WO1993013217A1

WO1993013217A1 - Antibiotic ge 21640a

Info

Publication number: WO1993013217A1
Application number: PCT/EP1992/002773
Authority: WO
Inventors: Sergio Stella; Nicoletta Montanini; Enrico Selva; Maurizio Denaro
Original assignee: Gruppo Lepetit S.P.A.
Priority date: 1991-12-27
Filing date: 1992-12-01
Publication date: 1993-07-08
Also published as: AU3083892A

Abstract

The present invention is directed to a new antibiotic substance denominated antibiotic GE 21640A, the addition salts thereof, the pharmaceutical compositions thereof and their use as medicaments, particularly in the treatment of infectious diseases involving microorganisms susceptible to them.

Description

ANTIBIOTIC GE 21640A

The present invention is directed to a new antibiotic substance denominated antibiotic GE 21640A, the addition salts thereof/ the pharmaceutical 0 compositions thereof and their use as medicaments, particularly in the treatment of infectious diseases involving microorganisms susceptible to them.

The compounds of the invention are also active as growth promotant agents in animals, such as poultry, g swine, ruminants, etc.

Another object of the invention is a process for preparing antibiotic GE 21640A which includes culturing Streptomyces sp. ATCC 55243 or an antibiotic GE 21640A _Q producing variant or mutant thereof and isolating the antibiotic of the invention from the mycelium and/or the fermentation broths.

Streptomyces sp. ATCC 55243 was isolated from a 5 soil sample and deposited on 24 October 1991 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 Maryland, U.S.A., under the provisions of the Budapest Treaty.

The strain has been accorded accession number ATCC ^{552 3«}

The production of antibiotic GE 21640A is achieved by cultivating a Streptomyces sp. strain capable of producing it, i.e. Streptomyces sp. ATCC 55243 or an antibiotic GE 21640A producing variant or mutant thereof, under aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic salts. -Many of the nutrient media usually employed in the fermentation art can be used, however certain media are preferred. Preferred carbon sources are glucose, mannose, galactose, starch, corn meal and the like. Preferred nitrogen sources are ammonia, nitrates, soybean meal, peptone, meat extract, yeast extract, tryptone, aminoacids, and the like. Among the inorganic salts which can be incorporated in the culture media there are the customary soluble salts capable of yielding sodium, otassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride, carbonate sulfate, phosphate, nitrate and the like ions. Ordinarily, the antibiotic-producing strain is pre-cultured in a shake flask, then the culture is used to inoculate jar fermentors for production of substantial quantities of the antibiotic substances. The medium used for the pre-culture can be the same as that employed for larger fermentations, but other media can also be employed. The antibiotic GE 21640A producing- strain can be grown at temperatures between 20 and 40°C, preferably between 24 and 35°C.

During fermentation, the antibiotic production can be monitored by testing broth or mycelial extract samples for antibiotic activity for instance by bio- assays or TLC or HPLC procedures.

Sensitive organisms to antibiotic GE 21640A such ^as Nβisseria caviae can be used as test organisms. The bioassay is conveniently performed by the agar diffusion method on agar plates. Maximum production of antibiotic activity generally occurs between the second and the fifth day of fermentation. Antibiotic GE 21640A is produced by cultivating the strain Streptomyces sp. ATCC 55243, or an antibiotic GE 21640A producing mutant or variant thereof, and is found both in the mycelium and in the filtered fermentation broth.

In the present disclosure when dealing with the compounds of the invention in relation to their physico- chemical or biological properties, the term "antibiotic GE 21640A" is generally considered to refer to antibiotic GE 21640A as well as the addition salts thereof.

Antibiotic GE 21640A may in fact be converted into a corresponding non-toxic pharmaceutically acceptable salt. Suitable salts include the alkali and alkaline earth metal salts, typically the sodium, potassium, calcium and magnesium salts, and the ammonium and substituted ammonium salts. Representative substituted ammonium salts include primary, secondary or tertiary (Cι-C₄)alkylammonium and hydroxy (Ci-C alkylammonium salts and, according to an embodiment of the present invention, the benzathine, procaine, hydrabamine and similar water insoluble, non-toxic, pharmaceutically acceptable salts. Another preferred class of salts of the compounds of the present invention is represented by the basic addition salts with basic aminoacids such as Arginine or Lysine, or aminosugars such as glucosamine and the like.

The alkali and alkaline earth metal salts are prepared according to the usual procedures commonly employed for preparing metal salts. As an example, antibiotic GE 21640A is dissolved into the minimum amount of a suitable solvent, typically a lower alkanol, the stoichiometric amount of a suitable selected base is gradually added to the obtained solution and the obtained salt is precipitated by the addition of a non- solvent. The alkali or alkaline earth metal salt which forms is then recovered by filtration or evaporation of the solvents.

Alternatively, these salts can be prepared in a substantially anhydrous form through lyophilization; in this case aqueous solutions containing the desired salts, resulting from the salification of antibiotic GE 21640A with a suitably selected alkali or alkaline 0 earth metal carbonate or hydroxide in such a quantity as to obtain a pH comprised between 7.0 and 8.5 are filtered from any insolubles and lyophilized.

The organic ammonium salts can be prepared substantially following the above procedure by adding the properly selected amine to a solution of antibiotic GE 21640A in a suitable solvent and then evaporating off the solvent and the excess of the amine reagent or by lyophilizing the concentrate solution.

