IE913058A1 - Antibiotic ge1655 complex and its factors a, b, and c - Google Patents

Abstract

The present invention is directed to a new antibiotic denominated antibiotic GE1655 complex, its factors antibiotic GE1655 factor A, antibiotic GE1655 factor B, and antibiotic GE1655 factor C, the pharmaceutical acceptable salts thereof, the pharmaceutical compositions thereof, and their use as medicaments against bacteria and fungi, particularly for the treatment of fungal infections in animals and human beings.

Description

The present invention concerns a new antibiotic substance arbitrarily denominated antibiotic GE1655 complex, its factors antibiotic GE1655 factor A, antibiotic GE1655 factor B, and antibiotic GE1655 factor C, a mixture of said factors in any proportion, pharmaceutical acceptable salts thereof, pharmaceutical compositions thereof, and the use of these new antibiotic substances as medicaments, particularly for the treatment of fungal infections in 15 animals and human beings. They can be also used for controlling phytopathogenic microorganisms in plants.

Another object of the present invention is a process for preparing antibiotic GE1655 complex and 20 its factors A, B, and C which consists in culturing the new strain Streptomvces hvgroscopicus ATCC 55085 or an antibiotic GE1655 producing variant or mutant thereof, recovering the complex from the fermentation broths, and separating the factors A, B, and C.

According to their physico-chemical properties antibiotic GE1655 complex and its factors A, B, and C can be assigned to the azalomycin class.

Azalomycin, the most representative antibiotic of this class, is a macrocyclic polyhydroxyl lactone type 2θ antibiotic produced by Streptomvces hvgroscopicus var. zalomvceticus as a mixture of at least three majorcomponents differing from each other in the substituents present on the nitrogens of the guanidino group (see U.S. patent N. 3076746 for the production of complex, J.Antib., Ser. A, 23 . 107 (1970) for the - 2 isolation of the factors, and Chem.Pharm.Bui1.,30, 4006 (1982) for the structure elucidation).

Other members of this class of antibiotics are, for example, guanidylfungin (Japan patent N. 83170482 and J.Antib., Ser. A, 37 . 1161, 1170 (1984)), and copiamycin (U.S. patent N. 3627880).

Streptomyces hvgroscopicus ATCC 55085 was isolated from a soil sample collected in Dehradun (India) and deposited on August 6, 1990 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 55085.

The production of the antibiotic GE1655 complex is achieved by cultivating a streptomyces strain capable of producing it, i.e. Streptomyces hvgroscopicus ATCC 55085 or an antibiotic GE1655 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, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride, carbonate, sulphate, 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 the 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 GE1655 producing strain can be grown at temperatures between 20° and 40°C, preferably between 24° and 35 °C.

During the fermentation, the antibiotic production can be monitored by testing broth samples for antibiotic activity, for instance, by bioassay or TLC or HPLC procedures.

Sensitive organisms to the antibiotic GE1655 such as Candida albicans 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 fifth day of fermentation.

Antibiotic GE1655 complex is produced by cultivating the strain Streptomvces hvgroscopicus ATCC 55085, or an antibiotic GE1655 producing mutant or variant thereof, and it is mainly found in the culture broths.

In the present disclosure when dealing with the compounds of the invention in relation to their physico-chemical or biological properties, the term antibiotic GE1655 is generally considered to refer to antibiotic GE1655 complex as recovered at the end of the fermentation process (see Example 2). > - 4 Morphological characteristics of Streptomvces hyqroscopicus ATCC 55085 Streptomvces hyqroscopicus ATCC 55085 grows well on most standard media.The vegetative mycelium, which has generally a thick and crusty surface, is composed of flexuous and branched hyphae with a diameter of about 0.8 micrometer. It exhibits a light cream to straw yellow color on most media.

The aerial mycelium is composed of long and branched hyphae with a diameter of 0.8 to 1.0 micrometer and forms sporophores arranged in very closed and short spirals from 2 to 5 turns, which are situated as dense clusters on the main stems of the aerial hyphae. The aerial mycelium which in other actinomycetes is generally very hydrophobic, on certain media (ISP N.3, N.4, N.7, oatmeal, Czapeck glucose agar and ISP N.8 agar, with the addition respectively of: mannitol, galactose, lactose, mannose, dextrose, inositol, fructose, ramnose, and ribose) becomes moistened and exhibits dark and glistening patches which, with the age, spread all over the surface. The spores are round to slightly oval with a diameter of 1-1.2x1.6 micrometer. Their color is ash grey but when wetted it becomes dark brown to almost black.

