NO832689L - ANTITUMOR ANTIBIOTICS. - Google Patents

Description

The present invention relates to a previously unknown anitumor antibiotic as well as its production and extraction.

Antitumor antibiotic, BBM-1644, according to the invention,

is a new protein antitumor antibiotic. Neocarzinostatin, macromomycin and auromomycin are examples of antibiotics of this type.

Neocarzinostatin (also called zinostatin) is an acidic protein macromolecule with a molecular weight of 10,700 and which consists of a single polypeptide chain with 109 amino acids, which is cross-linked by means of two disulphide bonds. Production of neocarzinostatin by fermentation of strains of Streptomyces carzinostaticus var. neocarzinostaticus is described in US Patent 3,334,022 and in the "Journal of Antibiotics",

18: 68-76 (1965). The amino acid sequence of neocarzinostatin is described in "Cancer Treatment Reviews" 6: 239-249 (1979).

Macromycin is a neutral or weakly acidic polypeptide with

an approximate molecular weight of approx. 15,000. The preparation of macromomycin by fermentation of Streptmyces macromomyceticus (NIHJ MC-8-42) is described in US Patent 3,595,954 and in "J. Antibiotics" 21: 44-49 (1968). Purification of macromomycin and characterization data for the purified compound are described in "J. Antibiotics" 29: 415-423 (1976).

Auromomycin is a weakly acidic polypeptide with a molecular weight

of approx. 12,500 and an isoelectric point at pH 5.4. It consists of 16 different amino acids. Isolation of auromomycin from the culture medium of Streptomyces macromomyceticus and characteristic properties of the purified product are described in "J. Antibiotics" 32, 330-229 (1979).

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

The present invention provides a so far

unknown protein antitumor antibiotic, here called BBM-1644, which antibiotic is produced by cultivating a new strain of Actinomadura, called Actinomadura sp. strain H710-49 (ATCC 39144) in an aqueous culture medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until the organism has produced a substantial amount of BBM-1644 in the culture medium, and if desired recovers BBM-1644 from the culture medium. The invention includes the BBM-1644 antibiotic in dilute solution, as crude concentrate, as crude solid and as purified solid.

In the attached drawing, Figure 1 shows the absorption spectrum

for BBM-1644 (KBr tablet), and Figure 2 shows ultraviolet absorption spectra for BBM-164 4 in water, 0.01 N HCI and 0.01 N NaOH.

The present invention relates to a previously unknown protein antitumor antibiotic called BBM-164 4, and its production by fermentation of a new strain of Actinomadura called Actinomadura sp. strain H719-4 9. The above-mentioned organism was isolated from a soil sample collected in West Germany. A biologically pure culture of the organism has been deposited at the American Type Culture Collection, Washington D.C., on July 1, 1982 and entered into the Permanent Microorganism Collection as ATCC 39144.

BBM-1644 inhibits the growth of various Gram-positive and acid-fast bacteria. The antibiotic also exhibits phage-inducing properties in lysogenic bacteria and inhibits the growth of lymphatic and solid tumors such as P388 leukemia in mice. The new antibiotic can therefore be used as an antibacterial agent or as an antitumor agent for inhibiting mammalian tumors.

> The microorganism.

Actinomycetes strain H710-49 was isolated from a soil sample

and prepared using conventional methods such as a

biologically pure culture for characterization. Strain H710-49

forms both substrate and aerial mycelium. The substrate mycelium is long, branched and not fragmented into short filaments. Short spore chains are borne on the tip of the aerial mycelium or monopodia-

branch. The spore chains contain 2 to 15 spores per chain (usually 4 to 8 spurs) and are straight, curved or curly. The spores have a warty surface and are oval to elliptical (0.5~0.6 x 0.7-1.2 ym) with a rounded or pointed end. Mature spores are often separated by empty hyphae. Swelling of the hyphal ends is occasionally observed on the substrate mycelium in Czapek's agar and Bennet's agar. Motile spores, sporangia or sclerotic grains are not seen in any of the investigated media.