0 Morphological characteristics of Streptomyces sp. ATCC 55243

Strain GE21640 grows well on most standard media.

Its vegetative mycelium is generally colorless and forms 5 long, slender and irregularly branched hyphae (0.8 to

1.0 μm in diameter).

The aerial mycelium forms chains of spores arranged in open spirals and hooks on the lateral branches of the main hypha. _Q The spore mass is light gray with dark gray spots.

The spores are round, non motile and have a diameter of

1.2 to 1.4 μm. Cultural characteristics of Streptomyces sp. ATCC 55243

For the examination of the cultural characteristics, strain GE21640 was cultivated on various standard media suggested by Shirling and

Gottlieb (Shirling E.D. and Gottlieb D.; 1966; Method for Characterization of Streptomyces species; Int. J. Syst. Bacteriol.; 16, 313-340) with the addition of several media recommended by aksman (Waksman S.A.; 1961; The Actinomycetes; The Williams and Wilkins Co., Baltimore; 2, 328-334).

Color determination was made, when necessary, by the method of Maerz and Paul (Maerz A. and M. Rea Paul; 1950; A Dictionary of Color; 2nd Edition McGraw-Hill Book Company Inc.; New York).

The ability of the organism to utilize carbon sources was investigated by the method of Shirling and Gottlieb.

The cultural and physiological characteristics and the carbon sources utilization were evaluated after 2 weeks of incubation at 28°C (Tables I, II, III).

TABLE I Cultural characteristics of strain Streptomyces sp.

ATCC 55243

Culture media Morphological characteristics

ISP n.2 (yeast Abundant growth with crusty extract-malt agar) surface, colorless, scarce formation of white aerial mycelium.

ISP n.3 (oatmeal agar) Abundant growth with crusty surface, colorless, moderate formation of ash gray spores

ISP n.4 (starch agar) Abundant growth with smooth surface, colorless, scarce formation of white aerial mycelium^"

ISP n.5 (glycerol- Abundant growth with slightly asparagine agar) crusty surface at the edges of the growth, traces of white aerial mycelium

ISP n.6 (peptone-yeast Moderate growth with slightly extract-iron agar) crusty surface, traces of white aerial mycelium

ISP n.7 (tyrosine Abundant growth with slightly agar) crusty surface at the edges of the growth, reddish-brown 8/E/10, moderate formation of graysh aerial mycelium, pinkish- brown to brown soluble pigment TABLE I (continued) Cultural characteristics of strain Streptomyces sp.

ATCC 55243

TABLE I (continued) Cultural characteristics of strain Streptomyces sp.

ATCC 55243

Culture media Morphological characteristics

Czapek-glucose agar Moderate growth with smooth and thin surface, colorless, scarce formation of white aerial mycelium

Czapek-sucrose agar Moderate growth with smooth and thin surface, colorless, scarce formation of white aerial mycelium

Glucose-asparagine Moderate growth with smooth agar and thin surface, colorless, moderate formation of white aerial mycelium

Calcium-malate agar Moderate growth with smooth and thin surface, colorless, moderate formation of white aerial mycelium

Skim-milk agar Abundant growth with smooth surface, rose 4/G/8, traces of white aerial mycelium, cinnamon- pink soluble pigment 5/D/10

Egg-albumine agar Moderate growth with smooth and thin surface, colorless, scarce formation of white aerial mycelium

Letter and numbers refer to the color determined according to Maerz and Paul (see above) TABLE II

Physiological characteristics of strain

Streptomyces sp. ATCC 55243

TABLE III Carbohydrate utilization

negative response

+/ doubtful response good positive response strong positive response For this test, medium ISP n. 8 is employed Sensitivity to temperature

The optimum growth temperature ranges from 28°C to 37°C. No growth is observed at 15°C and 50°C. Moderate growth at 20°C.

Chemotaxonomical characteristics

Cell wall analysis

The analysis of the whole-cell hydrolysate, carried out using the method of Staneck and Roberts (Staneck J.L. and Roberts G.D.; 1974, Simplified Approach to Identification of Aerobic Actinomycetes by Thin-Layer Chromatography; 28, 226-231), revealed the presence of LL-Diaminopimelic acid.

Whole cell sugar pattern

The analysis of the sugar content in the whole cell hydrolysate was carried out using the method of Saddler et al (Saddler G.S., Tavecchia P., Lociuro S., Zanol M., Colombo L. and Selva E.; 1991; Analysis of Madurose and Other Actinomycete Whole Cell Sugars by Gas Chromatography; Journal of Microbiological Methods (in press) .

Briefly, strain GE 24640A was examined using a modification of a technique described by Englyst, H.N. and Cummings, J.H. (1984) Analyst, 109, 937-942.

