CA1215337A - Antitumor antibiotic - Google Patents

Abstract

ABSTRACT

A novel antitumor antibiotic designated herein as BBM-1644 is produced by fermentation of Actinomadura sp. strain H710-49 (ATCC 39144 ). BBM-1644 inhibits gram-positive and acid-fast bacteria, induces prophage in lysogenic bacteria and inhibits the growth of tumors such as P388 leukemia in mice.

Description

lZ~5at~7 ( BACKG~OUND ~)F THE INVENTION

(1) Field of the Invention This invention relates to a new antitumor antibiotic and to its production and recovery.

(2) Description of the Prior Art The antitumor antibiotic of the present invention, BBM-1644, is a new member of the protein antitumor antibiotics exemplified by neocarzinostatin, macromomycin and auro~omycin.
Neocarzinostatin (also called zinostatin) is an acidic protein macromolecule of molecular weight 10,700 c~nsisting of a single polypeptide chain of 109 amino acids cross-linked by two disulfide bridges. Production of neocarzinostatin by fermentation of strains of Streptomyces carzinostaticus ~ar.
neocarzinostaticus is disclosed in U.S, P~tent 3,334,022 and in _ Antibiotics 18: 68-76 (1965). The amino acid sequence of neocarzinostatin is disclosed in Cancer Treatment Reviews 6: 239-~49 (1979).
Macromomycin is a neutral or weakiy acidic polypeptide with an approximate molecular weight of about 15,00~. Production of macromomycin by fermentation of Streptomyces macromomYceticus (NIHJ MC-8-42) is disclosed in U.S. Patent 3,595,954 and in J. Antibiotics 21: 44-49 (1968). Purification of macromomycin -and characterization data for the purified compound are disclosed 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 of pH 5.4. It consists of 16 different amino acids. Isolation of aur~momycin from the culture broth of Streptomyces macromomyceticus and characterizing properties of the purified product are dis-closed in J. Antibiotics 32: 330-339 (1979).

iZ15337 BBM-1644 may be differentiated from known polypeptide antitumor antibiotics such as neocarzinostatin, macromomycin and auromomycin by physico-chemical properties such as molecular weight, amino acid content and paper electrophoresis.

SUMMARY OF THE INVENTION

There is provided by the present invention a new protein antitumor antibiotic designated herein as BBM-1644, said antibio~ic being prepared by culti~ating a new strain of Actinomadura such as the one designated Actinomadura sp. strain H710-49 (ATCC 39144) in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a surbstantial amount of BBM-1644 is produced by said organism in said culture medium and optionally recovering the BBM-1644 from the culture medium. The invention embraces the BBM-1644 antibiotic in dilute solution, as a crude concen-trate, as a crude solid and as a purified solid.

The ir.ventioll further relates to the antibiotic ~ BB.~-1644 which:
(a) is effective in inhibiting the growth of gram-positive and acid-fast bacteria;
(b) is effective in inhibiting the growth of P388 leukemia in mice;
(c) induces prophage in lysogenic bacteria;;
(d) is soluble in water but practically insoluble in methanol, ethanol, acetone, ethyl acetate and n-hexane;
(e) exhibits an infrared absorption spectrum (KBr) substantially as shown in FIG. 1;
tf) exhibits ultraviolet absorption spectra in water, OoOlN
HCl and 0.0lN naOH substantially as shown in FIG 2;
(g) has an optical rotation of [a~D -75.6 in 0.25~ aqueous solution;

, ~2~S337 -3a-(h) has no definite melting point but gradually decomposes above about 240C;
(i) moves about 8.7 cm toward the anode during paper electro-phoresis at 4500 VO for 1 hour using 0.05M barkital buffer of pH 8.6;
(j) has the following elemental analysis: C, 46.60%; H, 6.45%;
N, 13.34%; S, 0.20% and O (by difference), 33.41%;
(k) is a high molecular weight peptide for which a molecular weight of about 22,000 is indicated;
(1) decolorizes potassium permanganate solution and gives positive Folin-Lowry, xanthoprotein, biuret and ninhydrin reactions and negati~e anthrone and Sakaguchi reactions; and (m) gives by hydrolysis the following relative amino acid composition based on the content of leucine being arbitrarily assigned as l o O alanine (8.8), aspartic acid (6.0), half-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 ( 1 1 0 1 ) O

The process for the production of the antibiotic, BBM-1644, which comprises cultivating a BBM-1644-producing strain of Actinomadura spO in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under The invention still further relates to a process for the production for of the antibiotic BBM-1644, which comprises cultivating a BBM-1644-producing strain of Actinomadura sp. in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1644 is produced by said organism in said culture medium.