Cultural characteristics of Streptomvces hyqroscopicus ATCC 55085 For the examination of the cultural characteristics, Streptomvces hyqroscopicus ATCC 55085 was cultivated on various standard media suggested by Shirling and Gottlieb (Shirling E.B. and Gottlieb D., 1966 - Method for Characterization of Streptomvces Species Int. J.Syst. Bacteriol. , 16., 313-340) with the addition of several media recommended by Waksman (Waksman, S.A. 1961 - The Actinomycetes - The William and Wilkins Co. Baltimore; Vol. 2, 328,334).

Color determination was made, when necessary, by the method of Maerz and Paul (A. Maerz 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 different carbon sources was investigated by the method described by Shirling and Gottlieb.

The cultural and physiological characteristics and the carbon sources utilization are reported in Tables I, II, and III.

The readings in Table I were taken after two weeks of incubation at 28 °C.

TABLE I Cultural characteristics of strain Streptomvces hvgroscopicus ATCC 55085 5 Culture media Morphological Characteristics ISP 2 (yeast extract malt agar) ISP 3 (oatmeal agar 20%) Oatmeal agar 60% ISP 4 (inorganic saltsstarch agar) Abundant growth with thick and crusty surface, light cream (10-C-3) with light brown reverse (12-1-10). Abundant formation of whitish aerial mycelium.

Moderate formation of light grey spores.

Abundant growth with smooth surface, light cream (10-C-3). Abundant formation of ash grey spores.

Abundant growth with smooth surface, light cream (10-C-3). Moderate formation of light grey spores.

Moderate growth with smooth surface, straw yellow (10-F-2). Abundant formation of ash grey spores.

TABLE I (continued) Cultural characteristics of strain Streptomyces hyqroscopicus ATCC 55085 5 Culture media Morphological Characteristics ISP 5 (glycerolasparagine agar) Moderate growth with thick and slightly crusty surface, light yellow (10-F-3). Moderate formation of whitish aerial mycelium.

ISP 6 (peptoneyeast extract agar) ISP 7 (tyrosine agar) Hichey and Tresner's agar Moderate growth with slightly wrinkled surface, iron colorless.

Abundant growth with thick and wrinkled surface, brown with dark brown reverse. Deep brown with a reddish tinge melanine pigment (8-H-12). Abundant formation of light grey spores.

Abundant growth with slightly, crusty surface, light cream (10-C-3) with deep amber reverse (12-F-9). Abundant formation of ash and grey spores.

TABLE I (continued) Cultural characteristics of strain Streptomvces hvgroscopicus ATCC 55085 5 Culture media Morphological Characteristics Czapeck glucose agar Glucose asparagine agar 25 Nutrient agar Potato glucose agar Moderate growth with smooth and thin surface colorless with lemon yellow edges. Moderate formation of light grey spores.

Abundant growth with smooth and thin surface straw yellow (10-F-2). Moderate formation of light grey spores.

Moderate growth with smooth surface, colorless. Moderate white aereal mycelium Abundant growth with thick and slightly crusty surface, light brown. Abundant formation of light grey spores.

TABLE I (continued) Cultural characteristics of strain Streptomyces hvoroscopicus ATCC 55085 5 Culture media Morphological Characteristics Calcium malate agar Good growth with smooth surface, colorless with yellowish reverse. Moderate formation of white aereal mycelium.

Dextrose triptone agar Abundant growth with slightly wrinkled surface, straw yellow (10-F-2) with deep golden reverse (10-G-7). Moderate formation of white aereal mycelium.

Egg albumine agar Moderate growth with smooth and thin surface colorless with lemon yellow edges. Moderate formation of ash grey spores.

Letter and numbers refer to the color determination according to Maerz and Paul (see above) - 10 TABLE II Physiological characteristics of Streptomyces hvgroscopicus ATCC 55085 Tests Results ___________________________________________ Starch hydrolysis positive (strong) 15 H2S formation negative on medium n. positive with lead acetate strips Tyrosine hydrolysis positive (strong) 20 Caseine hydrolysis positive (weak) Gelatine liquefaction positive (weak) 25 Calcium malate hydrolysis positive (strong) Litmus milk coagulation negative Litmus milk peptonization positive 30 Nitrate reduction positive (strong) Melanine production positive (strong) - 11 TABLE III Carbohydrate utilization Carbon source Growth Arabinose +++ Dextrose +++ Fructose +++ Galactose +++ Mannose +++ Ramnose +++ Ribose +++ Xylose +++ Lactose +++ Maltose +++ Sucrose +++ Raffinose +++ Inositol +++ Mannitol +++ Cellulose - Salicin + +++ abundant growth + moderate growth no growth For this test medium ISP 8 (T.G.Pridham,D. Gottlieb J.Bacteriol., 56. 107-114, 1948) is used and the results are evaluated after 2 weeks of incubation at 28°-30°C.