Unlike the usual species of the genus Streptomyces, strain H710-49 grows slowly and forms poor aerial mycelium in chemically defined media and naturally organic media. The color of the aerial mycelium is white and it acquires a pink tone after sporulation in oat agar, inorganic salt-starch agar and glycerol-asparagine agar. The mass color of the substrate mycelium is colorless, yellow, reddish brown or dark gray brown. No melanoid pigment is produced, but a lemon yellow diffusible pigment is seen 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 or D -mannitol. Strain H710-49 grows at 20°C, 28°C and 37°C, but not at 10°C and 41°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

of the tribe. Cultivational and physiological characteristics

for strain H710-4 9 is shown in Tables 1 and 2. The pattern of the strain's carbon source utilization is shown in Table 3.

Purified cell wall from strain H710-49 contains meso-diamino-pimelic acid, but lacks glycine. The whole-cell hydrolyzate shows the presence of madurose (3-O-methy1-D-galactose), glucose, ribose and some mannose. The cell wall preparation and the whole cell sugar components of strain H710-49 indicate that the strain belongs to

cell wall type III,.

The aforementioned characteristics of strain H710-49 are similar to those of the genus Actinomadura. According to Goodfellow et al's numerical taxonomy of Actinomadura and related actinomycetes in J. Gen. Microbiol. 112: 95-111 (1979), most Actinomadura species of soil origin are classified in group no. 7 (Cluster No. 7) among the 14 described groups. Strain H710-49 is most closely related to the species in group 7. Nonomura and Ohara have in J. Perment. Technol. 49: 904-912 (1971) referred 5 saprophytic species of the genera Actinomadura and Nonomura have in J. Ferment. Technol. 52: 71-77 (1974) and Preobrazhenskaya et al in Actinomycetes and Related Organisms 12: 30-38 (1977) described the identification and classification of Actinomadura species. After comparison with known Actinomadura species described in the literature, strain H710-4 9 is considered to belong to a new species of Actinomadura similar to A. roseola, A. salmonea, A. vinacea or A. corallina, which

is described in the above-mentioned Preobrazhenskaya et al. reference and in Japanese Kokai 55/94391.

It should be clear that for the production of BBM-1644 the present invention, although described in detail with reference to the particular strain Actinomadura sp. strain H710-49 (ATCC 39144), not limited to this microorganism or to microorganisms fully described by the culture characteristics shown herein. In particular, it is intended that the invention includes strain H710-49 and all natural and artificial BBM-1644 producing variants and mutants thereof.

Production of antibiotics.

The BBM-1644 antibiotic according to the invention can be prepared by cultivating a BBM-1644 producing strain of the genus Actinomadura, preferably a strain of Actinomadura sp. with 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 nutrient sources for actinomycetes. that is, assimilable sources of carbon and nitrogen as well as possibly inorganic salts and other known growth factors. Submerged aerobic conditions are preferably used for the production of large quantities of antibiotics, although surface cultures and flasks can also be used for the production of limited quantities. The most common methods used for the cultivation of other actinomycetes can be used in the present 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 nitrogen sources, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc. can be used either alone or combined with organic nitrogen sources, such as peptone, meat extract, yeast extract, corn slurry, soybean powder, cottonseed meal, etc. Nutritive inorganic salts can also be added if necessary for to provide sources for sodium, potassium, calcium, ammonium, phosphate, sulphate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt, iron etc.

Production of the antibiotic BBM-1644 can be carried out

at any temperature which contributes to satisfactory growth of the producing organism, e.g. 20-37°C, and preferably carried out at a temperature of approx. 27-32°C. Usually, optimal production is achieved in shake flasks after incubation periods of approx. 6 to 7 days. When tank fermentation is to be carried out, it is desirable to provide a vegetative inoculum in a nutrient substrate by inoculating the substrate culture with a slant substrate or soil culture or a lyophilized culture of the organism. After an active inoculum has been obtained in this way, it is aseptically transferred to the fermentation tank medium. Antibiotic production can be monitored by paper disk agar diffusion assay using Bacillus subtilis M 45 (Ree mutant: Mutation Res. 16: 165-174 (1972)) as test organism.