-3b-DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the infrared absorption spectrum of BBM-1644 (KBr pellet).
FIG. 2 shows the ultraviolet absorption spectra of BBM-1644 in water, 0.0lN BCl and 0.0lN NaOH.

DETAILED DESCRIPTION

This invention relates to a novel protein antitumor antibiotic designated herein as BBM-1644 and to its preparation by fermentation of a new strain of Actinomadura designated , ~

- ` ` ~2~S337 A'ctinomadura sp. strain H710-49. The above organism was isolated from a soil sample collected in West Germany. A
biological,ly pure culture of the organism has been deposited with the American Type Culture Collection, Washington, D.C., and added to its permanent collection of microorganisms as ATCC 39144.
BBM-1644 inhibits the growth of various gram-positive and acid-fast bacteria. The antibiotic also exhibits phage inducing pxoperties in lysogenic bacteria and inhibits growth of lymphatic and solid tumors such as P388 leukemia in mice. The new antibiotic, therefore, may be used as an anti-bacterial agent or as an antitumor agent for inhibiting mammalian tumors. ' The'Microorganism The actinomycete strain No. H710-49 was isolated from a soil sample and prepared by conventional procedures as a biologically pure culture for characterization. Strain H710-49 forms both substrate and aerial mycelia. The substrate mycelium is long, branched and not fragmented into short filaments. Short spore-chains are born on the tip or monopodial branch of aerial mycelium. The spore-chains contain 2--'to i~''s'po'res in a chain (mostly i to' 4-spores) and are straight, hooked or lo'oped in shape. The spores have a warty surface and are oval to elliptical (O.5~0.6 x O.7~1.2~m) in shape with a round or p,ointed end. Mature spores are often separated by empty hyphae.
Terminal swellings of hyphae are occasionally observed on the substrate mycelium in Czapek's agar and Bennett's agar. Motile spores, sporangia or sclerotic granules are not seen in any media examined.

~;~i5;~37 -s Unlike ordinary species of the genus Streptomyces, strain H710-49 grows slowly and forms poor aerial mycelium in chemically defined media and natural organic media. The color of aerial mycelium is white and turns to a pinkish shade after sporulation in oatmeal agar, inorganic salts-starch agar and glycerol-asparagine agar. Mass color of the substrate mycelium is colorless, yellow, reddish brown or dark grayish brown. Melanoid pigment is not produced, but a lemon yellow diffusible pigment is seen in glycerol-asparagine agar, tyrosine agar and Pridham-Gottlieb's basal agar supplemented with any one of glycerol, L-arabinose, D-xylose, L-rhamnose, D-glucose, D-fructose, trehalose and D-mannitol. Strain H710-49 grows at 20C, 28C and 37C, but not at 10C or 41C. 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. Cultural and physiological characteristics of strain H710-49 are shown in Tables 1 and 2, respectively. The pattern of carbon source utilization by the strain is shown in Table 3.

Table 1 Cultural characteristics* of strain H710-49 _ Tryptone-yeast extract G** poor to moderate; floccose, broth (ISP No. 1) sedimented and not pigmented.

Sucrose-nitrate agar G scant (Czapek's agar) R colorless to pale orange yellow (73)***
A scant; white (263) D none l~lS~37 Table 1 - continued _ Glucose-asparagine agar G poor R yellowish white (92) to deep orange yellow (69) A very scant; white (263) D none Glycerol-asparagine agar G poor to moderate (ISP No. 5~ R pale yellow (89) to dark orange yellow (72) A poor; white (263) to pale yellowish pink (31) D brilliant yellow (83) Inorganic salts-starch G poor to moderate agar (ISP No. 4). R colorless to deep yellow (85) A poor; pinkish white (9) to pale yellowish pink (31) D none ~yrosine agar (ISP No. 7) G moderate R brownish orange (54) to moderate reddish brown (43) A poor; white (263) to pale yellow (89) D strong yellow (84) Nutrient agar G poor to moderate R yellowish white (92) to moderate yellowish brown (77) A poor; white (263) D none i~7 Table 1 continued Yeast extract-malt extract G moderate agar (ISP No. 2) R dark yellow (88) to dark brown ~59) A scant; white (263) D light olive brown (94) Oatmeal agar (ISP No. 3) G poor R colorless A poor; white (263) to pinkish white (9) D none Bennett's agar G moderate R grayish yellowish brown (80) to dark grayish brown (62) A very scant; white (263) D moderate olive brown (95) Peptone-yeast extract- G poor iron agar (ISP No. 6) R grayish yellow (90) to dark grayish brown (62) A poor; white (~63) D none to moderate yellowish brown (77) * observed after incubation at 28~C for 3 weeks ** Abbreviation: G-- Growth; R - Reverse color; A - Aerial myc~lium;
D - Diffusible pigment *** Color and number in parenthesis follow the color standard in "Kelly, K. L. & D. B, Judd: ISCC-NBS color-name charts illustrated with Centroid Colors. U.S. Dept. o~ Comm. Circ. 553, Washington, D.C., Nov., lg75"