CHEMOTAXONOMICAL CHARACTERISTICS - 12 Cell wall analysis The analysis of aminoacids present in the cell wall was carried out by the HPLC method described by M.Zanol and L.Gastaldo '· HPLC Separation of the Three Stereoisomers of Diaminopimelic Acid in Hydrolyzed Bacterial Cells XIV International Symposium on Column Liquid Chromatography, Boston U.S.A, 1990.

Whole cell sugar pattern The analysis of the sugar content in the whole cell was carried out after hydrolysis of the freezedried biomass in 0.5 N HCI at 170°C for 70 min. in autoclave.

The resultant cell suspension was passed through a 0.77 micron Millipore filter, sugars were then derivatized using the method published by H.N.Englyst and J.H.Cummings: Analyst, 109. p.937-942 (1984).

Identity of Streptomvces hyqroscopicus ATCC 55085 The strain producing antibiotic GE1655 was assigned to the genus streptomyces and classified as Streptomvces hyqroscopicus because of the following chemical and morphological characteristics: a) The presence of L,L-diaminopimelic acid in the cell wall b) The absence of diagnostic sugars in the whole cell hydrolyzate - 13 c) The differentiation of the aereal mycelium in chains of spores arranged in short and very closed spirals d) The presence on the aereal mycelium of black, glistening and moistened spots due to high hygroscopicity of the spores.

As with other microorganisms, the characteristics of antibiotic GE1655 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 U.V. rays, X-rays, high freguency waves, radioactives rays, and chemicals such as nitrous acid, N-methyl-N1-nitro-N-nitrosoguanidine, and many others. All natural and artificial variants and mutants which belong to a species of the genus Streptomvces hygroscopicus and produce antibiotic GE1655 are deemed eguivalent to strain Streptomvces hvgroscopicus ATCC 55085 for the purpose of this invention and are contemplated to be within the scope of this invention.

As mentioned above, antibiotic GE1655 complex is generally found mainly in the fermentation broths.

The recovery of antibiotic GE1655 complex from the fermentation broths of the producing microorganism is conducted according to known per se technigues which include extraction with solvents, precipitation by adding non-solvents or by changing the pH of the solution, partition chromatography, reverse phasepartition chromatography, ion-exchange chromatography, affinity chromatography, and the like.

A procedure for recovering the antibiotic of the invention from the fermentation broths include - 14 10 extraction with a water-immiscible organic solvent of the antibiotic or of its salts with counter ions designed to form ion pair soluble in 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.

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 pratically 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 of the invention from the fermentation broths are: alkanols of at least four carbon atoms which may be linear, branched or cyclic such as n-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-l-hexanol, 2- ethyl-l-hexanol, 2-methyl-3-hexanol, l-octanol, 2- octanol, cyclopentanol, 2-cyclopentylethanol, 3- cyclopentyl-1-propanol, cyclohexanol, cycloheptanol, cyclooctanol, 2,3-dimethylcyclohexanol, 4- ethylcyclohexanol, cyclooctylmethanol, 6-methyl-5-hepten-2-ol, 1-nonanol, 2-nonanol, 1-decanol, 2-decanol, and 3-decanol? ketones of at least five carbon atoms such as methyl isopropyl ketone, methyl propyl ketone, methyl isobutyl ketone, methyl n-amyl ketone, methyl isoamyl ketone and mixtures or functional derivatives thereof.

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

When, following an extraction, an organic phase is recovered containing a substantial amount of water, 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 precipitating agents are petroleum ether, lower alkyl ethers, such as ethyl ether, propyl ether, and butyl ether, and lower alkyl ketones such as acetone.

According to a preferred procedure for recovering antibiotic GE1655 complex, the filtered fermentation broths can be contacted with an adsorption matrix followed by elution with a buffered polar water-miscible solvent or a mixture thereof, concentration under reduced pressure, extraction with water-immiscible solvents, and precipitation with a precipitating agent of the type already mentioned above.

Examples of adsorption matrixes that can be conveniently used in the recovery of the antibiotic substance of the invention, are polystyrene or mixed polystyrene-divinylbenzene resins such as S112 (Dow - 16 Chemical Co.), Amberlite XAD2 or XAD4 (Rohm and Haas), 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) . 2 ranging between 1 and 5, surface area (m /g) ranging between 1 and 100, apparent density (g/ml) ranging between 0.15 and 0.50, average pore diameter (Angstrom units) ranging between 100 and 3000 and particle size distribution where at least 40 percent of the particle size is lower than 300 micrometers, such as Polyamide-CC 6, Polyamide-SC 6, Polyamide-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); and charcoal.