Isolation and Purification.

When the fermentation is finished, BBM-1644 is mainly found in the liquid part of the fermented substrate after separation of the solid part by filtration or centrifugation. The harvested substrate can thus be separated into mycelial cake and substrate supernatant by centrifugation. The filtrate is then concentrated and dialyzed against tap water with a semipermeable membrane such as a cellophane tube to remove permeable impurities. The internally retained solution (after removal of solubles) containing BBM-1644 can then be saturated with a salting-out reagent such as ammonium sulfate to precipitate BBM-1644 as a solid feedstock. This solid substance can be dissolved in water and desalinated by dialysis against tap water.

Further purification of the impure BBM-1644 can be accomplished by conventional procedures used with other acidic polypeptides. E.g. the aqueous solution containing BBM-1644 can be absorbed on an ion exchanger such as "DEAE-Sephadex", "DEAE-Cellulose", "CM-Sephadex" or "CM-cellulose" and eluted with a neutral salt solution. Successive chromatographic steps are preferably used

with a gradient concentration of the salt solution used as eluent. Aqueous fractions containing the purified BBM-1644 are then concentrated to dryness, e.g. by lyophilization.

Physico-chemical properties of BBM-1644.

BBM-1644 is isolated as an amorphous white powder after lyophilization. When examined by high voltage paper electrophoresis (4500 V in 0.05 M barbital buffer pH 8.6), ^BBM-16 44 migrates as an acid migrating 8.7 cm toward the anode after 1 hour. BBM-1644 does not have a well-defined melting point and it gradually decomposes above 240°C. It is soluble in water, but practically insoluble in common organic solvents, such as methanol, ethanol, acetone, ethyl acetate and

n-hexane. The antibiotic exhibits an optical rotation of

[c] 2q = -75.6° in 0.25% aqueous solution. As shown in Figure 2, the UV spectrum of BBM-1644 shows absorption maxima at 275 nm (E'° 8.2) and 310 nm (E° 4.6, shoulder) in aqueous

1 cm ^ 1cmv twist. It exhibits an almost identical UV spectrum in water and in 0.01 N HCl solution, but only a single maximum at 285 nm (E^m8.9) in 0.01 N NaOH solution. The IR spectrum for BBM-1644, measured in KBr, is shown in figure 1. The spectrum indicates the presence of NH and OH groups (3300-2980 cm~^) and amide groups (1650 and 1540 cm"''). The antibiotic gives positive reactions with Folin-Lowry, xanthoprotein, biuret and ninhydrin reagents and decolorizes potassium permanganate solution. It is negative to anthrone and Sakaguchi reactions. When BBM-1644 is co-chromatographed on "Sepahdex G-75" with ovalbumin down molecular weight 43,000, chymotrypsinogen with molecular weight 25,000 and ribonuclease A with molecular weight 13,700, it elutes just after chymotrypsinogen and the molecular weight is therefore estimated to be about 22,000. Elemental analysis for BBM-16 44 shows 46.60% carbon, 6.45% hydrogen, 13, 34% nitrogen and 0.20% sulfur The presence of 13 different amino acids in the BBM-1644 molecule was demonstrated by amino acid analysis as listed in Table 4. Basic amino acids such as lysine, histidine and arginine are found in BBM-1644.

BBM-16 44 is fairly stable in the pH range 2-9, but stability drops rapidly outside this pH range. The aqueous solution of BBM-1644 is stable for 2 hours at 50°C at neutral pH. After exposure to ultraviolet light, BBM-1644 loses its antibiotic activity within 20 minutes.