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~;~15337 Table 3 Utilization of caxbon sources* by strain H710-49 Glycerol +
D(-)-Arabi~ose L(+)-Arabinose +
D-Xylose +
D-Ribose L-Rhamnose +
D-Glucose +
D-Galactose D-Fructose +
D-Mannose L(-)-Sorbose Sucrose Lactose Cellobiose +
Melibiose Trehalose +
Raffinose D(+)-Melezitose Soluble starch +
Cellulo~e - -Dulcitol Inositol D-Mannitol +
D-Sorbitol Salicin * Observed after incubation at 28C for 3 weeks.
Basal medium: Pridham-Gottlieb inorganic medium ~:~lS337 Purified cell-wall of strain H710-49 contains meso-dial[linopimelic acid but lacks glycine. The whole ce11 hydroly-zate sl~ows the presence of madurose (3-0-methyl-D-galactt~se), glucose, ribose and a small amount of mannose. The cell-wall composition and whole cell sugar components of strain H710-49 indicate that the strain belongs to cell-wall Type IIIB.
The above-described characteristics of strain H710-49 resemble those of the genus Actinomadura. According to the numerical taxonomy of Actinomadura and related actinomycetes by Goodfellow et al. in J. Gen. Microbiol.~112 95-111 (1979), mos~ Actinomadura species of soil origin are classified into Cluster No. 7 among the 14 clusters described. Strain No.
H710-49 is most related $o the species of Cluster 7. Nonomura and Ohara in J. Ferment. Technol. 49: 904-912 (1971) reported -five saprophytic species of the genus Actinomadura and t~onomura [_ Ferment. Technol. 52: 71-77 (1974)] and Preobrazhenskaya et al. ~Actinomycetes and Related Organisms _: 30-38 (1977)]
described the identification and classification of Actinomadura species. As a result of comparison with known Actinomadura species described in the literature, strain H710-49 is considered to belong to a new species o E Actinomadura similar to A. roseola, A. salmonea, A. ~rinacea or A. corallina described in the Preobrazhenskaya et al. reference above and in Japanese Kokai 55/94391.
It is to be understood that for the production of BBM-1644, the present invention, though described in detail with reference to the particular strain Actinomadura sp. strain H710-49 (ATCC 39144 ), is not limited to this microorganism or to micro-organisms fully described by the cultural characteristics dis-closed herein. It is specifically intended that the invention embrace strain H710-49 and all natural and artificial BBM-1644-producing variants and mutants thereof, ~2~5337 -~; Antibiotic Production ~

The BBM-1644 antibiotic of the present invention may be prepared by cultivating a BBM-1644-producing strain of the genus Actinomadura, preferably a strain of Actinomadura sp. having the identifying characteristics of ATCC 39144 or a mutant thereof, in a conventional aqueous nutrient medium.
The organism is grown in a nutrient medium containing known nutritional sources for actinomycetes, i.e. assimilable sources of carbon and nitrogen plus optional inorganic salts and other known growth factors. Submerged aerobic conditions are preferably employed for the production of large quantities of antibiotic, although for production of limited amounts, surface cultures and bottles may also be used. The general procedures used for the cultivation of other actinomycetes are applicable to the present invention.
The nutrient medium should contain an appropriate assimilable carbon source such as glycerol, L(+)-arabinose, D-xylose, D-ribose, D-glucose, D-fructose, soluble starch, D-mannitol or cellobiose. As nitrogen sources, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc.
may be used either alone or in combination with organic nitrogen sources such as peptone, meat extract, yeast extract, corn steep liguor, soybean powder, cotton seed flour, etc. There may also be added if necessary nutrient inorganic salts to provide sources of sodium, potassium, calcium, ammonium, phosphate, s~ulfate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt, iron, and the like.
Production of the BBM-1644 antibiotic can be effected at any temperature conduci~e to satisfactory growth of the producing organism, e.g. 20-37C, and is conveniently carried out at a temperature of around 27-32C Ordinarily, optimum production is obtained in shaker flasks after incubation periods of about 6-7 days~ When tank fermentation is to be carried out, .