In the case of polystyrene resins, polystyrene-divinylbenzene resins or acrylic resins a preferred eluent is a buffered mixture of water-miscible solvents; in the case of a polyamide resin the eluent is preferably an aqueous mixture of water-miscible solvents, while for charcoal a preferred eluent is a lower ketone such as acetone or a lower alcohol such as methanol.

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 elution of the antibiotic substances of the invention are: lower alkanols, e.g. - 17 (C^-C-j)alkanols such as methanol, ethanol, and propanol; phenyl(C^-C^j)alkanols such as benzyl alcohol; lower ketones, e.g. (C3~C4)ketones such as acetone and ethylmethylketone; cyclic ethers such as dioxane and tetrahydrofuran; 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; acetic acid, dimethylsulfoxide and acetonitrile.

The further purification of a crude preparation of antibiotic GE1655 complex can be accomplished by any of the known techniques but it is preferably conducted by means of chromatoqraphic procedures.

Examples of these chromatographic procedures are those reported above in relation to the recovery step and include also chromatography on stationary phases such as silica gel, allumina, Florisil, and the like, with an organic eluting phase made of mixtures of solvents including halogenated hydrocarbons, ethers, ketones of the type already mentioned above or reverse phase chromatography on silanized silica gel 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, controllled 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. - 18 The fractions recovered from the purification step (e.g. exclusion chromatography on Sephadex LH-20) are pooled together, concentrated under reduced pressure, and precipitated by addition of a precipitating agent selected from those mentioned above (e.g. petroleum ether), giving pure antibiotic GE1655 complex.

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, e.g. Candida albicans, or TLC or HPLC procedures, which may involve a U.V. or microbial detection 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-8 alkyl groups having an p uniform diameter (such as 5 micrometer Bakerbond Octyl (C8) supplied by J.T.Baker Research Products Phillisburg, New Jersey 08865 USA), a pre-column which is a silica gel functionalized with C-8 alkyl groups (such as octasilane silica gel 5 micrometer RP 8 Brownlee Labs.), and an eluent which is a linear gradient mixture of polar water miscible solvents, such as one of those described above, in an aqueous buffered solution. Preferably this solution is adjusted to pH 5-7. A most preferred eluent is represented by a linear gradient from 40 to 60% of eluent B in eluent A (v/v) wherein eluent B is a mixture of acetonitrile/tetrahydrofuran/isopropyl alcohol/aqueous buffer, pH 5-7, 35: 25 : 10 : 30, and eluent A is a mixture of acetonitrile/aqueous buffer, pH 5-7, 20 : 80. - 19 The separation of factors A, B, and C may be conveniently carried out by preparative HPLC methods which utilize essentially the same conditions described above for the analytical monitoring.

A preferred preparative HPLC technique is represented by a reverse phase HPLC chromatographic system equipped with a silanized silica gel column functionalized with C-18 alkyl groups having an uniform diameter (such as 5 micrometer Nucleosil C18, 250 x 40 mm, Stagroma, CH-8304 Wallisellen), and an eluent which is a linear gradient mixture of polar water miscible solvents, such as those described above, in an aqueous buffered solution.

A most preferred eluent is represented by a linear gradient from 5% to 10% of eluent B in eluent A wherein eluent B is a mixture of acetonitrile/tetrahydrofuran/aqueous buffer, pH 6.5, 60 : 20 : 20 (v/v) and eluent A is a mixture of acetonitrile/aqueous buffer, pH 6.5, 40 : 60 (v/v).

The column is connected to a Shimadzu SPD-6 AV U.V. detector at 254 nm.

The fractions of various chromatographic runs with homogeneous antibiotic content are pooled together and concentrated thus obtaining separated residual solutions.

The different residues are lyophilizated to give, respectively, antibiotics GE1655 factor A, GE1655 factor B, and GE1655 factor C.

Since antibiotic GE1655 complex and its factors A, B, and C are amphoteric, containing both basic, and acid groups, they are capable of forming salts with suitable acids and bases according to conventional procedures. The pharmaceutically acceptable salts so formed are also part of this invention. < » - 20 Pharmaceutically acceptable salts are salts which are useful in the therapy of warm-blooded animals. Representative and suitable salts of the antibiotics of the invention include those acid addition salts formed by standard reaction with both organic and inorganic acids such as, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, trichloroacetic, succinic, citric, ascorbic, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, glutamic, camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic acid, and the like.

Representative examples of these bases are: alkali metal or alkaline-earth metal hydroxides such as sodium, potassium, and calcium hydroxide; ammonia and organic aliphatic, alicyclic or aromatic amines such as methylamine, dimethylamine, trimethylamine, and picoline.

