +

p-glucose + D-galactose

D-fructose +

D-Mannose -

L {-) - sorbose

Sucrose -

Lactose

Cellobiose + melibiose

Trehalose +

Raffinose D (+) - melezitose

soluble starch +

Cellulose -

Dulcit ¹

Inositol -

D-mannitol +

D-sorbitol

Salicin -

* Observed after 3 weeks of incubation at 28 ⁰ C. basal medium: Pridham-Gottlieb inorganic medium

The purified cell walls of organisms of strain H710-49 contain meso-diaminopimelic acid but do not contain glycine. The whole cell hydrolyzate gives "the presence of madurose (3-O-methyl-D-galactose), glucose, ribose, and a small amount of mannose, and this is due to the composition of cell walls and whole cell sugar components of strain H710-49 the cell wall type IIL · assign.

The above-described characteristics of the strain H 710-4 9 are similar to those of the genus Actinomadura. According to Goodfellow et al. in J. Gen. Microbiol. 112 :

95-111 (1979) of Actinomadura and related Actinomycetes, most Actinomydura species isolated from soil are classified as Cluster No. by the 14 described clusters. The relationship of the strain H 710-49 is greatest with the species of the cluster 7. Nonomura and Ohara report in J. Ferment. Technol. 4_9_, 904-912 (1971) on five saprophytic species of the genus Actinomadura; Nonomura (J. Ferment, Technol., 52, 71-77

J (1974)) and Preobrazhenskaya et al. (Actinomycetes and Related Organisms, 1 2 30-38 (1977)) describe the identification and classification of Actinomadura species. By comparison with known Actinomadura species described in the literature, strain H710-49 is considered to be a new Actinomadura species, similar to those described in the above-cited publication by Preobrazhenskaya et al. and Japanese Kokai 55/9 43 91 described A. roseola, S. salmones, A. vinacea or A. corallina.

It should be noted that the present invention is not limited by the preparation of BBM-1644, which is detailed with reference to the strain Actinomadura sp. H710-49 (ATCC 39144) is limited by the aid of this microorganism or by means of microorganisms which are described by the characteristics given in the context of the present application. Rather, the invention encompasses strain H710-49 and all natural and conversion-produced BBM1644-producing variants and mutants thereof.

Preparation of the antibiotic

The antibiotic BBM-1644 according to the invention can be prepared by using a BBM-16 44 producing strain of the genus Actinomadura, preferably an Actinomadura sp. Strain having the characteristics of strain ATCC 39144, or a mutant thereof, in a conventional, aqueous Culture medium breeds. The nutrient medium contains known nutrient sources for actinomycetes, i. assimilable carbon and nitrogen sources, and optionally inorganic salts and other known growth factors. For the production of large quantities of antibiotic it is preferred to use aerobic submersion conditions, while for the production of small quantities it is also possible to use surface cultures and bottles. The general methods suitable for the cultivation of other actinomycetes are also applicable within the scope of the invention.

The nutrient medium should contain a suitable, assimilable carbon source, such as glycerol, L - (+) - arabinose, D-xylose, D-ribose, D-glucose, D-fructose, soluble starch, D-mannitol or cellobiose. As the nitrogen source, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate and the like may be used either alone or in combination with organic nitrogen sources such as peptone, meat extract, yeast extract, corn steep liquor, soybean powder, cottonseed meal and the like. If necessary, inorganic nutrient salts may also be added to provide sodium, potassium, calcium, ammonium, phosphate, sulfate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt - to provide iron sources und.dergleichen _f.

The preparation of the antibiotic BBM-1644 can be carried out at any temperature which ensures satisfactory growth of the organisms, for example - at 20 to 37 ⁰ C. It is advisable to operate at a temperature around 27 to 3 2 ⁰ C. An optimal production is usually obtained in shake flasks after an incubation period of approximately 6 to 7 days. For a tank fermentation, it is desirable to produce a vegetative inoculum in a nutrient broth by inoculating the broth culture with a slant or soil culture or a lyophilized culture of the organism. After having prepared an active inoculum in this way, it is added aseptically to the medium of the fermentation tank. Antibiotic production can be performed using the paper disc agar diffusion assay using Bacillus

subtilis M45 [Rec Mutant; Mutation Res. J_6, 165-174 (1972)] as a test organism.