Freeze-dried biomass (50-100 mg) contained in 15 ml Pyrex tubes fitted with PTFE (polytetrafluoroethylene) lined caps was hydrolyzed in 1 ml of 0.5M HCl at 121°C for 20 min. The resultant acid hydrolyzate, after cooling to room temperature, was filtered (MILLEX-HA 0.45 μ ; Millipore Inc.) and 0.2 ml of 17M ammonium hydroxide added to the filtrate. After the addition of 0.1 ml of a freshly prepared solution of 10% sodium borohydride (W/v; Aldrich) in 3M ammonium hydroxide the 5 tube was shaken and incubated at 37°C for 1 h. Glacial acetic acid was then added dropwise until effervescence was no longer visible. An aliquot (0.2 ml) was then transferred to a fresh tube, cooled to°C and kept on ice, and 0.3 ml of 1-methylimidazole (Sigma Inc.) and 2 _Q ml of acetic anhydride were slowly added. Tubes were shaken and allowed to stand for a further 15 min after which time 5 ml of distilled H₂O was added. After cooling to room temperature 1 ml of dichloro ethane was added and the tube shaken vigorously for 2-3 min. Phase 5 separation was achieved by centrifugation at low speed, the upper aqueous layer was aspirated and discarded. The organic phase was placed in a small glass vial, sealed and stored at -80°C until analysis.

A standard mixture was prepared using commercially 0 available sugars (Sigma Inc.) Aqueous sugar solutions were reduced by sodium borohydride and derivatised as outlined above.

Alditol acetate solutions (1 μl) were analyzed using a model 5880 GC (Hewlett-Packard) fitted with 5 flame ionisation detection. Separation was achieved using a 0.25 mm X 30m SP 2380 (Supelco Co.) fused silica capillary column, the film thickness being 0.25 μm. The initial column temperature, 230°C was maintained for 5 min after which time it was increased to 270°C at 10°C _Q per minute. The final temperature being reached, was maintained for 20 min. The injector and detector temperatures were set at 200°C and 300°C, respectively.

Alditol acetate solutions (1 μl) were analyzed using a model 5985B GC/MS (Hewlett-Packard). Separation was achieved using the same column as above. The initial column temperature was held at 180°C for 2 min. increased to the final temperature (270°C) at 10°C per minute and held for 10 min. The temperature of the transfer line was set at 280°C. Detection by the mass spectrometer, following gas chromatographic separation, was achieved by total ion monitoring (TIM) operating in electron impact mode (70eV; scan range 33-500 m/z). Selected ion initoring (SIM) was used to detect the presence of the peracetylated madurose, diagnostic fragments monitored were 129 and 261 m/z. These values were selected from literature data (Fukuda, K. and Hamada, A. (1978) Biophys. Acta 544, 445-447).

It revealed major amounts of Glucose, Galactose and Ribose and a minor quantity of Mannose. This sugar pattern is considered to be "non characteristic" and typical for the genus Streptomyces.

Identity of strain GE 21640

This strain was assigned to the genus Streptomyces because of the following morphological and chemical characteristics:

- the formation of aerial hyphae bearing long uniserial chains of non motile spores

the presence of LL-Diaminopimelic acid in the whole cell (chemotype I)

the absence of sugars of diagnostic value in the whole cell hydrolisate.

As with other microorganisms, the characteristics of the GE 21640A producing strains are subject to variation. For example, artificial variants and mutants of the strain can be obtained by treatment with various known mutagens, such as O.V. rays. X-rays, high frequency waves, radioactive rays, and chemicals such as nitrous acid, N-methyl-N'-nitro-N-nitrosoguanidine, and many others. All natural and artificial variants and mutants which belong to a species of the genus Streptomyces sp. and produce antibiotic GE 21640A are deemed equivalent to strain Streptomyces sp. ATCC 55243 for the purposes of this invention and are contemplated to be within the scope of this invention.

As mentioned above, antibiotic GE 21640A is generally found mainly in the broth of the producing strain, while a minor amount of substance is found also in the mycelium.

The recovery of antibiotic GE 21640A from the mycelium or the fermentation broths of the producing microorganism is conducted according to known per se techniques such as extraction with solvents, precipitation by adding non-solvents or by changing the pH of the solution, partition chromatography, reverse- phase partition chromatography, ion-exchange chromatography, molecular exclusion chromatography and the like.

A preferred procedure for recovering the antibiotic substance of the invention from the mycelium includes extracting the filtered or centrifugated mycelium with a water-miscible organic solvent, concentrating the extracts and recovering the crude antibiotic substance by precipitation, optionally with the addition of a precipitating agent, by extraction of the aqueous residue with a water-immiscible organic solvent or by adsorption chromatography followed by elution of the desired product from the adsorption matrix.

A preferred procedure for recovering the antibiotic substance of the invention from the fermentation broth, includes extraction with a water- immiscible organic solvent, followed by precipitation from the concentrated extracts possibly by adding a precipitating agent or further extraction of an aqueous residue thereof with a water-immiscible solvent. Alternatively, the fermentation broth can be contacted with an adsorption matrix followed by elution with a polar elution mixture. This chromatographic procedure can also be applied to concentrated extract obtained from the fermentation broth instead of on the broth itself.

The term "water-miscible solvent" as used in this application, is intended to have the meaning currently given in the art to this term and refers to solvents that, at the conditions of use, are miscible with water in a reasonably wide concentration range.

Examples of water-miscible organic solvents that can be used in the extraction of the antibiotic substance of the invention from the mycelial mass are: lower alkanols, e.g. (Cι~C₃)alkanols such as methanol, ethanol and propanol; phenyl(C₁-C₃)alkanols such as benzyl alcohol; lower ketones, e.g. (C₃-C₄) etones such as acetone and ethylmethylketone; cyclic ethers such as dioxane and tetrahydrofurane; glycols and their products of partial etherification, such as ethylene glycol, propylene glycol and ethylene glycol monomethyl ether; lower amides such as dimethylformamide and diethylformamide. The term "water-immiscible solvent" as used in this application, is intended to have the meaning currently given in the art to this term and refers to solvents that at the conditions of use are slightly miscible or practically immiscible with water in a reasonably wide concentration range, suitable for the intended use.