The transformation of the non-salt compounds of the invention into the corresponding addition salts, and the reverse, i.e. the trasformation of an addition salt of a compound of the invention into the non-salt form are within the ordinary technical skill and are encompassed by the present invention. - 21 Physico-chemical characteristics of antibiotic GE1655 complex A) Ultraviolet absorption spectrum, which is shown in Figure 1 of the accompanying drawings, and exhibits the following absorption maxima: 0.1 M HCI 0.1 M KOH 15 Phosphate buffer pH 7.4 Methanol Lambda max (nm) 230 (shoulder) 240 268 240 268 235 (shoulder) 240 268 240 260 (shoulder) B) Infrared absorption spectrum in KBr pellet which is shown in Figure 2 of the accompanying drawings and exhibits the following absorption maxima (cm-1) : 3398; 2926; 2854; 1705; 1640; 1458; 1419; 1383; 1299; 1279; 1263; 1230; 1141; 1069; 1005; 970; 918; 856; 837; 662; 604; 559 - 22 C) ^H-NMR spectrum which is shown in Figure 3 and exhibits the following groups of signals (in ppm) at 500 MHz recorded in DMSO-d^ (hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00 ppm); the multiplicity of the signals is indicated between parenthesis where: (S) indicates singlet (d) indicates doublet (m) indicates multiplet (br s) indicates broad singlet 7. 5. 4 . 3. , 1. 0. 55 (br s) ; 7. , 16 (rn) ? 6. .00-5.75 (m)7 95- -5.80 (m) ; 5.38 (m) ; 5.08 (m)7 4.68 (m) 7 02 (m) ; 3.90 (m) ; 3.83 (m) 7 3.67 (m) 7 3.55 (m) 09 (m) ; 3.04 (S) 7 2.95 (t) 7 2.73 (s) 7 2.68 (s) 50 (DMS Od6) ; 2.38 (m) ; 2.00-1.90 (m) ; 1.82 (m) 74 (m); 1.59 (S) ; 1.02 (d); 0.93 (d) ? 90- -0.75 (d) .

D) Positive ion FAB spectrum was obtained on a Kratos MS-50 double focusing mass spectrometer, using 8 kV accelerating voltage. A saddle field -5 atom gun was used with Xe gas (2 X 10 torr pressure indicated on the source ion gauge) at 6 kV voltage and 1 mA current.

The samples were studied in a glycerol matrix acidified with 0.1N CHgCOOH in positive ionization mode. FAB-MS analysis shows three protonated molecular ions M + 1 at m/z 1082,1068, and 1054 E) Retention-times (Rfc) were analyzed by reverse phase HPLC under the following conditions: - 23 R* column: Bakerbond Octyl (C8) (reverse phase silanized silica gel; 5 micrometer) 4.6 x 250 mm (*) Bakerbond is a trademark of HPLC columns supplied by J.T. Baker Research Products Phillisburg, New Jersey 08865 USA precolumn: Brownlee Labs RP 8 (octasilane silica gel; 5 micrometer) eluent A: CH3CN - 6.6mM ammonium citrate 20:80(v/v) eluent B: CH3CN - tetrahydrofuran - isopropyl alcohol - 6.6 mM ammonium citrate 35:25:10:30(v/v) elution mode: linear gradient from 40% to 60% of eluent B in eluent A in 20 min. flow rate: 1.8 ml/min U.V. detector: 254 nm Antibiotic GE1655 shows the following Rt values: 10.3, 10.56, 11.12 min.

Physico-chemical characteristics of antibiotic GE1655 factor A A) Ultraviolet absorption spectrum which exhibits the following absorption maxima: - '24 Lambda max (nm) Phosphate buffer pH 7.4 240 268 Methanol 240 260 B) XH-NMR spectrum which exhibits the following groups of signals (in ppm) at 500 MHz recorded in DMSO-άθ (hexadeuterodimethylsulfoxide) using TMS the internal standard (0.00 ppm)): 45 (br s); 7. 17 (m); 6. 30-6.15 (m); 95-5.80 (m); 5.39 (m); 5.08 (m); 4.72 (m); 02 (m); 3.83 (m) ; 3.67 (m); 3.3 (H2O) t 09 (m); 2.73 (s); 2.50 (DMSOdg); 2.38 (m) ? 00-1.90 (m) ; 1.73 (m) ; 1.59 (s); 1.02 (d) ; 95 (d); 0.90- 0.75 (d) C) Positive ion FAB spectrum was obtained on a Kratos MS-50 double focusing mass spectrometer, using 8 kV accelerating voltage. A saddle field . -5 atom gun was used with Xe gas (2 X 10 torr pressure indicated on the source ion gauge) at 6 kV voltage and 1 mA current. The samples were studied in a glycerol matrix acidified with O.l N CH COOH in positive ionization mode.