Insulation and cleaning

After the end of the fermentation and after separation of the solid components of the fermentation broth by filtration or centrifugation, BBM-1644 is found mainly in the liquid part of the fermentation broth. Thus, the broth can be separated by centrifuging into a mycelial cake and supernatant broth. The filtrate is then concentrated and dialyzed to remove permeable contaminants against tap water using a seraipermeable membrane such as a cellophane tube.

The retained in the interior of solution (after removal of insoluble materials) containing the _BBM-f 1644, can be saturated with a suitable reagent for salting out such as ammonium sulfate then to precipitate BBM-1644 as a crude solid. This solid can then be dissolved in water and desalted by dialysis against tap water.

Further purification of the crude BBM-16 44 can then be accomplished by conventional methods used for other acidic polypeptides. For example, the aqueous solution containing BBM-I644 may be adsorbed on an ion exchanger such as DEAE-Sephadex, DEAE-cellulose, CM-Sephadex or CM-cellulose and eluted with a neutral salt solution. You can

35 preferably uses cascaded

chromatographic stages and as eluent a saline solution with a concentration gradient. The fractions containing the purified BBM-1644 are then concentrated to dryness by lyophilization.

Physicochemical properties of BBM-1644

BBM-1644 is isolated as an amorphous white powder after lyophilization. Using high-voltage paper electrophoresis (4500 V in 0.05 M barbital buffer at pH 8.6) BBM-1644 moves 8.7 cm towards the anode after one hour due to its acid nature. BBM-1644 shows no pronounced melting point and decomposes gradually above 240 ^° C.

The antibiotic is soluble in water but practically insoluble in common organic solvents such as methanol, ethanol, acetone, ethyl acetate and η-hexane. The antibiotic exhibits an optical rotation of [ct] _D = -75.6 in a 0.25% aqueous solution.

Its illustrated in Figure 2 UV spectrum in aqueous

1% solution has an absorption maximum at 275 nm (E. 8.2)

λ 9. ^{1 cm}

and 310 nm (E ° 4.6, shoulder). The UV spectra of an aqueous and a 0.01 N HCl solution are almost identical, while the UV spectrum of a 0.01 N NaOH solution

30 solution only a single maximum at 285 nm

1% (E 8.9). The IR spectrum of BBM-1644 measured in KBr is shown in FIG. The spectrum indicates the presence of NH and OH groups (3300, 2980 cm " ¹ ) and amide groups (1650 and 1540 cm" ¹ ).

The antibiotic gives positive reactions with Folin-Lowry7 xanthoprotein-biuret and ninhydrin reagents and decolorizes potassium permanganate solution. It behaves negatively against the Anthron and Sakaguchi reaction. In co-chromotography on Sephadex G-7 5 with ovalbumin (MW 4300), chymotrypsinogen (25000) and ribonuclease A (13700), BBM-1644 is eluted shortly after chymotrypsinogen so that its molecular weight is about 22,000 is appreciated. Elemental analysis of BBM-1644 yields 46.60% hydrogen for carbon. 6.45%, nitrogen 13.34% and sulfur 0.20%. The presence of 13 different amino acids in the BBM 1644 molecule was found from the amino acid analysis compiled in Table 4. Amino acids such as lysine, histidine and arginine are in

20 BBM-1644 not available.

20

25

Table 4 Amino acid composition of BBM-1644

amino acid

alanine

aspartic acid

half-cystine

glutamic acid

glycine

isoleucine

leucine

phenylalanine

proline

serine

threonine

tyrosine

Valine Relative composition * of the amino acids

8.8 6.0 1 .0 5.7 8.7 2.3 1.0 1 .4 4.1 1 .6 7.2 0.6 11.1

The content of leucine was arbitrarily set to 1.0.

ΒΒΜ-1644 is relatively stable in the pH range of 2 to 9, but outside of this range, the stability drops sharply. An aqueous solution of BBM-1644 is stable at 50 ^° C. and neutral pH for 2 hours. When irradiated with ultraviolet light, the antibiotic activity of BBM-1644 is lost within 20 minutes.