lZ~S337 it is desirable to produce a vegetative inoculum in a nutrien~
~roth by inoculating the broth culture with a slant or soil culture or a lyophilized culture of the organism. After obtaining ~n active inoculum in this manner, it is transferrea aseptically to the fermentation tank medium. Antibiotic production may be monitored by the paper disc-agar diffusion assay using Bacillus subtilis M45 ~Rec mutant; Mutation Res.
16: 165-174 (1972)~ as the test organism.

.
Isolation and Purificati~n .

When fermentation is complete, BBM-1644 exists mainly in the liquid part of the fermented broth after separation of the solid part by filtration or centrifugation. Thus, the harvested broth may be ~eparated into mycelial cake and broth supernatant by centrifugation. The filtrate is then concentrated and dialyzed against tap water by a semipermeable membrane such as a cellophane tube to remove permeable impurities. The inside-retained solution (after remoYal of insoluble materials) contain-ing the BBM-1644 may then be saturated with a salting out reagent such as æmmonium sulfate to precipitate out BBM-1644 as a crude solia. This solid may be dissolved in water and desalted by dialysis against tap water.
Further purification of the crude BBM-1644 may be accomplished by conventional procedures used with other acidic polypeptides. For example, the aqueous solution containing BBM-1644 may be adsorbed on an ion exchanger such as DEAE-5ephadex, DEAE-Cellulose* CM-Sephadex*or CM-cellulose* and eluted with a neutral ~alt solution. Successive chromatographic steps ar~ pre~erably employed with a gradient concentration of salt solution used as the eluant. Aqueous fractions containing the purified BBM-1644 axe then concentrated to dryness as by lyophilization.
*Trade Mark LS~37 .
- Physico-chemical'Properties of BBM-1644 BBM-1644 is isolated as an amorphous white powder up~n lyophilizationO When examined by high voltage paper electrophoresis (4500 V in 0.05M-bar~ital buffer at pH 8.6~, ~BM-1644 migrates as an acid travelling 8.7 cm toward the anode after one hour. BBM-1644 does not show a definite melting point and gradually decomposes above 240C. It is soluble in water, but practically insoluble-in common organic sol~ents such as methanol, ethanol, acetone, ethyl acetate and n-hexane. The antibiotic shows an optical rotation of ~j26=
-7~.6 in 0.25% aqueous solution.--As depictéd'in ~IG. 2, the W spectrum of B8M-1644 shows absorption maxima at 275 nm (ElCm 8.2) and 310 nm (ElCm 4.6, shoulder) in aqueous solution.
It exhibits a nearly identical W spectrum in water and in o.oiN HCl solution, but only a single maximum at ~B5 nm (El%m 8.9) in 0.01N NaOH solution. The IR spectrum of BBM-1644 measured in KBr is shown in FI G . 1. 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 to Folin-L~wry, xanth~protein, biuret and ninhydrin reagents and de~olorizes potassium permanganate solution. It is negative to anthrone and Sakaguchi reactions. When co-chroma't'o'g'raphëd on Sephadex G,-75 with o~albumin (MW 43,000), chymotrypsinogen (25,000) and ribonuclease A (13,700), BBM-1644 is eluted just after chymotrypsinogen and therefore its molecular weight is ~stima~ed to be hround 22,000. Elemental analysis of BBM-1644 indicates carbon 46.60~, hydrogen 6.45%, nitrogen 13.34% and sulfur 0.20~. The presence o~ 13 kinds of amin~ acids in the BBM-1644 molecule was re~ealed by amino acid analysis as shown in Table 4. asi~ amino acids such as lysine, histidine and arginine are not present in BBM-1644.

*Trade Mark ~.