The above-described physicochemical properties of BBM-1644 indicate that it belongs to the protein antitumor antibiotic group that includes neocarzinostatin, macromomycin and auromomycin. However, BBM-1644 can be separated from the known protein antitumor antibiotics due to molecular weight, amino acid content and paper electrophoresis. The paper electrophoretic mobilities for BBM-1644, neocarzinostatin and macromomycin are shown in Table 5.

The neocarzinostatin group of antibiotics usually exhibits

two UV absorption maxima at approx. 275 and 250 nm, while BBM-1644 has a UV maximum at approx. 275 and 310 nm. It is

recently reported in Biochem. Res. Commun. 95: 1351-1356

(1980) that the maximum at 350 nm for neocarzinostatin antibiotics may be due to the non-protein chromophores essential to their biological activity. It is noted that BBM-1644 has a chromophore that is different from the known neocarzinostatin group of antibiotics.

Biological properties of BBM-1644.

BBM-1644's antibacterial activity was determined by twofold agar serial dilution. A nutrient agar medium was used for gram-positive and gram-negative bacteria, with nutrient agar medium containing 4% glycerol being used for acid-fast bacteria and Sabouraud agar medium for fungi. The activity was expressed as minimal inhibitory concentration, MIC, in the agar medium and the results are shown in Table 6 together with the results for neocarzinostatin. BBM-1644 showed strong inhibitory activity against gram-positive and acid-fast bacteria, but did not inhibit the growth of gram-negative bacteria and fungi. BBM-1644's antibacterial spectrum is similar to that of neocarzinostatin, while the intrinsic activity of BBM-1644 is more potent than the last-mentioned in any of the test organisms.

The ability of BBM-1644 to induce prophage in lysogenous bacteria, ILB, was determined by the method of Lein et al (Nature 196:783-784, 1962) using neocarzinostatin as the reference compound. Plate counts were made on agar plates containing test materials, T, and controls, C. A T/C ratio at plate counts of more than 3 was considered significant and the ILB activity was expressed by the minimal inducing concentration of the test compound. As shown in Table 7, the ILB activity of BBM-1644 is similar to that observed with neocarzinostatin, the minimal inducing concentration being 1 mcg/ml.

BBM-1644's antitumor activity is determined in mice (BDF^ strain) against lymphocytic leukemia P388. Each mouse was inoculated intraperitoneally with 3x10 3 cells of the tumor. Graduated doses of the antibiotic were administered intraperitoneally to mice 2 4 hours after tumor transplantation. The treatment was given once daily on days 1, 4 and 7 (q3d x 3 schedule) or 9 consecutive days (qd 1-»9 schedule). Neocarzinostatin was tested for comparison as a reference antitumor agent and the results are summarized in table 8. BBM-1644 was highly active against mouse leukemia in a dose interval from 0.03 to 1.0 mg/kg/day in both treatments. BBM-1644's antitumor activity was approximately the same as (qd 1 -»9 scheme) or 3 times more potent than (q3d x 3 scheme) neocarzinostatins in terms of minimal effective dose.

BBM-1644's antitumor activity was also demonstrated in another trial against P388 leukemia in mice, the results of which are shown below in Table 9. Details of the methods used in this trial are described in Cancer Chemother. Rep. 3: 1-87 (Part 3), 1972.

The acute toxicity of BBM-1644 was determined in mice (dd Y strain) by single intraperitoneal administration and the LD^-g was calculated to be 5.8 mg per kg.

i As shown above, BBM-1644 has strong antibacterial activity against gram-positive and acid-fast bacteria and is thus applicable for the therapeutic treatment of mammals, including

humans, and other animals against infectious diseases caused by such bacteria. In addition, it can be used for other conventional application areas for antibacterial agents, such as disinfection of 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-16 44 is also therapeutically useful for inhibiting the growth of mammalian tumors.

The present invention therefore provides a method for the therapeutic treatment of a human or animal host that is attacked by a bacterial infection or a malignant tumor, which method comprises administering to the host an effective antibacterial or tumor-inhibiting dose of BBM-1644 or a pharmaceutical preparation thereof.