The above-described physicochemical properties of BBM-16 44 show that it belongs to the group of protein antitumor antibiotics comprising neocarzinostatin, macromomycin and auromomycin. However, among the known protein antitumor antibiotics, BBM-1644 can be distinguished by molecular weight, amino acid content and paper electrophoresis. The paper electrophoretic mobilities of BBM-1644, neocarzinostatin and macromomycin are summarized in Table 5.

Table 5

25 paper electrophoresis *

Mobility (mm from the application spot)

BBM-1644 +87

30 neocarzinostatin +31

Macromomycin -20

* 4 500 V, 1 hour; Barbital buffer pH 8.6. 35

The neocarzinostatin antibiotic group usually exhibits two UV absorption maxima at approximately 275 and 350 nm, whereas BBM-1644 has UV maxima at approximately 275 and 310 nm. Recently, Biochem. Res. Commun. 9_5, 1351-1356 (1980) that the maximum of the neocarzinostatin antibiotics at 350 nm is due to non-protein chromophores which are crucial for biological activity, V. It should be noted that BBM-1644 is a chromophore. which is different from that of the known neocarzinostatin antibiotics group

   15

Biological properties of BBM-1644

The antibacterial activity of BBM-1644 was determined by the 2-fold serial agar dilution method. For gram-positive and gram-negative bacteria used agar media, for acid-resistant bacteria Agarnährmedium with 4% glycerol and fungi

J Sabouraud Agar medium. The activity was expressed as minimal inhibitory concentration (MIC) in agar medium, the results in Table 6 are compared to those of neocarzinostatin. BBM-1644 shows a strong inhibitory activity against Gram-positive and acid-resistant bacteria, it inhibits. However, the growth of gram-negative bacteria and fungi does not. The antibacterial spectrum of BBM-1644 is similar to that of neocarzinostatin, whereas the activity of BBM-16 44 (intrinsic activity) is larger with respect to some test organisms.

Table 6 In vitro antimicrobial activity

MIC in mcg / ml test organism

Staphylococcus aureus FDA 209P

("~ ^s ) Staphylococcus aureus Smith - ._ '

Streptococcus pyogenes A20 201

Micrococcus luteus PCI 1001 Micrococcus flavus D12 Bacillus subtilis PCI 219

Escherichia coli NIHJ

Klebsiella pneumoniae D-11

Proteus vulgaris A943 6 Pseudomonas aeruginosa A9930 Mycobacterium smegmatis 607 D8

Mycobacterium phlei D88

Candida albicans IAM 4888

The ability of BBM-1644 to induce prophage in lysogenic bacteria (ILB) was determined by the method of Lein et al. (Nature 196 , 783-784, 1962) using Neocarzinostatin as reference compound. The determination of the number of plaques took place

30 for agar plates containing test material (T) and control plates (C). A T / C ratio of more than 3 was considered significant. ILB activity was expressed as the minimum inducing concentration of test compound. As from Table 7

As can be seen in Figure 35, the ILB activity of BBM-1644 is similar to that of neocarzinostatin with the minimum inducing concentration being 1 mcg / ml.

BBM-16 44 neocarzinostatin 0.4 1 .6 0.2 0.8 6.3 3.1 1 .6 1.6 1 .6 1 .6 0.8 3. 1 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 12.5 50 3.1 12.5 7100 > 100

Table 7 5

Induction of lysogenic bacteria by BBM-I644

ILB activity (T / C) * 100 10 1 0.1 mcg / ml

BBM-1644 10.4 10.3 6.0 1.2 Neocarzinostatin 28.6 20.4 10.8 0.7

15 * significant activity: T / C of> 3.0

The antitumour activity of BBM-1644 was determined in mice (BDF ₁ strain) against lymphocytic leukemia P3. Each mouse was inoculated intraperitoneally with 3x10 tumor cells. The antibiotic was administered intraperitoneally to mice in graded doses 24 hours after tumor implantation. Treatments were made once a day on the 1st.