~5337 Table 4 :: Amino acid comeosition of BBM-1644 Relative composition*
Amino acid of amino acids Alanine 8,8 Aspartic acid 6.0 half-Cystine 1.0 Glutamic acid 5.7 Glycin 8,7 Isoleucine 2.3 Leucine 1.0 Phenylalanine 1.4 Proline 4.1 Serine 1.6 Threonine . 7.2 Tyrosine O.6 Valine 11.1 * Content of leucine was arbitrarily assigned as 1Ø

~Z~LS33~

BBM-1644 is fairly stable in the pH range of 2~9, b~t the stability declines sharply beyond this pH r~nge.
The aqueous solution of BBM-1644 is stable for 2 hours at 50C at neutral pH. Upon exposure to ultraviolet li~ht, the antibiotic activity of BBM-1644 is lost within 20 minutes.
The physico-chemical properties of BBM-1644 described above indicate that it is a member of the protein antitumor antibiotic group which includes neocarzinostatin,~macromomycin and auromomycin. BBM-1644, however, can be differentiated from the known protein antitumor antibiotics by its molecular weight, amino acid content and paper electrophoresis, The paper electrophoretic mobilities of BBM-1644, neocarzinostatin and macromomycin are shown in Table 5.

Table 5 Paper ele_trophoresis*

Mobility (mm from the ~oadinq spot) BBM-1644 +87 Neocarzinostatin ~31 Macromomycin -20 * 4,500 V, 1 hour; Barbital buffer p~ 8.6 The neocarzinostatin group of antibiotics commonly exhibit two W absorption maxima at around 275 and 350 nm, while BBM-1644 has the W maxima at around 275 and 310 nm. It has recently been reported in Biochem. Res. Commun. 95: 1351-1356 (1980) _ that the maximum at 350 nm of the neocarzinostatin antibiotics might be due to the non-protein chromophores which are essential for their'biological activity. It is noted that BBM-1644 has a chromophore different from that of the known ne~carzinostatin group of antibiotics.

~ S~37 Biological Properties of BBM-1644 .,- .
The antibacterial activity of BBM-1644 was determined by the serial two-fold agar dilution method. Nutrient agar medium was used for gram-positive and gram-negative bacteria;
nutrient agar medium containing 4% glycerol for acid-fast bacteria and Sabouraud agar medium for fungi. The activity was expressed as minimum inhibitory concentration (MIC~ in the agar medium and the results are shown in Table 6 along with those of neocarzinostatin. BBM-1644 showed p~tent inhibitory activity against gram-positive and acid-fast bacteria but did not inhibit the growth of gram-negative-bacteria and fungi, The antibacterial spectrum of BBM-1644 is similar to that of neocarzinostatin, while the intrinsic activity of BBM-1644 is more potent than the latter in some o~ the test organisms.

Table 6 In vitro antimicrobial activity MIC in mcq/ml Test organismBBM-1644 Neocarzinostatin StaPhylococcus aureus FDA 209P 0.4 1.6 Staphylococcus aureus Smith 0.2 0.8 -Streptococcus pyogenes A20201 6.3 3.1 Micrococcus luteus PCI 1001 1.6 1.6 Micrococcus flavus D12 1.6 1.6 Bacillus subtilis PCI 219 0.8 3.1 Escherichia coli NIHJ >100 >100 Klebsiella pneumoniae D-ll >100 >100 Proteus vulqaris A9436 >100 >100 Pseudomonas aeru~inosa A9930 >100 >100 Mycobacterium smegmatis 607 D8712.5 50 Mycobacterium phlei D88 3.1 12.5 Candida albicans IAM 4888 ~100 >100 ~215337 The ability of BBM-1644 to induce prophAge--~n lys~genic bacterium (I~B) w~s determined by the method o~ ~ein et.al. (Nature 196: ~83-7B4, 1962) using neo-carzinostatin as a reference compound. The plaque count was made on agar plates containing test material (T) and control (C). A ~/C ratio of the plaque counts of greater than 3 was considered significant and the ILB activity was expressed by the minimum inducing concentration of the test compound. As shown in Table 7, the ILB activity of BBM-1644 is similar ~o that observed with neocarzinostatin, the minimum inducing concentration being 1 mcg/ml.

Table 7 Induction of lysogenic ~acterium bY BBM-1644 I~B activitY (T/C)*
100 10 1 0.1 mcq/ml _ BBM-1644 10.4 10.3 6.0 1.2 Neocarzinostatin 28.6 20.4 10.8 0.7 * significant activity: T/C O~ ~3 !

The antitumor acti~ity of BBM-1644 was determined in mi~e (BDFl strain) against lym~hocytic leukemia P388. Each mouse was inoculated intraperitoneally with 3 x 105 cells of the ~umor.
Graded doses of the antibi~tic were administered t~ mice intr~-peritcneally 24 hours a~ter tumor implantation. The treatments were given on~e a day on days 1.4 ~nd 7 (q3d x3 schedule~ or 9 consecutive days ~qd 1~9 schedule). Neocarzinostatin was comparatively tested as ~ reference antitumor agent and the results are summarized in Ta~le 8. BBM-1644 was highly active against the mouse leukemia in a dose range of 0.03~1~0 mg/kg/day .