Examples of water-immiscible organic solvents that can be used in the extraction of the antibiotic substance of the invention from the fermentation broth are: the usual hydrocarbon solvents which may be linear, branched or cyclic such as hexane or cyclohexane; halogenated hydrocarbons such as chloroform, carbon tetrachloride, dichloroethane, fluorobromoethane, dibromoethane, trichloropropane, chlorotrifluorooctane and the like; aromatic hydrocarbons such as benzene, toluene, xylene and the like; esters of at least four carbon atoms, such as ethyl acetate, propyl acetate, ethyl butyrrate, and the like; alkanols of at least four carbon atoms which may be linear, branched or cyclic such as butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1- hexanol, 2-hexanol, 3-hexanol, 3,3-dimethyl-l-butanol, 4-methyl-l-pentanol; 3-methyl-l-pentanol, 2,2-dimethyl- 3-pentanol, 2,4-dimethyl-3-pentanol, 4,4-dimethyl-2- pentanol, 5-methyl-2-hexanol, 1-heptanol, 2-heptanol, 5- methyl-1-hexanol, 2-ethyl-l-hexanol, 2-methyl-3-hexanol, 1-octanol, 2-octanol, cyclopentanol, 2- cyclopentylethanol, 3-cyclopentyl-l-propanol, cyclohexanol, cycloheptanol, cyclooctanol, 2,3- dimethylcyclohexanol, 4-ethy1cyclohexanol, cyclooctylmethanol, 6-methyl-5-hepten-2-ol, 1-nonanol, 2-nonanol, 1-decanol, 2-decanol and 3-decanol; straight or branched alkyl ethers and mixture thereof such as petroleum ether, ethyl ether, propyl ether, butyl ether, etc; and mixtures or functional derivatives thereof.

As known in the art, phase separation may be improved by salting.

When following an extraction an aqueous phase is recovered containing a substantial amount of an organic solvent, it may be convenient to azeotropically distill water from it. Generally, this requires adding a solvent capable of forming minimum azeotropic mixtures with water, followed by the addition of a precipitating agent to precipitate the desired product, if necessary. Representative examples of organic solvents capable of forming minimum azeotropic mixtures with water are n- butanol, benzene, toluene, butyl ether, carbon tetrachloride, chloroform, cyclohexane, 2,5- dimethylfurane, hexane and m-xylene; the preferred solvent being n-butanol.

Examples of chromatographic systems that can be conveniently used in the recovery of the antibiotic substance of the invention, are polystyrene or mixed polystyrene-divinylbenzene resins such as Amberlite XAD2 or XAD4 (Rohm and Haas), S112 (Dow Chemical Co.) and Diaion HP 20 (Mitsubishi); acrylic resins such as XAD7 or XAD8 (Rohm and Haas); polyamides such as polycaprolactames, nylons and cross-linked polyvinylpyrrolidones generally having a pore volume (ml/g) ranging between 1 and 5, surface area (m²/g) ranging between 1 and 100, apparent density (g/ml) ranging between 0.15 and 0.50, average pore diameter (A°ngstrom units) ranging between 100 and 3000 and particles size distribution where at least 40 percent of the particle size is lower than 300 micrometers, such as Polyamide-CC 6, Polyamide-SC 6, Polya ide-CC 6.6, Polyamide-CC 6AC and Polyamide-SC 6AC (Macherey-Nagel & Co., West Germany), the polyvinylpyrrolidone resin PVP- CL (Aldrich Chemie GmbH & Co., KG, West Germany), the polya ide resin PA 400 (M. Woelm AG, West Germany); and carbon.

In the case of polystyrene or acrylic resin a preferred eluent is a polar solvent mixture of water- 0 miscible solvents such as those reported above; in the case of a polyamide resin the eluent is preferably an aqueous mixture of a water-miscible solvent, such as the ones mentioned above, while for carbon a preferred eluent is a lower ketone such as acetone or a lower 5 alcohol such as methanol.

The further purification of a crude preparation of antibiotic GE 21640A can be accomplished by any of the known techniques but is preferably conducted by means of _Q chromatographic procedures.

Examples of these chromatographic procedures are those reported above in relation to the recovery step and include also chromatography on stationary phases 5 such as silica gel, allu ina, Florisil and the like, with an organic eluting phase made of mixtures of solvents including halogenated hydrocarbons, ethers, higher ketones of the type already mentioned above or reverse-phase chromatography on silanized silica gel _Q having various functional derivatizations and eluting with an aqueous mixture of water-miscible solvents of the kind mentioned above.

Conveniently, also the so-called steric exclusion chromatographic technique can be employed with good purification results. In particular, controlled pore cross-linked dextrans in which most hydroxyl groups have been alkylated, e.g. Sephadex LH-20 (Pharmacia Fine Chemicals, Ab), are usefully employed in this technique.

As usual in this art, the production as well as the recovery and purification steps may be monitored by a variety of analytical procedures including bioassays such as paper disc or agar diffusion assays on sensible microorganisms or TLC or HPLC procedures, which may involve a DV or icrobial detention step.