4th and 7th day (g3d χ 3 plan) or nine consecutive days (qd 1 -> 9 plan). Neocarzinostatin was used as a comparison, the results are shown in Table 8. BBM-1644 was highly effective against leukemia in mice in a dose range of

30 0.03 ~ / 1 _r 0 mg / kg / day for both types of treatment.

The antitumor activity of BBM-1644 was approximately the same (gd 1 - *> 9 plots) or 3 times higher (q3d χ 3 plots) relative to the minimum effective dose than the activity of neocarcinostatin.

Table 8

Antitumour activity against leukemia P388

T / C (%) in MST

Dose in mg / kg / day, ip. q3d χ

BBM-1644 188

Neocarzinostatin 163

0/3

188

150

138 125

125 113

(λ, 1 0, 3, 0, 01

113

in T / C (%) in MST 1 -> 9 dose 0 mg / kg / day , ip, qd 0.3 , 1 0.03 0.01

BBM-1644 tin 1 25 1 75 1 38 1 13 Neocarzinosta 1 63 1 38 1 25 1 13

* mean survival time; significant activity: T / C of Λ 125%

The anti-tumor activity of BBM-16 44 was also demonstrated by another test for P388 leukemia in mice. The results are summarized in Table 9 below. Details of this test are in Cancer Chemother. Rep. 3_, 1-8 7 (Part 3), 1972 described.

Table 9 material Dose ip MSl .0 leukemia AWC over Effect ^ Neocarzino D.1.4 & 7 mg / kg / inj .5 effect gm live de statin 1.6 .5 MST d-6 / on BBM-1644 to P388 0.8 days .0; % T / C -4.8 6.6 treatment 0.4 8th .5 89 -4.3 6.6 plan 0.2 10 .0 117 -3.2 6.6 0.1 15 .0 172 -2.5 6.6 BBM-1644 D.1 12:05 15 .0 167 -1.2 6.6 2:56 13 .5 150 -1.7 6.6 1.28 12th .5 133 - 4.6 0.64 9 .0 100 -4.1 6.6 12:32 14th , 0 156 -4.9 6.6 12:16 14th , 5 161 -4.8 6.6 12:08 16th , 0 183 -4.1 6.6 12:04 14th 5 156 -2.8 6.6 D.1,447 12:02 12 .0. 133 -2.7 6.6 1.28 10th 5 117 -2.5 6.6 0.64 11th 5 122 -3.3 6.6 12:32 23rd 0 261 -3.5 6.6 12:16 19th 0 211 -4.3 6.6 12:08 18th 0 206 -3.8 6.6 12:04 14th 0 161 -3.3 6.6 12:02 12th 0 133 -2.9 6.6 QD 14 9 12:01 12th 0 133 -2.5 6.6 0.64 10th ο '' ' 111 -1.9 6.6 12:32 10th 0 111 -5.2 6.6 12:16  8th. 5 89 -4.2 6.6 12:08 10th 0 111 -4.3 6.6 12:04 19th 0 211 -4.2 6.6 12:02 18th 0 200 -3.5 6.6 12:01 15th 172 -3.3 6.6 0005 13th 144 -2.2 6.6 12th 133 -1.1 6.6 11th 122

Table 9, continued

material

Treatment plan

Dose, ip, MST mg / kg / Inj days

Effect MST% T / C

Over-AWC live

gm on the 5th d.6 day (3 0]

Control 10 ⁷ Saline 8.0 10/10 10 ^δ Saline 9.0 -0.3 20/20 10 ⁵ Saline 11.0 10/10 10 ⁴ Saline 14.0   - 10/10

Tumor inoculumj_ 10 ascites cells, ip (plus titration)

Host:

Toxic:

Rating:

Effect:

Evaluation:

CDF

mice

<4/6 mice alive on day 5 MST = median survival% T / C = (MST treated / MST control) x100% T / C> 125 considered significant antitumor activity.

The acute toxicity of BBM-1644 was determined in mice (dd Y strain) after a single intraperitoneal administration, the LD ^ _n being 5.8 mg / kg.