~i5337 by both treatments. The antitumor activity of BBM-1644 was about the same as (qd 1~9 schedule~ or 3 times more potent (q3d x 3 s~chedule) than that of neocarzinostatin in terms of minimum effective dose.

Table 8 Antitumor activity aqainst leukemia P388 .

T/C (%) in MST*
Dose in mg/k~/day, ip, q3d x 3 1 0.3 0.1 0.03 0.01 Neocarzinostatin -163 15~ --125- 113 T/C (%) in MST
Dose in mg/kg/day, ip, qd 1~9 0.3 0.1 0.03 0.01 BBM-1644 125~ 5 ;138 - 113 Neocarzinostatin 163- 138 ----125--~ 113 .
* median survival time;-, signi-ficant activity: T/C of _125%

Antitumor activity of B~M-1644 was also indicated by a second test against P388 leukemia in mice, the results of which are shown below in Table 9. Details of the methods used in this test have been described in Cancer Chemother. Rep. 3: 1-87 tPart 3), 1972.

~2~S;~3~

Table 9 Effect of BBM-1644 on P388 Leukemla Effect AWC
Treatment Dose, IP MST MST sm Survivors Material Schedule mg/kg/inj Days %T/C d.6 Day 5(303 Neocarzino- D.1,4&7 1.6 8.0 89 -4.8 6/6 statin 0.8 10.5 117 -4.3 6/6 0.4 15.5 172 -3.2 6/6 0.2 15.0 167 -2.5 6/6 0.1 13.5 150 -1.2 6/6 0.05 12.0 133 -1.7 6/6 BBM-1644 D.l 2.56 9.0 100 - 4/6 1.28 14.0 156 -4.1 6/6 0.64 14.5 161 -4.9 6/6 0.32 16.5 183 -4.8 6/6 0.16 14.0 156 -4.1 6/6 0.08 12.0 133 -2.g 6/6 0.04 10.5 117 -2.7 6/6 0.02 11.0 122 -2.5 6/6 D.1,4&7 1.28 23.5 261 -3.3 6/6 0.64 19Ø 211 -3.5 6/6 0.32 18.5 206 -4.3 6/6 0.16 14.5 161 -3.8 6/6 0.08 12.0 133 -3.3 6/6 0.04 12.0 133 -2.9 6/6 0.02 10.0 111 -2.5 6/6 0.01 10.0 111 -1.9 6/6 QD 1~9 0.64 8.0 89 -5.2 6/6 0.32 10.0 111 -4.2 6/6 0.16 19.0 211 -4.3 6/6 0.08 18.0 200 -4.2 6/6 0.04 15.5 172 -3.5 6/6 0.02 13.0 144 -3.3 6/6 ~ 0.01 12.0 133 -2.2 6/6 0.00511.0 122 -1.1 ~/6 ~S;~37 ~ 20 -Table 9 continued Effect A~C
Treatment Dose, IP MST MST gm Survivors Material _Schedule mg/k~/ini Days ~T/C d.6 Day 5(30) .
Control~107 Saline 8.0 - - 10/10 6 Saline-` 9.0 - -0.3 20/20 105 Saline 11.0 - _ 10/10 104 Saline 14.0 - _ 10/10 .

Tumor inoculum: 106 ascites cells, ip ~plus titration) Host : CDFl ~ mice.
Tox : <4/6 mice ali~e on Day 5.
Evaluation : MST = median survival time, Effect : % T/C = (MST treated/MST control) x 100.
Criteria : % T/C _ 125 considered significant antitumor activity.

