GB2134119A

GB2134119A - Luzopeptin E2

Info

Publication number: GB2134119A
Application number: GB08402410A
Authority: GB
Inventors: Charles A Claridge; Henry Schmitz; Terrence W Doyle; Marianne F Kammer
Original assignee: Bristol Myers Co
Current assignee: Bristol Myers Co
Priority date: 1983-01-31
Filing date: 1984-01-30
Publication date: 1984-08-08
Also published as: SE8400450D0; KR840007106A; IT8419348A0; AU2372184A; IT1175927B; SE464582B; BE898807A; CA1220746A; NL8400237A; JPS59173091A; FR2540116B1; KR880001638B1; ZA84613B; JPH0645638B2; DE3403125A1; MY8800015A; SE8400450L; GB2134119B; CH663415A5; GR81749B

Abstract

A cyclic depsipeptide antibiotic designated luzopeptin E2 is produced by fermentation of Actinomedura luzonensis ATCC 31491 and isolation of the antibiotic free of substances coproduced therewith. Luzopeptin E2 exhibits antimicrobial activity and inhibits the growth of mammalian tumors.

Description

SPECIFICATION Luzopeptin E2 Background of the invention (1) Field of the invention This invention relates to a new cyclic depsipeptide antibiotic and a method for its production and recovery. Also provided are pharmaceutical compositions containing the new antibiotic and methods for using said antibiotic (and compositions thereof) as an antimicrobial and antitumor agent.

(2) Description of the prior art There is disclosed in U.K. Patent Application 2,050,384A an antitumor antibacterial complex designated BBM-928 and its production by fermentation of a new strain of actinomycetes designated strain G455--101 (ATCC 31491). This reference discloses that the BBM-928 complex has at least six components designated BBM-928 A, B, C, D, E and F. Components A, B, C and D were fully characterized in the reference while components E and F are characterized only by their Rf values in two silica gel TLC systems and by in vitro antimicrobial activities against certain aerobic bacteria.

The BBM-928-producing organism strain G455--101 was later determined to be a new species of the genus Actinomadura and designated Actinomadura luzonensis nov. sp. (see J.

Antibiotics 33(10): 1098-1102, 1980).

J. Am. Chem. Soc. 103: 1241 (1981) discloses the structures of BBM-928 A, B and C as does Pep tide Chemistry 1980, K. Okawa (Ed.), Protein Research Foundation, Osaka, Japan, pg. 11 9-1 24 (1981) and J. Antibiotics 34 (2): 148-159(1981).

The production, isolation, characteriziation and antitumor activity of the BBM--928 components are disclosed in J. Antibiotics 33 (10): 1087-1097(1980). As in U.K. Application 2,050,384A, components E and F are characterized only by their Rf values in two silica gel TLC systems.

A proposed mechanism of the antitumor activity of BBM-928 is disclosed in Biochemistry 19: 5537 (1980).

The BBM-928 antibiotics are presently named the luzopeptin antibiotics after the producing species. As used herein, therefore, luzopeptin A, B, C, etc. are identical to BBM-928A, B, C, etc.

previously used in the literature.

Description of the drawings Figure 1 shows the infrared absorption spectrum of luzopeptin E2 when pelleted in KBr.

Figure 2 shows the proton magnetic resonance spectrum of luzopeptin E2 dissolved in CDCI3.

Figure 3 shows the carbon 3 nuclear magnetic resonance spectrum of luzopeptin E2 dissolved in CDCI3.

Summary of the invention This invention relates to a novel antitumor antibiotic designated luzopeptin E2 and to a process for its preparation and isolation in a purified state free of co-produced substances. The antibiotic is obtained by cultivating a luzopeptin E2-producing strain of Actinomadura luzonensis, preferably Actinomadura luzonensis ATCC 31491 or a mutant thereof, in an aqueous medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of luzopeptin E2 is produced by said organism in said culture medium and recovering the luzopeptin E2 from the culture medium in a purified form substantially free of co-produced substances.

Luzopeptin E2 exhibits both antimicrobial and antitumor activities.