A preferred HPLC technique is represented by a reverse-phase HPLC using a column with porous and spheric particles of silanized silica gel, e.g. silica gel functionalized with C-18 alkyl groups having a uniform diameter (such as 5 micrometer Ultrasphere ODS Altex; Beckman Co.), a pre-column which is a silica gel functionalized with C-18 alkyl groups (such as RP 18 Brownlee Labs) and an eluent which is a linear gradient mixture of a polar water miscible solvent, such as one of those described above, in an aqueous buffered solution. Preferably this solution is adjusted to pH 4-7. A most preferred eluent is represented by a linear gradient from 30 to 90% of eluent A in eluent B wherein eluent B is a mixture of acetonitrile/aqueous buffer, pH 4-7, 20:80 and eluent A is a mixture of acetonitrile/aqueous buffer, pH 4-7, 80:20. Phisico-chemical characterization of antibiotic GE

21640A

A) ultraviolet absorption spectrum registered with a PE-320 PERKIN-ELMER spectrophotometer that exhibits the following absoption maxima:

0

5

0

0.1N KOH 289

B) retention-time (R_t) of 7.90 min and retention time relative to efrotomycin (Merck Co; R 9.21 min) of 5 0.86 when analized by reverse phase HPLC under the following conditions:

Column: ϋltrasphere ODS (5 μm) 4.6 mm (i.d.) x 250 mm (reverse phase silanized silica gel; _Q ALTEX-Beckman Co.)

Phase A: CH₃CN : 22 mM ammonium phosphate buffer pH 4.0 80:20 (v/v)

Phase B: CH₃CN : 22 mM ammonium phosphate pH 4.0 20:80 (v/v) Elution mode: linear gradient from 30% to 90% Phase A in 15 min Flow rate: 1.5 ml/min Detection: DV 230 nm C) R_f value of 0.33 in the following TLC chromatographic system: CH2CI₂ : methanol, 85:15 (v/v); using silica gel plates (silica gel 6OF₂₅₄, Merck Co.) Visualization UV light at 254 nm; In the same system efrotomycin showed a R_f of 0.59,

D) infrared absorption spectrum registered in nujol mull with IFS-48 Fourier Transform spectrophotometer which is shown in Figure 1 of the accompanying drawings that exhibits the following absorption maxima (cm-l): 3398, 2922 (nujol), 2854

(nujol) 1690, 1641, 1618, 1549, 1462 (nujol), 1377

(nujol), 1302, 1259, 1219, 1186, 1165, 1103, 1022,

1005, 974, 945, 937, 883, 806, 721 (nujol), 669

E) main FAB-MS peak of GE21640 A at 964.5, corresponding most likely to the lowest isotope of the protonated molecule upon analysis on a Kratos MS-50 double focusing mass spectrometer, using 8 kV accelerating voltage and a saddle field atom gun with Xe gas at 6 kV voltage and 0.23 mA current; test substance pre-mixed with a glycerol matrix;

F) iH-NMR spectrum, which is shown in Figure 2, that exibits the following groups of signals (in ppm) at 500 MHz recorded in DMSO-dβ (hexadeutero- dimethylsulfoxide) with a BROKER AM-500 spectrophotometer using TMS as the internal standard (0.00 ppm); (s = singlet, d = doublet; dd = doublet of doublets, m = multiplet; brs = broad singlet): 8.60, t; 7.20, dd; 6.55, s; 6.02-5.85, d and dd; 5.67-5.53, dd and m; 5.43, m; 4.82, brs; 4.75, brs; 4.47 and 4.44, m; 4.30-4.02, m; 3.90, m; 3.80- 3.65, m; 3.60, m; 3.50-3.12, m and s; 3.05, s; 1.90-1.70, m; 1.68, d; 1.56, s and m; 1.06, d; 0.82, s; 0.61, d;

G) 13C-NMR spectrum, which is reported in Figure 3 of the accompanying drawings, that exhibits the following groups of signals (ppm) at 500 MHz recorded in DMSO-dβ with a BROKER AM-500 spectrophotometer using TMS as the internal standard (0.00 ppm):

174.5, 167.6, 144.2, 140.2, 136.9, 135.7, 130.5, 130.0, 129.4, 129.3, 128.5, 126.8, 125.9, 125.2; 121.7, 98.7, 96.0, 89.0, 83.1, 76.7, 75.3, 74.7, 73.4, 73.3, 71.6, 70.3, 66.5, 66.2, 63.5, 55.6, 55.6, 54.4, 48.8, 39.6, 31.4, 30.9, 29.8, 29.8, 24.2, 24.1, 17.2, 17.1, 15.7, 13.2, 10.8, 10.1

H) pK of about 6 upon titration with 0.01N KOH in methylcellosolve: ater, 4:1

On the basis of the physico-chemical data and by reference to compounds of known structure the following formula is tentatively attributed to antibiotic GE 21640A:

The antimicrobial activity of the compounds of the invention can be demonstrated by a series of standard tests in vitro. MIC are determined by microbroth dilution (inocula 10* to 105 CFO/ml). Incubation times are 24 h, except for N. qonorrhoeae, H. influenzae, P. acnes, and B. fraqilis (48h). All organisms are incubated at about 37°C except for Candida albicans (30°C). N. qonorrhoeae and H. influenzae are incubated in a 5% C0₂ atmosphere, anaerobes in an anaerobic gas mixture. Media used are: Iso-Sensitest broth (Oxoid) (staphylococci, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruqinosa, Proteus vulqaris); Todd-Hewitt broth (Difco) (streptococci); GC base broth (Difco) + 1% IsoVitalex (BBL) (N. qonorrhoeae) ; brain heart infusion broth (Difco) + 1% Supplement C (Difco) (H. influenzae); Wilkins-Chalgren broth (Oxoid) (anaerobes) (T.D. Wilkins and S. Chalgren, Antimicrob. Ag. Chemother. 10, 926, 1976); phosphate-buffered yeast nitrogen broth (Difco) (Candida albleans).