In another aspect, the present invention provides a pharmaceutical composition comprising an effective antibacterial or tumor-inhibiting amount of BBM-1644 in combination with an inert pharmaceutically acceptable carrier or an inert pharmaceutically acceptable diluent. These preparations may be formulated in any pharmaceutical form suitable for parenteral administration.

Preparations according to the invention for parenteral administration comprise sterile aqueous or non-aqueous solutions, suspensions or emulsions. They can also be prepared in the form of sterile solid preparations that can be dissolved in sterile water, physiological saline or another sterile injectable agent immediately before use.

As will be appreciated, the actual preferred amounts of BBM-1644 antibiotic used will vary according to the particular preparation being formulated, the method of application and the particular situs, host or disease being treated. The person skilled in the art will take many factors which modify the drug's effect into account, e.g. age, body weight, sex, diet, administration time, administration method, excretion rate, host condition, drug combinations, reaction sensitivities and the severity of the disease. The administration can be carried out continuously or periodically within the maximum tolerated dose. Optimum levels of application for a given set of conditions can be determined by the person skilled in the art using conventional dose determination tests taking into account the guidelines set out above.

The invention is described in more detail below using examples. "DEAE cellulose" is a diethylaminoethyl ion-exchanged cellulose. "SEPHADEX G-50" is a filtration gel. "DEAE SEPAHDEX A50"

is a diethylaminoethyl anion exchange gel. "SEPADEX" is a registered trademark.

Example 1:

Fermentation of BBM-164 4

An agar slant substrate with well-established growth of Actinomadura

sp. H710-4 9 was used for inoculation of seed medium

(100 ml in a 500 ml Erlenmeyer flask) containing 1% mannitol, 2% peptone and 1% yeast extract, the pH of which was adjusted to 7.2 before sterilization. The seed culture was incubated at 32°C for 72 hours on a rotary shaker (250 rpm)

and 5 ml of the culture was transferred to the second inoculum medium

(100 ml) with the same composition as the first. It was grown under the same conditions used for the first culture. Five ml of the inoculum growth thus obtained was used to initiate fermentation in 500 ml Erlenmeyer flasks containing 100 ml of fermentation medium containing 2.5% mannitol, 0.5% glucose, 1% soybean meal, 0.5% peptone,

1% meat extract, 0.3% CaCO3 and 0.2% NaCl. The fermentation was carried out at 28 C on a rotary shaker at 250 rpm. minute. The production of antibiotic was monitored by paper disc agar diffusion assay using Bacillus subtilis M45 (Ree mutant) as the test organism. The antibiotic

activity in the culture substrate gradually increased as the fermentation progressed and reached approx. 300 me per ml after 6-7 days.

Example 2:

The harvested substrate in an amount of 18 L obtained by the fermentation in Example 1 was separated into a mycelial cake and a substrate supernatant using a Sharpless centrifuge ("Kokusan No. 4A"). The filtrate

was concentrated below 40°C to 1/10 of the original volume and the concentrate was dialyzed using cellophane tubing against tap water in a cold room. The internally retained solution was concentrated to approx. 1.5 1 which was centrifuged at 8000 G to remove insoluble material. The clear supernatant was saturated with ammonium sulfate and left for 5 hours at 5°C. The formed precipitate was collected by centrifugation, dissolved in 300 ml of water and desalted by dialysis against tap water. The dialyzed solution in an amount of 700 ml contained 22 g of solid raw material of BBM-1644 which was detected by lyophilization of a portion of the solution. The remainder of the solution was used for subsequent purification without concentration to avoid decomposition. The antibiotic solution was passed through a column of "DEAE-cellulose"

(Cl , 400 ml), and the column was washed with 1 liter of water and developed with 1/15 M phosphate buffer (pH 7.0) containing 0.3 M sodium chloride. The active factions were combined