As shown above, BBM-1644 has potent anti-bacterial activity against gram-positive and acid-resistant bacteria and is thus useful in the therapeutic treatment of infectious diseases caused by such bacteria in mammalian animals, including humans, and other animals. About that

BBM-1644 also be used for other common types of antibacterial agents, for example, for the disinfection of medical and dental material.

Because of the induction of prophage in lysogenic bacteria and because of the _: strong antitumor activity against P388 leukemia in mice

BBM-1644 is also therapeutically useful for inhibiting the growth of tumors in mammals, including humans

 15

The treatment of an antibacterial infection or a malignant tumor is carried out by administering an effective antibacterial or tumor inhibiting dose of BBM-1644 or a pharmaceutical agent based on BBM-1644.

The invention also relates to a pharmaceutical agent, ^ containing an effective antibacterial or tumor-inhibiting amount of BBM J-1644 in combination with an inert pharmaceutically acceptable carrier or diluent. The compositions of the invention may be prepared in any form suitable for parenteral administration.

Such preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. The compositions of the invention may also be prepared in the form of sterile solid compositions which are in sterile water, more physiologically, immediately before use

Saline or other sterile injectable medium.

The amounts of BBM-I644 preferred for administration will vary depending on the particular formulation, the mode of administration

10 spot and disease and the one to be treated

Diseased. One of ordinary skill in the art will consider many factors that may modify the effect of the drug, such as age, body weight, sex, diet, time of administration, route of administration, excretion rate and condition, drug combinations, responsiveness and severity of the disease. Administration may be continuous or periodic within the maximum tolerated dose.

Based on certain conditions, the optimal application rate may be determined by one skilled in the art based on the above guidelines and using standard dosing testing.

The following examples serve to illustrate the invention without limiting it. DEAE cellulose is a diethylaminoethyl ion exchange cellulose. SEPHADEX G-50 is a filtration gel manufactured by Pharmacia Fine Chemicals, Inc. DEAE SEPHADEX A-50 is a diethylaminoethyl anion exchange gel manufactured by Pharmacia Fine Chemicals, Inc. SEPHADEX is Chemicals, Inc.

SEPHADEX ^ is a trademark of Pharmacia Fine

ta ''"'ο°' · 3 ^Λ

5 Example 1

Fermentation of BBM-16 44

An agar slant culture with Actinomadura sp. H710-49 colonies were used to inoculate a seed medium *) (100 ml in a 500 ml Erlenmeyer flask) containing 1% mannitol, 2% peptone and 1% yeast extract with the pH adjusted to 7.2 before sterilization has been. The seed culture was on a shaking apparatus (250 ÜPM) 7 for 2 hours at 32 ⁰ C inkubiert- 5 ml of this culture were transferred to a second seed medium (100 ml) of the same composition as the first seed medium. The cultivation was carried out under the same conditions as for the first seed culture. 5 ml of the inoculum thus prepared was used to initiate the fermentation in 500 ml Erlenmeyer flasks containing ~ 100 ml of fermentation medium containing 2.5% of mannitol, 0.5% of glucose, 1% of soybean meal, 0, 5% peptone, 1% meat extract, 0.3% CaCO ₃ and O ', 2% NaCl. The fermentation was carried out at 28 ⁰ C on a shaker (rotary shaker) at 250 rpm. Antibiotic production was monitored by the paper disc agar diffusion assay using Bacillus Subtilis M45 (Rec Mutant) as a test organism. The antibiotic activity of the culture broth gradually increased as the fermentation progressed, reaching 6 ~ 7

Days about 300 meg / ml. 35

Example 2

The fermentation broth (18 liters) obtained according to Example 1 was separated into mycelial cakes and supernatant using a sharpless centrifuge apparatus (Kokusan No. 4A). The filtrate was below 40 ⁰ C and concentrated to 1/10 of its original volume and then using a cellophane tube (Union cabide) dialysed in a cold room against tap water. The internally retained solution was concentrated to approximately 1.5 L to remove insoluble material