The minimal inhibitory concentrations (MIC, micrograms/ml) for some microorganisms are reported below in Table IV.

TABLE IV (M.I.C. (mcg/ml)

TABLE I (continued) (M.I.C. (mcg/ml)

The antimicrobial activity of the compound of the invention is confirmed in an experimental septicemia in mice. Briefly, male and femal CD-I mice (Charles River) weighing 18-22 g are infected intraperitoneally with 5xl0⁴ CFO of S. pyogenes in 0.5 ml of sterile saline + 1% peptone. This challenge corresponds to 125 times the LD₅₀ for the infecting organism, untreated animals die between 48 and 72 h after infection. Tratment is given subcutaneously (sc) within 10 min and at 3, 6 and 24 after infection. Four groups of 5 mice are treated with different dose levels of GE 21640A. The 50% effective dose was calculated by the Spearman and Kaerber method from the percentages of animals surviving to day 7 at each dose. (Finney, D.J., The Spearman-Kaerber method, pp. 524-530, In: D.J. Finney, Statistical method in biological assay 1952, Charles Griffin & Company Limited, London). The ED₅₀ of GE 21640A (sc) was 24 mg/kg/dose.

In view of its properties, the compound of the invention can be used as an active ingredient in the preparation of medicaments for human or animal therapy.

In particular, antibiotic GE 21640A is an antimicrobial agent mainly active against gram positive and gram negative anaerobic and aerobic bacteria. It appears to be particularly active against Propiobacterium acnes and Neisseriae.

In view of its antimicrobial activity the compound of the. invention can effectively be employed as the active ingredients of antimicrobial preparations used in human an veterinary medicine either for the prevention or the therapy of infectious diseases caused by susceptible pathogenic bacteria.

The main therapeutic indication of the antibiotic substance of the invention is thus in the treatment of infections related to the presence of a microorganism susceptible to it.

The term "treatment" is intended to encompass also prophylaxis, therapy and cure. The patient receiving this treatment is any animal in need thereof, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or in admixture with pharmaceutically acceptable carriers and can also be administered in conjunction with other antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy, thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.

In addition to its use in pharmaceutical formulations suitable for the prophilaxis and cure of acne, such as lotions, creams etc., antibiotic GE 21640A or a pharmaceutically acceptable salt thereof may find application as an antimicrobial agent of primary choice in the treatment of gonorrhea. Gonorrhea is presently being treated with a number of different antibiotics, primarily with penicillin and spectinomycin and alternatively with tetracycline or ampicillin. However, as the incidence of gonorrhea has risen steadily in the last 15-20 years, the widespread use of these antibiotics for treatment of gonorrhea has resulted in an increasing frequency of drug resistance. Because of this, the development of new antibacterial compounds which are remarkably active against the microorganism responsible for gonorrhea, including also some resistant to drugs in current therapy, represents an advance in the treatment of this disease.

In general, for antibacterial treatment antibiotic GE 21640 as well as its non-toxic pharmaceutically acceptable salts can be administered by different routes such as topically or parenterally. However, the parenteral administration is the preferred route of administration. Compositions for injection may take such forms as suspensions, solutions, or emulsions in oily or aqueous vahicles, and may contain adjuvants such as suspending, stabilizing and/or dispersing agents.

Alternatively, the active ingredients may be in powder form for reconstitution at the time of delivery when a suitable vehicle, such as sterile water for injections, is added thereto.

Depending on the route of administration, these compounds can be formulated into various dosage forms. In some instances, it could be possible to formulate the compounds of the invention in enteric- coated dosage forms for oral administration which may be prepared as known in the art (see for instance "Remington's Pharmaceutical Sciences", fifteenth edition. Mack Publishing Company, Easton, Pa, OSA, page 1614).

This could be especially the case when it. is desired that the antimicrobial be particularly active or adsorbed in the enteric tract, while passing unaltered through the gastric tract.

The amount of active principle to be administered depends on various factors such as the size and condition of the subject to be treated, the route and frequency of administration, and the causative agent involved. The antibiotic substance of the present invention, namely antibiotic GE 21640A and the physiologically acceptable salts thereof, are generally effective at a daily dosage comprised between about 5 and about 100 mg of active ingredient per kg of patient body weight, optionally divided in 2 to 4 administrations per day.

Particularly desirable compositions are those prepared in dosage units containing from about 100 to about 5,000 mg per unit.

However, when used for the treatment of gonorrhea, where because of practical problems a single dose therapy is highly preferred, higher minimum doses of antibiotic GE 21640A generally ranging between 10 and 100 mg/kg, should be employed, in order to maintain an effective blood level of the drug over an extended period of time.