J + FAB-MS analysis showing the M+H peak at m/z 1082 D) Retention-time (R^) was analyzed by reverse phase HPLC under the following conditions: p* column: Bakerbond Octyl (C8) (reverse phase silanized silica gel? 5 micrometer) 4.6 x 250 mm - 25 (*) Bakerbond is a trademark of HPLC columns supplied by J.T. Baker Research Products Phillisburg, New Jersey 08865 USA precolumn: Brownlee Labs RP 8 (octasilane silica gel; 5 micrometer) eluent A: CH3CN - 6.6mM ammonium citrate 20:80(v/v) eluent B: CH3CN - tetrahydrofuran - isopropyl alcohol - 6.6 mM ammonium citrate 35:25:10:30(v/v) elution mode: linear gradient from 40% to 60% of eluent B in eluent A in 20 min. flow rate: 1.8 ml/min U.V. detector: 254 nm Antibiotic GE1655 factor A shows an Rt value of 11.12 min.

Physico-chemical characteristics of antibiotic GE1655 factor B : 3Q A) Ultraviolet absorption spectrum, which is shown in Figure 4 of the accompanying drawings, and exhibits the following absorption maxima: - ‘26 Lambda max (nm) Phosphate buffer 240 pH 7.4 268 Methanol 240 260 B) ...

H-NMR spectrum which exhibits the following groups of signals (in ppm) at 500 MHz recorded in DMSO-d6 (hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00 ppm)): 57 (br s); 7.16 (m); 6.30-6.15 (m) ; 95- 5.80 (m) ; 5.39 (m); 5.08 (m) ; 4.72 (n) ; 02 (m) ; 3.90 (m) 3.83 (m) ; 3.67 (m) ; 3.3 06 (m) ; 2.70 (S)? 2.50 (DMSOdg); 2.37 (m) ; 00- 1.90 (m) ; 1.73 (m) ; 1.59 (s) ; 1.02 (d) ; 94 (d) ; 0.90-0.75 (d) C) Positive ion FAB spectrum was obtained on a Kratos MS-50 double focusing mass spectrometer, using 8 kV accelerating voltage. A saddle field atom gun was used with Xe gas (2 x 10 torr pressure indicated on the source ion gauge) at 6 kV voltage and 1 mA current. The samples were studied in a glycerol matrix acidified with 0.1 N CH_COOH in positive ionization mode. FAB-MS 3 + analysis shows the M+H peak at m/z 1068 D) Retention-time HPLC under the (R^.) was analyzed by reverse phase following conditions: R* column: Bakerbond Octyl (C8) (reverse phase silanized silica gel; 5 micrometer) 4.6 X 250 mm - '27 (*) Bakerbond is a trademark of HPLC columns supplied by J.T. Baker Research Products Phillisburg, New Jersey 08865 USA precolumn: Brownlee Labs RP 8 (octasilane silica 5 gel; 5 micrometer) eluent A: CH^CN - 6.6mM ammonium citrate 20:80(v/v) eluent B: CH3CN - tetrahydrofuran - isopropyl alcohol - 6.6 mM ammonium citrate 35:25:10:30(v/v) elution mode: linear gradient from 40% to 60% of 15 eluent B in eluent A in 20 min. flow rate: 1.8 ml/min U.V. detector: 254 nm Antibiotic GE1655 factor B shows a Rfc value of .56 min. - 28 Physico-chemical characteristics of antibiotic GE1655 factor C : A) Positive ion FAB spectrum was obtained on a 5 Kratos MS-50 double focusing mass spectrometer, using 8 kV accelerating voltage. A saddle field . —5 atom gun was used with Xe gas (2 X 10 torr pressure indicated on the source ion gauge) at 6 kV voltage and 1 mA current. The samples were studied in a glycerol matrix acidified with 0.1N CH COOH in positive ionization mode. FAB-MS J , + analysis shows the M+H peak at m/z 1054 B) Retention-time (Rt) was analyzed by reverse phase HPLC under the following conditions: p* column: Bakerbond Octyl (C8) (reverse phase silanized silica gel; 5 micrometer) 4.6 X 250 mm (*) Bakerbond is a trademark of HPLC columns supplied by J.T. Baker Research Products Phillisburg, New Jersey 08865 USA 25 precolumn: Brownlee Labs RP 8 (octasilane silica gel; 5 micrometer) eluent A: CH.jCN - 6.6mM ammonium citrate ,n 20:80(v/v) eluent B: CH3CN - tetrahydrofuran - isopropyl alcohol - 6.6 mM ammonium citrate 35:25:10:30(v/v) - 29 elution mode: linear gradient from 40% to 60% of eluent B in eluent A in 20 min. flow rate: 1.8 ml/min U.V. detector: 254 nm Antibiotic GE1655 factor C shows a R^. value of .3 min.