The acute toxicity of BBM-1644 was determined in mice (dd Y strain) by single intraperitoneal administration, the L~50 being calculated as 5.8 mg/kg.
As shown above, BBM-1644 possesses potent antibacterial activity against gram-positive and acid-fast bacteria and is thus useful in ~he therapeutic treatment of mammals and other animals for infectious diseases caused by such bacteria. Additionally, it may be utilized for other conventional applications of anti-bacterial agents such as disinfecting medical and dental equipment.
The induction of prophage in lysogenic bacteria and the marked antitumor activity shown against P388 leukemia in mice indicate that BBM-lÇ44 is also therapeutically useful in inhibiting the growth of mammalian tumors.
The present invention, therefore, provides a method for therapeutically treating an animal host affected by a bacterial infection or by a malignant tumor which comprises administering ~iL5;~37 to said host an effective antibacterial or tumor-inhibiting dose of BBM-1644 or a pharmaceutical composition thereof.
~ n another aspect, the present in~ention provides a pharmace.utical composition which comprises an effective antibacterial or tumor-inhibiting amount of BBM-1644 in combination with an inert pharmaceutically acceptable carrier or diluent. These compcsitions may be made up in any pharma-ceutical form appropriate for parenteral administrati~n.
Preparations according to the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. They may also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, physiological saline or some other sterile injectable medium immediately before use.
It will be appreciated that the actual preferred amounts of the BBM-1644 antibiotic used will vary according to the particular composition formlllated, the mode of application and the particular situs, host and disease being treated, Many factors that modify the action of ~he drug will be taken into account by those skilled in the art, for example, age, body weight, sex, diet, time o~ administration, route of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease.
Administration can be carried out continuously or periodically within the maximum tolerated dose. Optimal application rates for a given set of conditions can be ascertained by those skilled in the art using conventional dosage determination tests in view of the above guidelines.
The following examples are pro~ided for illustratiYe purposes only and are not intended to limit the scope of the invention. DEAE Cellulose is a diethylam'noethyl ion exchange cellulose. SEPHADEX G-50 is a filtration gel manufactured by Pharmacia Fine Chemicals, Inc. DEAE SEPHADEX A-S0 is a diethyl-aminoethyl anion exchange gel manufactured by Pharmacia Fine ~ , Chemicals, Inc. SEPHADEX is a trademark of Pharmacia Fine Chemicals, Inc. -Fermentation of BBM-1644 An agar slant with-well-established growth of Actinoma-d _ sp'.'H710-49-was u'sed to inoculate seed mediu~ (100 ml in a 500-ml Erlenmyer flask) containing 1% mannitol, 2% peptone and 1~ yeast extract, the pH being adjusted to 7.2 before steriliza-tion. The seed culture was incubated at 32C for 72 hours on a rotary shaker (250 rpm) and 5 ml of the culture was transferred to the second seed medium (100 ml) composed of the same composition as the fi'rst~seed medium.~~~'It was cultiYated under the same condition as that used for the first seed culture.
Five ml of the inoculum growth thus prepared was employed to start fermentation in ~00-ml Erlenmyer flasks which contained 100 ml of fermentation medium having composition of 2.5% mannitol, O.5% glucose, 1% soybean meal, 0.5~ peptone, 1% meat extract, 0.3~ CaCO3 and 0.2~ NaCl. Fermentation was carried out at 28C
on a rotary shaker with 250 rpm rotation. The antibiotic production was monitored by the paper disc-agar diffusion assay using Bacillus subtilis M4~ (Rec mutant) as the test organism.
. _ The antibiotic activity in the culture broth gradually increased with the progress of fermentation and reached about 300 mcg/ml after 6~7 days.
. . .

~ .

The harves~ed broth (18 liters~ obtained from the Example 1 fermentation was separated to mycelial cake and broth supernatant by using a sharpless centrifuge apparatus ~Kokusan No. 4~). The filtrate was concentrated below 40C to one-tenth ~2153:~7 the original volume and the concentrate was dialyzed by c~ phane tubing (Union Car~ide) against tap water ln a cold room. The inside-retained solution was concentrated to about 1.5 liters which was centrifuged (8,000 G) to remove insoluble materials. The clear supernatant was saturated with ammonium sulfate and allowed to stand for 5 hours at 5C. The precipitate formed was collected by centrifugation, dissolved in water (300 ml) and desalted by dialysis against tap water. The dialyzed solution (700 ml) contained 22 grams of crude solid of BBM-1644 as revealed by lyophilization of a part of the solution. The rest of the solution was used for subsequent purification without concentration in order to avoid decomposition. The antibiotic solution was passed through a column of DEAE-cellulose (Cl , 400 ml) and the column was washed with water (1 liter) and developed with 1/15M phosphate buffer (pH 7.0) containing 0.3M sodium chloride. The active fractions were combined (300 ml), dialyzed for 18 hours against tap watex and chromatographed on a column of DEAE-cellulose (400 ml) which had been equilibrated with l/lSM phosphate buffer (pH 7.5).
The column was developed with the same buffer solution containing an increasing amount of sodium chloride (0~0.2M). The active eluate was desalted by dialysis and charged on a column of DEAE-Sephadex A-50 (17 ml). The column was developed with 1/15M
phosphate buffer (pH 7.5) containing a gradient concentration of sodium chloride (0~0.3M). The active fractions as determined by B. subtilis M45 assay were pooled and dialyzed against running water for 18 hours. The desalted solution was chromatographed on a column of DEAE-Sephadex A-50 (18 ml) using a 1/15M phosphate buffer (pH 7.0)-NaCl (0~0.3M) system as an eluant. The appropriate fractions were collected, concentrated to 10 ml and applied on a column of Sephadex G-50 for desalting. The column was eluted by deionized water and the active eluate lyophilized to afford 120 mg of white powder. The sample of BBM-1644 thus obtained was homogeneous as revealed ~y polyacrylamide gel electrophoresis.