Furthermore, in the treatment of gonorrhea, a sustained-action parenteral dosage form is preferably employed. Sustained-action formulations can be prepared based on different mechanisms and methods, as known in the art. A preferred method for preparing a sustained- action formulation containing antibiotic GE 21640A involves the use of a water insoluble salt of this antibiotic substance, suspended in an aqueous or oily medium.

A unit dosage form for intramuscular injection prepared with 250 mg of antibiotic GE 21640A dissolved in 5 ml of a solvent having the following composition: propylene glycol 40%, ethanol 10%, trimethoxymethylamine (w/v)n 50%.

A unit dosage form for intramuscular injection is prepared with 5 ml of sterile suspension OSP containing 8% propylene glycol and 3500 mg of antibiotic GE 21640A. A unit dosage form for intramuscular injection is prepared with 2000 mg of antibiotic GE 21640A sodium salt suspended in 5 ml of sterile water for injection.

The following examples further illustrate the invention and have not to be interpreted as limiting it in any way.

Example 1

Production of antibiotic GE 21640A

A culture of strain GE 21640 was grown on an oatmeal agar slant for two weeks at 28-30°C and then was used to inoculate one 500 ml flask containing 100 ml of a medium with the following composition:

The flask was incubated on a rotary shaker for 92 h at 28°C and 200 rpm stirring. The obtained culture was then inoculated into a jar fermenter containing 4 liters of the same medium. The culture was incubated at 28°C with 900 rpm stirring and aeration (about one standard liter of air per volume per minute). After 48 hours the broth culture was transferred into a 2001 fermenter containing the above described medium. The culture was incubated for about 72 hours at 28°C with 150 rpm stirring and aeration (about 0.5 standard liter of air per volume per minute).

Antibiotic production was monitored by HPLC analysis and.by paper disc agar assay using Neisseria caviae ATCC 14659 grown overnight at 35°C on Todd-Hewitt medium.

Example 2

Recovery of crude antibiotic GE 21640A

The broths of two fermentations (400 1) were harvested after 72 hours and were pooled. The mycelium was removed by filtration with Hyflo filter aid.

Antibiotic GE21640 A was found both in the filtrate and in the mycelium cake. a The antibiotic of the invention was recovered from the filtrate by batch adsorption (pH 4.5; 3 hours stirring) on 121 of S112 polystyrene resin (The Dow Chemical Company). The resin was then filtered, washed and eluted with a mixture of acetone : water : n-butanol - 8:1:1 (v/v). The eluates were concentrated under reduced pressure and the aqueous residue was extracted with ethyl acetate at pH 4.5. Crude antibiotic GE 21640A precipitated upon addition of petroleum ether to the concentrated organic phase. The precipitate was collected by filtration and was dried yielding 10.7 g of crude antibiotic GE 21640A.

b) The mycelium was extracted with acetone (100 1) and the extract was concentrated to an aqueous residue which was extracted twice with ethyl acetate at pH 4.5. Crude antibiotic GE 21640A (15.5 g) precipitated from the concentrated organic phase upon addition of petroleum ether.

Example 3

Purification of GE 21640A

A portion (4.4 g) of the crude preparation from the filtered broth was dissolved in methanol and was loaded on a 60x8 cm column of LH-20 Sephadex resin swollen in methanol. The chromatographic fractions eluted with methanol were analyzed by HPLC and by agar diffusion assay on Neisseriae caviae ATCC 14659. The fractions containing the antibiotic of the invention were pooled and were concentrated under vacuum. The oily residue was dissolved in dichloromethane (CH₂CI₂) and was applied to a silica gel column containing 100 g of silica gel swollen in CH₂CI₂- The column was eluted with a series of mixtures of methanol and CH₂CI₂ having the methanol content increasing from 4% to 80%. The antibiotic of the invention was found mainly into chromatographic fractions eluted with a 30% methanol mixture (HPLC analysis and agar diffusion assay on N. caviae ATCC 14659). The pooled fractions were then concentrated to dryness. The residue was dissolved in t-butanol and was freeze dried yielding 220 mg of purified antibiotic GE 21640A.

Example 4

Purification of GE 21640A

A portion (3 g) of the crude preparation from the filtered broth was dissolved in 60 ml of acetonitrile (CH₃CN) and 80 ml of 40 mM ammonium formate buffer (4.0). The solution was applied to a chromatographic column containing 300 g of silanized silica gel (E. Merck; Darmstadt F.R. Germany) swollen in a mixture of 40 mM ammonium formate buffer (pH 4.0) : CH₃CN - 80:20 (v/v). The column was eluted with a series of mixtures of 40 mM ammonium formate (pH 4.0) and CH₃CN having the CH₃CN content increasing from 20% to 80%. The antibiotic of the invention was found mainly (HPLC analysis and agar diffusion assay on N. Caviae ATCC 14659) into fractions eluted with a 35% CH₃CN mixture.

These fractions were pooled and acetonitrile was removed by distillation. The resulting aqueous solution was extracted twice with ethyl acetate. The organic solutions were pooled and were concentrated under reduced pressure. The residue was dissolved in methanol and was applied to a 30x2.5 cm column of LH-20 Sephadex resin swollen in methanol.