Biological activity of the antibiotic products The antimicrobial activity of the the invention can be demonstrated by a standard tests in vitro. compounds of series of Minimal inhibitory concentrations (MIC) are determined by microbroth dilution methodology. Inocula 4 5 are 10 -10 cfu/ml for yeasts, Aspergillus mger. and bacteria? about 1% of a standardized aqueous (0.2% v/v) Tween 80 suspension containinq a mixture of mycelia and conidia for dermatophytes. Funqi are grown at 30°C, MIC are read at 20-24 h for Candida albicans, at 48 h for Cryptococcus neoformans and Aspergillus niger. at 72 h for Trichophyton mentagrophytes and Microsporum canis. Media used are pH 7 phosphate-buffered yeast nitrogen base broth (Difco), supplemented with glucose (1% w/v) and L-asparagine (0.15% w/v) for yeasts and Aspergillus niger. YM broth (Difco) for dermatophytes.

Bacteria are cultured at 37°C, MIC are read at 20-24 h except for Neisseria gonorrhoeae. Haemophilus influenzae, Propionibacterium acnes. and Bacteroides fragilis which are read at 48 h. Neisseria gonorrhoeae and Haemophilus influenzae are incubated in a 5% C02 atmosphere? anaerobes are incubated in an anaerobic gas mixture. Media used are Oxoid Iso-Sensitest broth (staphylococci, Enterococcus faecalis. Escherichia coli. Proteus mirabilis. Pseudomonas aeruginosa), Difco Todd-Hewitt broth (streptococci), Difco GC Base broth with 1% BBL Isovitalex for Neisseria gonorrhoeae, Difco Brain Heart Infusion broth with 1% Difco supplement C for H. influenzae. Oxoid Wilkins-Chalgren broth for anaerobes. - 31 Minimal inhibitory concentrations for Gardnerella vaginalis are determined by agar dilution on Difco Casman medium base with 5% rabbit blood and 0.15% . 4 5 lysed rabbit blood. Inocula are 10 -10 cfu/spot; microorganisms are incubated at 37’C in an anaerobic gas mixture; MIC are read at 48 h.

The minimal inhibitory concentrations (MIC) in micrograms/ml for some fungi and bacteria are reported in table IV, V, VI, and VII.

CQ Ch O +J rd υ E (0 \ Ct, Ul e (C Ch cn o < CH □ CH •h o E -p — υ • (0 CJ Cn - '32 Τ M3 rH <0 r-( M3 (N M) CO CM CO CM co Tf rH w E-< W Q in CD 1 in in rH 1 • io α ι CO O] CO (M O r-l Ε 1 m w O 1 o ο 1 1 rH m CN o rH rH rH Cn CO o o rH n CM CM a CM rH Cn u Pl * CQ Eh < Ul m in c (0 M rH J E rH rH tJ 0 Ul P Ch Ul MH o c Φ c Φ <0 Ul <0 c o •rH Φ o rH Ul Λ C Λ P rH •H rH o rtj > as o (0 o Λ CO o o P P o •rH <¥> •rH 4-) P O P 0 c cn c > <0 (0 o + u a Aspergillus niger L 53 ATCC 10535 Trichophyton mentagrophytes L634 SKF17410 - 33 Table V ANTIBACTERIAL M.I.C. DETERMINATION Strain M.I.C. (mcg/ml) Antibiotic GE1655 complex Staph. aureus L165 Tour 8 + 30% bovine serum 64 10 Staph. epidermidis L147 ATCC 12228 4 Staph. haemolvticus L602 8 Strep. pyogenes L49 C203 8 15 Strep. pneumoniae L44 UC41 8 Strep. faecalis L149 ATCC 7080 32 Propionibacterium acnes L1O14 ATCC 6919 4 Bacteroides fragilis L1010 ATCC 23745 8 Neisseria gonorrhoeae L997 ISM68/126 32 Haemophilus influenzae L970 type b ATCC 19418 16 Escherichia coli L47 SKF12140 >128 Proteus vulgaris L79 ATCC 881 >128 Pseudomonas aeruginosa L4 ATCC 10145 >128 - '3 4 Tabella VI ACTIVITY OF GE1655 COMPLEX AGAINST CANDIDA SPECIES mcg/ml Species N.strain MIC range MIC5QMIC9o C. albicans * 24 2-8 4 4 C. maris L1660 1 2 C. qlabrata L1661 1 4 C. kruzei L244 1 4 C. trooicalis L243 1 2 * The test was carried out on a series of 24 Candida albicans strains of the applicant's collection identified with the following internal code numbers : L1404, L1405, L1406, L1407, L1408, L1409, L1410, L1411, L1412, L1413, L1414, L1415, L1416, L1417, L1418, L1429, L1430, L1431, L1432, L1632, L1633, L1662, L1664, L1665. - 35 Table VII ACTIVITY OF GE1655 COMPLEX AGAINST GARDNERELLA VAGINALIS (CLINICAL ISOLATES) N. strains MIC range mcg/mlMIC50MIC90 14* 32->128 64 128 * The test was carried out on a series of 14 Gardnerella vaginalis strains of the applicant's collection identified with the following internal code numbers: L332, L529, L530, L531, L1622, L1623, L1624,L1625, L1626, L1627, L1628, L1629, L1630, L1631. - ‘36 Fungicidal activity of GE1655 complex A logarithmically growing culture of C. albicans SKF 2270 in buffered supplemented yeast nitrogen broth (30C, with agitation) was exposed to concentrations of GE1655 complex above the MIC value and sampled at intervals. The initial inoculum was about 106 cfu/ml. Suitably diluted aliquots were plated in duplicate by inclusion in 2.5 ml of Sabouraud soft agar (0.7%) on Sabouraud agar plates for determination of viable colony forming units (cfu). Plates were incubated for two days at 30 °C.