Detailed description Luzopeptin E2 provided by the present invention has been found to be a minpr component of the luzopeptin (or BBM-928) antibiotic complex disclosed in U.K. Application 2,050,384A. That application discloses the fermentation of Actinomadura luzonensis sp. nov. to produce a luzopeptin antibiotic complex and the separation of such complex into six bioactive components now called luzopeptins A, B, C, D, E and F. There is no disclosure, however, of the antibiotic component luzopeptin E2 which has now been found to be coproduced in the fermentation ofActinomadura luzonensis.

The new antibiotic of the present invention has been isolated from the Actinomadura luzonensis fermentation broth in an essentially pure form and has been characterized by its physical, chemical and biological properties as described in more detail below.

Studies carried out by colleagues of the present inventors after their isolation and characterization of luzopeptin E2 have established that luzopeptin E (see, for example, U.K. Application 2,050,384A) is not in fact a single antibiotic component but is instead a mixture of components including luzopeptin E2.

Properties of luzopeptin E2 Luzopeptin E2 is a cyclic depsipeptide antibiotic containing a quinoline nucleus as the chromophore. From spectral and chemical analysis, luzopeptin E2 has been determined to have the structural formula

Luzopeptin E2 is a white solid having a melting point of 201-2030C, a molecular weight of 1315.3732 and a molecular formula C60H78N14020. Elemental analysis indicates the following average percentages by weight: carbon-53.1 9 hydrogen--5.40 nitrogen-1 2.92 oxygen (by difference)-28.49 The infrared absorption spectrum of luzopeptin E2 when pelleted in KBr is shown in Figure 1 of the accompanying drawings.Characteristic infrared absorption bands are exhibited at the following frequencies expressed in reciprocal centimeters: 3470, 3370, 2980, 2983, 2850, 1 740, 1645, 1 520, 1418,1350,1285,1230, 1195, 1150,1128,1105,1065, 1025,905,885,795,785and750.

Proton magnetic resonance and carbon 3 nuclear magnetic resonance spectra of luzopeptin E2 were determined with a Bruker Model WM-360 spectrometer operating at 360 MHz and 90 MHz respectively. The PMR and CMR spectra are shown as Figures 2 and 3 of the drawings.

In a solvent system consisting of xylene-methyl ethyl ketone-methanol (5:5:1 v/v), luzopeptin E2 exhibits an Rf value of 0.33 as determined by silica gel thin layer chromatography.

The key structural feature of luzopeptin E2 which distinguishes it from known luzopeptin components is the presence of a hexahydropyridazine moiety instead of the tetrahydropyridazine group.

Preparation of luzopeptin E2 Luzopeptin E2 may be produced by cultivating a luzopeptin E2-producing strain of Actinomadura luzonensis, preferably a strain of Actinomadura luzonensis having the identifying characteristics of ATCC 31491 or a mutant thereof, under submerged aerobic conditions in an aqueous nutrient medium.

The producing organisms for luzopeptin E2 and the fermentation conditions for obtaining it are as disclosed in U.K. Patent Application 2,050,384A.

While the preferred producing organism isActinomadura luzonensis strain G455-1 01 (ATCC3 1491), any luzopeptin E2-producing strain or mutant of the preferred organism which can be produced from such organism by conventional means such as x-radiation, ultraviolet radiation, treatment with nitrogen mustards, phage exposure, and the like, is intended to be included within the scope of the present invention.

The producing organism is grown in a conventional nutrient medium containing an assimilable carbon source such as starch, glucose, dextrin, maltose, lactose, sucrose, fructose, mannose, molasses, glycerol and the like. The nutrient medium should also contain an assimilable nitrogen source such as protein, protein hydrolysate, polypeptides, amino acids, corn steep liquor, casein, urea and the like as well as nutrient inorganic salts which provide inorganic anions and cations such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, nitrate, and the like.

While not essential for production and recovery of luzopeptin E2, it is preferred to add a sulfurcontaining compound selected from the group consisting of D,L-methionine, S-methyl-cysteine, S ethyl-L-cysteine. D,L-ethionine, D-methionine, L-methionine, acetyl-methionine, 2-aminoethanethiol, 2-hydroxy-4-(methylthio)butyric acid, D,L-O-methyl-serine, L-cysteine and L-methionyl glycine to the nutrient medium. The preferred sulfur-containing compounds are D,L-methionine and S-methylcysteine. Such sulfur-containing compounds may be added for example, in a concentration of from about 0.05% to about 0.5% (wt/vol) of the fermentation medium with the most preferred concentrations for D,L-methionine and S-methyl-cysteine being 0.15% and 0.1%, respectively.