(300 ml), dialyzed for 18 hours against tap water and chromatographed on a column of "DEAE cellulose" (400 ml) equilibrated with 1/15 M phosphate buffer of pH 7.5. The column was developed with the same buffer solution containing increasing amounts of sodium chloride (0-0.2M). The active eluate was desalted by dialysis and loaded onto a column of "DEAE-Sephadex A-50" (17 ml). The column was developed with 1/15 M phosphate buffer of pH 7.5 containing a graded concentration of sodium chloride (0-0.3 M). The active fractions detected by B. subtilis M45 analysis were mixed together and dialyzed against running water for 18 hours. The desalinated up-

solution was chromatographed on a column of "DEAE Sephadex A-50" (18 ml) using a 1/15 M phosphate buffer (pH 7.0)-NaCl (0-0.3 M) system as eluent. The appropriate fractions were pooled, concentrated to 10 ml and loaded onto a Sephadex G-50 column for desalting. The column was eluted with deionized water and the active eluate was lyophilized, whereby 120 mg of white powder was obtained. The sample of BBM-1644 thus obtained was homogeneous, as shown by polyacrylamide gel electrophoresis.

Claims (9)

1. Antibiotic with the condition BBM-1644, characterized in that it (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 insoluble 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 Figure 1; (f) exhibits ultraviolet absorption spectra in water, 0.01 N HCl and 0.01 N NaOH which are substantially as shown in Figure 2; (g) has an optical rotation of W ^ -75.6° in 0.25% aqueous solution, (h) has no well-defined melting point, but it gradually composes over approx. 24 0°C, (i) moves approx. 8.7 cm towards the anode during paper electrophoresis at 4,500 V for 1 hour using 0.05 M barbital buffer with pH 8.6, (j) has the following elemental analysis: C, 46.60%, H, 6.45%, N, 13.34%, S, 0.20% and 0 (by difference calculation), 33.41%. (k) is a high molecular peptide, for which it indicates a molecular weight of approx. 22,000, (1) decolorizes potassium permanganate solution and gives positive Folin, Lowry, xanthoprotein, biuret and ninhydrin reactions and negative anthrone and Sakaguchi reactions, and (m) on hydrolysis gives the following relative amino acid composition, based on the content of leucine, which is arbitrarily set to 1.0: alanine (8.8), asparagine- acid (6.0), half-cystine (1.0), glutamic acid (5.7), glycine (8.7), isoleucine (2.3) leucine (1.0) phenylanine (1.4), proline (4,1), serine (.1,6), threonine (7,2), tyrosine (0,6) and valine (11,1). 2. Method for producing the antibiotic BBM-16 44, characterized in that a BBM-16 44-producing strain of Actinomadura sp is cultivated. in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until the organism has produced a substantial amount of BBM-1644 in the culture medium. 3. Method according to claim 2, characterized in that BBM-1644 is extracted from the culture medium. 4. Method according to claim 2 or 3, characterized in that the BBM-1644-producing organism has the identifying characteristics of Actinomadura sp. H710-49 (ATCC 39144). 5. Biologically pure culture of the microorganism Actinomadura sp. ATCC 39144, which culture is capable of producing the antibiotic BBM-16 44 in a recoverable amount after cultivation in an aqueous nutrient medium containing assimilable sources of nitrogen and carbon. 6. Procedure for therapeutic treatment of a human or animal host affected by a bacterial infection, characterized in that the host is administered an effective antibacterial dose of BBM-1644. 7. Method for the therapeutic treatment of a human or animal host which is attacked by a malignant tumor, which is sensitive to BBM-1644, characterized in that a tumor-inhibiting dose of BBM-1644 is administered to the host. 8. Pharmaceutical preparation, characterized in that it contains an effective antibacterial amount of BBM-1644 in combination with a pharmaceutical carrier or a pharmaceutical diluent. 9. Pharmaceutical preparation, characterized in that it contains an effective tumor-inhibiting amount of BBM-1644 in combination with a pharmaceutical carrier or a pharmaceutical diluent.