The column was then eluted with methanol. The fractions containing antibiotic GE 21640A (HPLC analysis) were pooled and were concentrated under vacuum. Antibiotic GE 21640A precipitated upon addition of diethylether and was collected by filtration yielding 363 mg of material containing mainly the antibiotic of the invention.

Example 5

Isolation of high purity antibiotic GE 21640A

A portion (15 mg) of preparation of antibiotic GE 21640A, obtained according to the procedure described above, was dissolved in 0.6 ml of CH₃CN and 1.4 ml of 22 mM ammonium phosphate buffer (pH 4.3). The solution was injected into a HPLC 250x20 mm Hibar column (E. Merck; Darmstadt F.R. Germany) packed with 7 μm silica gel functionalized with octadecylsilane groups. The column was equilibred with a mixtures of 22 mM ammonium phosphate buffer (pH 4.3) : CH₃CN - 65:35 (v/v) and was eluted with the same mixture. Eluted fractions were collected according to the elution profile recorded by OV detection at 230 nm. The fractions from 12 subsequent chromatographic runs which contained the pure antibiotic of the invention were pooled and were concentrated under reduced pressure to remove CH₃CN. The resulting aqueous solution was extracted twice with an equal volume of ethyl acetate. The organic phases were pooled and were concentrated to dryness under reduced pressure. The oily residue was then dissolved in t-butanol and was freeze dried yielding 21 mg of pure antibiotic GE 21640A.

Claims

1. Antibiotic GE 2164OA having the following characteristics, in the non-salt form:

0.1N KOH 289

B) retention-time (R_t) of 7.90 min and retention time relative to efrotomycin (Merck Co; R_t 9.21 min) of 0.86 when analized by reverse phase HPLC under the following conditions:

Column: Oltrasphere ODS (5 μm) 4.6 mm (i.d.) x 250 mm (reverse phase silanized silica gel; ALTEX-Beckman Co.)

Phase A: CH₃CN : 22 mM ammonium phosphate buffer pH 4.0 80:20 (v/v)

Phase B: CH₃CN : 22 mM ammonium phosphate pH 4.0 20:80 (v/v) Elution mode: linear gradient from 30% to 90% Phase A in 15 min Flow rate: 1.5 ml/min Detection: OV 230 nm C) R_f value of 0.33 in the following TLC chromatographic system: CH2CI₂ : methanol, 85:15 (v/v); using silica gel plates (silica gel 6OF₂₅₄, Merck Co.) Visualization OV light at 254 nm; In the same system efrotomycin showed a R_f of 0.59,

D) infrared absorption spectrum registered in nujol mull with IFS-48 Fourier Transform spectrophotometer that exhibits the following absorption maxima (cm-i): 3398, 2922 (nujol), 2854 (nujol) 1690, 1641, 1618, 1549, 1462 (nujol), 1377 (nujol), 1302, 1259, 1219, 1186, 1165, 1103, 1022, 1005, 974, 945, 937, 883, 806, 721 (nujol), 669

F) 1H-NMR spectrum that exibits the following groups of signals (in ppm) at 500 MHz recorded in DMSO-d₆ (hexadeutero-dimethylsulfoxide) with a BROKER AM- 500 spectrophotometer using TMS as the internal standard (0.00 ppm);

(s = singlet, d = doublet; dd = doublet of doublets, m = multiplet; brs = broad singlet): 8.60, t; 7.20, dd; 6.55, s; 6.02-5.85, d and dd; 5.67-5.53, dd and m; 5.43, m; 4.82, brs; 4.75, brs; 4.47 and 4.44, m; 4.30-4.02, m; 3.90, m; 3.80- 3.65, m; 3.60, m; 3.50-3.12, m and s; 3.05, s; 1.90-1.70, ΠL; 1.68, d; 1.56, s and m; 1.06, d; 0.82, s; 0.61, d; G) 13Q-NMR spectrum that exhibits the following groups of signals (ppm) at 500 MHz recorded in DMSO-dβ with a BROKER AM-500 spectrophotometer using TMS as the internal standard (0.00 ppm):

H) pK of about 6 upon titration with 0.01N KOH in methylcellosolve:water, 4:1

2) Antibiotic GE 21640A which is an antibiotic substance obtainable upon recovery from the fermentation broth of Streptomyces Sp. ATCC 55243.

3) A compound according to claims 1 or 2 having the following structure formula:

4) A process for preparing a compound of claims 1, 2 or 3 which comprises cultivating Streptomyces sp. ATCC 55243 or a GE 21640A producing variant or mutant under submerged aerobic conditions in the presence of assimilable sources of carbon, nitrogen and inorganic salts and recovering the produced antibiotic therefrom.

5) A pharmaceutical composition which contains a compound of claims 1, 2 or 3 in admixture with a pharmaceutically acceptable carrier.

6) A compound of claims 1, 2 or 3 for use as a medicine.

7) Use of a compound according to claims 1, 2 or 3 for preparing a medicament for use as an antibiotic. 8) Method of using a compound of claims 1, 2 or 3 as an antimicrobial agent which comprises administering an antimicrobially effective amount to a patient in need thereof.

9) Streptomyces sp. ATCC 55243.

10) A biologically pure culture of Streptomyces sp. ATCC 55243 or a GE 21640A producing mutant or variant thereof, capable of producing antibiotic GE 21640A under aerobic fermentation conditions in the presence of assimilable sources of carbon, nitrogen and inorganic salts.