Addition of the above-mentioned sulfur-containing compound is found to increase the production of luzopeptin E2 so that it can be more readily assayed and isolated.

In producing luzopeptin E2, any temperature conducive to satisfactory growth of the producing organism may be employed. Temperatures ranging from about 200 to 450C are operable with a temperature range of 27 to 350C most preferred. Maximum production of luzopeptin E2 is generally obtained after about 72 to 96 hours.

Conventional methods are employed in the fermentation process. For example, preparation of small amounts is conveniently carried out in shake flasks or by surface cultures. Preparation of large amounts is preferably carried out under submerged aerobic conditions in sterile tanks. With tank fermentation a vegetative inoculum is first produced in a nutrient broth by inoculating the broth culture with a spore from the organism to provide a young active seed culture which is then aseptically transferred to the fermentation tank medium. Aeration in tanks and bottles may be provided by forcing sterile air through or onto the surface of the fermenting medium with further agitation in tanks provided by a mechanical impeller. Anti-foaming agents such as silicone oil, soybean oil and lard oil may be added as needed.

The course of the fermentation can be followed by assaying the fermentation medium from time to time against an organism sensitive to luzopeptin E2 such as Bacillus subtilis ATCC 6633. Luzopeptin E2 may also be detected by UV observation at 360 nm or by its Rf value of 0.37 in the silica gel TLC system xylene-methyl ethyl ketonemethanol (9:9:1 v/v).

Isolation of luzopeptin E2 Luzopeptin E2 may be isolated from the fermentation broth by conventional procedures such as solvent extraction procedures and chromatographic procedures as more fully described in Example 1.

Such procedures allow the luzopeptin E2 component to be recovered in a purified form substantially free of co-produced products.

Biological activity data The in vitro minimum inhibitory concentrations (MIC) of luzopeptin E2 were determined by the serial agar dilution method. Mueller-Hinton agar was generally used for gram-positive and gramnegative bacteria except for acid-fast bacteria in which case No. 1001 medium (3% glycerol, 0.3% sodium L-glutamate, 0.2% peptone, 0.31% Na2HPO4, 0.1% KH2PO4, 0.005% ammonium citrate, 0.001% MgSO4 and 1.5% agar) was used. The results are shown in Table 1 in comparison with the activity of luzopeptin A.

Table 1 In vitro antimicrobial activity of luzopeptin E2 MIC in mcg/ml Test organism Luzopeptin E2 Luzopeptin A Staphylococcus aureus 209P 0.4 0.4 Staphylococcus aureus Smith 0.4 0.8 Streptococcus pyogenes S-23 < 0.05 0.2 Sarcina lutea PCI 1001 0.1 0.2 Micrococcus flavus D12 0.2 0.2 Corynebacterium xerosis 53K-I 0.8 0.8 Bacillus subtllis PCI 219 0.2 0.8 Bacillus megaterium D-2 0.2 0.2 Bacillus anthracis A9540 0.1 0.2 Escherichia coli N I H J > 100 > 100 Klebsiella pneumoniae D-1 1 > 100 > 100 Proteus vulgaris A9436 > 100 > 100 Pseudomonas auruginosa A9930 > 100 > 100 Mycobacterium smegmatis 607 0.8 0.4 Mycobacterium phlei D88 1.6 0.4 The antitumor activity of luzopeptin E2 against leukemia P388 was evaluated in BDF, mice by previously reported procedures (Can. Chem. Rep. 50: 479, 1966 and Can. Chem. Rep. 3: 1 972).

Results of this test are shown below in Table 2 along with results for luzopeptin A.