15 centrifuged (8000 G). The clear supernumerary

Liquid was saturated with ammonium sulfate and allowed to stand at 5 ⁰ C for 5 hours. The precipitate formed was isolated by centrifugation, dissolved in water (300 ml) and desalted by dialysis against tap water. The dialyzed solution (700 ml) contained 22 g of BBM-1644 as a crude solid; this could be demonstrated by lyophilization of part of the solution. The remaining portion of the solution was used for subsequent purification without constriction to avoid decomposition. The antibiotic solution was passed through a DEAE-cellulose column (Cl, 400 ml). The column was washed with water (1 liter) and eluted with 1/15 M phosphate buffer (pH 7.0) containing 0.3 M sodium chloride. The drug-containing fractions were pooled (300 ml), dialyzed against tap water for 18 hours and chromatographed on a DEAE-cellulose column (400 ml) equilibrated with 1/15 M phosphate buffer (pH 7.5). The column was developed with the same buffer solution containing an increasing amount of sodium chloride (0 to about 0.2 M). The

Active ingredient-containing eluate was desalted by dialysis 5 and on a DEAE-Sephadex A-50 column

(17 ml). The column was eluted with 1/15 M phosphate buffer (pH 7.5) containing a sodium chloride concentration gradient (0 to about 0.3 M). The drug-containing fractions determined by the B. subtilis M45 assay were pooled and dialyzed against running water for 18 hours. The desalted solution was chromatographed on a DEAE-Sephadex A-50 column (18 mL) using a 1/15 M phosphate buffer (pH 7, O) -NaCl (0 to about 0.3 N) system as the eluent. The appropriate fractions were collected, concentrated to 10 ml and desalted on a Sephadex G-50 column, the column was eluted with deionized water and lyophilized eluate containing drug f.

20 to obtain 120 mg of a white powder.

Polyacrylamide gel electrophoresis was used to show that the resulting BBM-1644 sample was homogeneous.

Claims (3)

_ Ji- '_ Claiming claim
5 1. A process for the preparation of the anti-tumor antibiotic BBM-16 44, the a) effectively inhibits the growth of gram-positive and acid-resistant bacteria; b) the growth of P388 leukemia in mice effectively inhibited;
c) inducing prophages in lysogenic bacteria; d) is water-soluble but practically insoluble in methanol, ethanol, acetone, ethyl acetate and η-hexane; 20 e) has in the infrared spectrum substantially maxima at 3250, 2980, 1650, 1540, 1450, 1400, 1240, 109 0 and 940 cm "; f) in the ultraviolet spectrum in water and 0.01N i%
HCl maxima at 275 nm (E ^ m 8.2) and 310 nm ^ m (Έ ^ 4.6) and a maximum in 0.01 N NaOH at 28 5 nm ( e] ^% 8.9); 1 cm g) in 0.25% aqueous solution an optical (2 Rotation of [α] _n = -75.6 °; h) does not have a defined melting point, but 35 gradually decomposes above 24 ° C. i) in paper electrophoresis at 4500 V, using a · 0.05 M barbital buffer of pH 8.6 after one hour, running approximately 8.7 cm towards the anode; j) has the following elementary analysis: 10 C, 46.60%; H, 6.45%; N ₇ 13.34%; S 0.20% and 0 (by difference), 33.41%; k) is a high molecular weight peptide having a molecular weight of about 22,000; 1) decolorizes potassium permanganate solution and gives positive Folin-Lowryr, xanthoprotein, biuret and ninhydrin reactions and negative anthrone and Sakaguchi responses; 20 and m) after hydrolysis has the following relative amino acid composition, based on the arbitrary lucin content of 1.0: alanine (8.8), aspartic acid (6.0), Hal-cystine (1.0), glutamic acid (5 , 7), glycine (8,7), isoleucine (2,3), leucine (1,0), phenylalanine (1,4), proline (4,1), serine (1,6), threonine (7, 2), tyrosine (0.6) and valine (11.1), characterized in that a BBM-1644 producing Actinomadura sp. Strain in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen 35 aerobic Submersbedingungen breeds. 2. Method according to item 1, characterized in that the antibiotic BBM-1644 is recovered from the culture medium. 3. The method according to item Ί or 2, characterized in that the B3M-1644 producing organism has the characteristics of Actinomadura Sp. H7t0j ^ 49 (ATCC 39144). HienuJLSeiien drawings 259 / Hh 35