Claims (6)

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

1. The process for the production of the antibiotic, BBM-1644, which comprises cultivating a BBM-1644-producing strain of Actinomadura sp. in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1644 is produced by said organism in said culture medium.

2. The process according to Claim 1 wherein the BBM-1644 is recovered from the culture medium.

3. The process according to Claim 1 wherein the BBM-1644-producing organism has the identifying characteristics of Actinomadura sp. H710-49 (ATCC 39144 ).

4. The process according to Claim 2 wherein the BBM-1644-producing organism has the identifying characteristics of Actinomadura sp. H710-49 (ATCC 39144 ).

5. The antibiotic BBM-1644 which:
(a) is effective in inhibiting the growth of gram-positive and acid-fast bacteria;
(b) is effective in inhibiting the growth of P388 leukemia in mice;
(c) induces prophage in lysogenic bacteria;
(d) is soluble in water but practically insoluble in methanol, ethanol, acetone, ethyl acetate and n-hexane;
(e) exhibits an infrared absorption spectrum (KBr) substantially as shown in FIG, 1;
(f) exhibits ultraviolet absorption spectra in water, 0.01N HCl and 0.01N NaOH substantially as shown in FIG. 2;
(g) has an optical rotation of [.alpha.]? -75.6° in 0.25%
aqueous solution;
(h) has no definite melting point but gradually de-composes above about 240°C;
(i) moves about 8.7 cm toward the anode during paper electrophoresis at 4500 V. for 1 hour using 0.05M
barbital buffer of pH 8.6;
(j) has the following elemental analysis: C, 46.60%;
H, 6.45%; N, 13.34%; S, 0.20% and O (by difference), 33.41%;
(k) is a high molecular weight peptide for which a molecular weight of about 22,000 is indicated;
(l) decolorizes potassium permanganate solution and gives positive Folin-Lowry, xanthoprotein, biuret and ninhydrin reactions and negative anthrone and Sakaguchi reactions; and (m) gives by hydrolysis the following relative amino acid composition based on the content of leucine being arbitrarily assigned as 1.0: alanine (8.8), aspartic acid (6.0), half-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), whenever prepared by the process of Claim 1, or by an obvious chemical equivalent thereof.

6. The antibiotic BBM-1644 which:
(a) is effective in inhibiting the growth of gram-positive and acid-fast bacteria;
(b) is effective in inhibiting the growth of P388 leukemia in mice;
(c) induces prophage in lysogenic bacteria;
(d) is soluble in water but practically insoluble in methanol, ethanol, acetone, ethyl acetate and n-hexane;

(e) exhibits an infrared absorption spectrum (KBr) substantially as shown in FIG, 1;
(f) exhibits ultraviolet absorption spectra in water, 0.01N HCl and 0.01N NaOH substantially as shown in FIG. 2, (g) has an optical rotation of [.alpha.]? -75.6° in 0.25%
aqueous solution;
(h) has no definite melting point but gradually de-composes above about 240°C;
(i) moves about 8.7 cm toward the anode during paper electrophoresis at 4500 V. for 1 hour using 0.05M
barbital buffer of pH 8.6;
(j) has the following elemental analysis: C, 46.60%;
H, 6.45%; N, 13.34%; S, 0.20% and O (by difference), 33.41%;
(k) is a high molecular weight peptide for which a molecular weight of about 22,000 is indicated;
(l) decolorizes potassium permanganate solution and gives positive Folin-Lowry, xanthoprotein, biuret and ninhydrin reactions and negative anthrone and Sakaguchi reactions; and (m) gives by hydrolysis the following relative amino acid composition based on the content of leucine being arbitrarily assigned as 1.0: alanine (8.8), aspartic acid (6.0), half-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), whenever prepared by the process of Claim 2, 3 or 4 or by an obvious chemical equivalent thereof.