Table 2 Effect of luzopeptin E2 on P-388 lymphatic leukemia Tumor inhibition Compound Dose (mg/kg/day) MST(% TIC) Luzopeptin E2 64 Toxic 32 144 16 167 8 122 4 122 2 100 1 100 Luzopeptin A 64 1 56 32 161 16 144 8 144 4 128 2 111 Treatment: Once daily for nine injections Host: BDF1 9 mice Evaluation: MST=median survival time % T/C=MST treated/MST controlx 100 Criterion: T/C > 125 considered significant antitumor effect Toxicity: < 4/6 survivors, day 5 In a second test the antitumor activity of luzopeptin E2 was evaluated against leukemia P388 in BDF1 mice by the method reported in J. Antibiotics 33:1087-1097 (1980). Luzopeptin A was comparatively tested as a reference compound.As shown in Table 3, luzopeptins A and E2 showed nearly equivalent antitumor activity. The intraperitoneal toxicity determined after multiple dosing (qd 1e9) of luzopeptin A and E2 is shown in the last line of Table 3. The toxicity of luzopeptin E2 is almost 1/2 that of luzopeptin A.

Table 3 Antitumor activity of luzopeptin A on P388 leukemia Dose* lipJ Antitumor activity {% T/C% in MUST) in mg/kg/day Luzopeptin A Luzopeptin E2 0.1 0.03 89 143 0.01 174 181 0.003 168 149 0.001 141 129 0.0003 121 117 MED* (mg/kg/day) 0.001 0.001 LD50* (mg/kg/day) 0.019 0.034 *dosing schedule:qd 1 o9 As indicated by the antimicrobial and mouse tumor data provided above, luzopeptin E2 is useful as an antibiotic and also as an antitumor agent for inhibition of mammalian malignant tumors such as P388 leukemia. The invention includes within its scope pharmaceutical compositions containing an effective antimicrobial or tumor-inhibiting amount of luzopeptin E2 in combination with an inert pharmaceutically acceptable carrier or diluent. Such compositions may also contain other active antimicrobial or antitumor agents and may be made up in any form appropriate for the desired route of administration. Examples of such compositions include solid compositions for oral administration such as tablets, capsules, pills, powders and granules, liquid compositions for oral administration such as solutions, suspensions, syrups or elixers and preparations for parenteral administration such as sterile 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.

According to another aspect of the invention, a method is provided for therapeutically treating a mammalian host affected by a microbial infection or by a malignant tumor (e.g. P388 leukemia) which comprises administering to said host an effective antimicrobial or tumor-inhibiting dose of luzopeptin E2 or a pharmaceutical composition thereof.

The foliowing example serves to illustrate the invention without in any way limiting it to the specific embodiment disclosed. Pharmamedia is a trademark of Buckeye Oilseed Products Co., Fort Worth, Texas, for cottonseed meal. Nutrisoy is a trademark of Archer Daniels Midland Co., Decatur, Illinois, for soybean meal ground into a flour. Fermo 30 is a trademark of Yeast Products Inc., Clifton, New jersey, for a yeast powder. Skellysolve B is a trademark of Skelly Oil Co., Kansas City, Missouri, for a petroleum solvent comprising isomeric hexanes and having a boiling point of 60"--680C. Nylon Copol 8 is a tubular piece of nylon used as a column in place of glass. The nylon can withstand certain solvents and, when the chromatography is completed, the column can be cut to obtain the fractions.

Example 1 Preparation of luzopeptin E2 A. Fermentation A well-grown vegetative preparation of Actinomadura luzonensis strain G455-1 01 was maintained frozen (-1 20C) in sucrose at a final concentration of 20%. This frozen vegetative preparation was used to inocculate a vegetative medium containing 3% cerelose,1 % Pharmamedia (cottonseed meal), 1% Nutrisoy (soybean meal powder) and 0.3% CaCO3. The seed culture was incubated at 350C for 48 hours on a rotary shaker (210 rpm) and 7 ml of growth was transferred to a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium composed of 3% cerelose, 2% Pharmamedia, 1% Fermo 30 (yeast powder), 0.5% CaCO3 and 0.15% DL-methionine, the pH being adjusted to 7.0 before sterilization.Production of luzopeptin E2 reached a maximum between 72 and 96 hours at the 350C incubation temperature.

B. Detection Luzopeptin E2 can be isolated from the fermentation mixture by dilution of the whole broth with an equal volume of pH 8, 0.5M Tris-HCI buffer followed by an extraction with two broth volumes of methylene chloride. After a brief centrifugation to break the emulsion, the solvent layer was spotted on Analtech silica gel plates and developed in a xylene-methyl ethyl ketone-methanol (9:9:1) system.

Luzopeptin E2 appears at Rf 0.37, between BBM-928A (Rf 0.57) and BBM-928B (Rf 0.27). All three compounds can be detected by UV observation at 360 nm or by bioautography employing agar adjusted to pH 8.0 and seeded with Bacillus subtilis ATCC 6633.

C Isolation The whole beer (10 from a fermentation of Actinomadura luzonensisstrain G455--101 grown in the presence of DL-methionine (1.5 g/l), was adjusted to pH 8 with sodium carbonate and stirred for 2 hours with 4 1 of n-butanol in the presence of diatomaceous earth as the filter aid. The resulting mixture was filtered, and the organic phase separated and concentrated under reduced pressure to an oily residue. Trituration of the oily residue with Skellysolve B (trademark of Skelly Oil Co. for petroleum solvent comprising isomeric hexanes and having a b.p. of 60--680C) and diethyl ether afforded, after filtration and drying, 4 g of enriched luzopeptin complex.The enriched luzopeptin complex was chromatographed on a dry silica gel column prepared according to Loev and Goodman (Progress in Separation and Purification, Vol. 3, p. 73, 1970). Dry column silica gel (Merck; 280 g) was deactivated by the addition of 40 ml of distilled water and vigorous shaking for 3 hours followed by equilibration for 3 additional hours with 1 8 ml of a mixture of xylenemethyl ethyl ketone (1 :1 by vol.). The silica gel was then used to pack the column using a 1 60 gauge Nylon Copol 8 (4x 100 cm) column with the help of glass wool.

The luzopeptin complex (4 g) was dissolved in 30 ml of xylene-methyl ethyl ketone (1 :1 by vol), filtered, coated on silica gel by removal of the solvent under reduced pressure and applied to the dry column. The column was developed with methyl ethyl ketone-xylene-methanol (5:5:1 by vol) until the eluent had just overrun the bed of column. Observing the column with a portable UV lamp at 360 nm, the yellow fluorescing zones were cut and the resulting silica gel sections eluted with chloroformmethanol (1 :1 by vol) and, after thin layer chromatography (TLC) analysis on SiO2 (methyl ethyl ketonexylene-methanol; 5:5:1 by vol), the solids containing luzopeptin E2 were combined (494 mg). The enriched luzopeptin E2 fraction (494 mg) was dissolved in methylene chloride, coated on silica gel and chromatographed on a 2x80 cm silica gel column slurry packed in toluene with a toluene-methanol gradient (0 to 10%). Fractions were collected and analyzed by TLC on silica gel (methyl ethyl ketonexylene-methanol; 5:5:1 by vol). Elution with 7% methanol yielded luzopeptin A and impurities followed by elution of luzopeptin A and luzopeptin E2 (Fractions 71-75) and pure luzopeptin E2 (Fractions 76-80). Crystallization of pure luzopeptin E2 from methylene chloride-toluene afforded 22 mg of crystalline luzopeptin E2.

Claims

1. The antitumor antibiotic luzopeptin E2 having the structural formula

2. The antitumor antibiotic luzopeptin E2, said antibiotic being substantially free of substances coproduced therewith.

3. A process for the preparation of antitumor antibiotic luzopeptin E2 having the formula

which comprises cultivatingActinomadura luzonensis ATTC 31491, or a luzopeptin E2-producing mutant thereof, in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of luzopeptin E2 is produced by said organism in said culture medium and isolating said luzopeptin E2 from the culture medium substantially free of substances co-produced therewith.

4. The process according to claim 3 wherein a sulfur-containing compound selected from the group consisting of D,L-methionine, S-methyl-cysteine, S-ethyl-L-cysteine, D,L-ethionine, Dmethionine, L-methionine, acetylmethionine, 2-aminoethanethiol, 2-hydroxy-4-(methylthio) butyric acid, D,L-O-methyl-serine, L-cysteine and L-methionyl glycin is added to the nutrient medium in the fermentation step.

5. The process according to claim 3 wherein D,L-methionine or S-methyl-cysteine is added to the nutrient medium in the fermentation step.

6. A process of the preparation of luzopeptin E2 substantially as hereinbefore described with reference to the Example.

7. Luzopeptin E2 for use in the treatment of a microbial infection or malignant tumor in a mammal.

8. Luzopeptin E2 substantially free of substances coproduced therewith, for use in the treatment of a microbial infection or malignant tumor in a mammal.