After 48 h at a concentration of GE1655 complex four times the MIC (32 mcg/ml) more than 99.5% of yeasts were killed. More than 99.9% killing was obtained in the first 7 hours with some regrowth between 7 and 24 h.

Ge 1655 complex is thus to be considered fungicidal for C. albicans.

The antibiotics of the invention are also active against phytopatogenic fungi.

In vivo antifungal activity of GE1655 complex The in vivo protective activity of the antibiotic GE1655 complex against Candida albicans in ovariectomized female CD rats (weighing 150-170 g, obtained from Charles River 7 days after surgery) was determined.

Candida albicans L145 was grown overnight in Sabouraud dextrose broth (Difco) at 30’C. The culture was centrifuged at 6000 r.p.m. for 20 min at 4‘C, the - 37 yeast was resuspended in nutrient broth N.2 (Oxoid) containing 15% (v/v) glycerol and stored at -80’C. After 7 days of acclimatization in a temperature and humidity controlled room, the animals received 4 mg of estradiol benzoate (Sigma) subcutaneously. Four days later they were infected by intravaginal (i.va.) instillation with 0.025 ml of thawed Candida albicans suspension (about 5xl06 cfu/rat).

GE1655 complex and miconazole (Sigma) were dissolved in dimethylsulfoxide (DMSO) and then diluted 1:10 with PEG 400; this risulted in homogeneous suspensions. Animals received 0.1 ml of one of these suspensions i.va. twice daily for three days starting 24 h after the infection. Control animals were treated with the vehicle (10% DMSO in PEG 400).

The presence of Candida was determined by inserting a disposable 0.01 ml calibrated loop into the vagina.

The sample was resuspended in 0.2 ml of sterile peptonized saline and cultivated on Sabouraud dextrose agar containing 100 mg/1 of rifampicin to inhibit growth of bacterial contaminants.

The results obtained in 2 different Candida vaginitis experiments (Table VIII) show that the animals treated daily with 10 mg of GE1655 complex or 4 mg of miconazole had vaginal load significantly lower (p<0.01) than the control group. - '38 Table VIII CANDIDA VAGINITIS IN OVARIECTOMIZED RATS.

Data from 2 experiments Therapy Dose Route Mean log1Q cfu ± SD/sample** mg/rat exp. 1 exp. 2 per treat.

Vehicle * ** i. va. 3.75+0.31 3.66+0.62 Miconazole 4 II 1.20+1.01 1.24+0.86 GE1655 10 II 1.94+0.51 1.90+0.60 complex *DMSO and PEG 400 (10:90) ** cfu/sample after 3 days of treatment - '39 Acute toxicity data The acute toxicity of antibiotic GE1655 complex in mice was determined upon intraperitoneal (i.p.), intravenous (i.v.), and oral (p.o.) administration.

Male and female CD.^ mice (weight 18-22 g, Charles River) were used. Antibiotic GE1655 was solubilized in DMSO and 5% glucose solution buffered at pH 7 (10:90). The animals were treated with a single dose of 20, 10 or 5 mg/kg i.v.; 40, 20 or 10 mg/kg i.p.; 500 or 250 mg/kg p.o.

The LDr<1 was estimated on the seventh day after OU treatment by the Spearman and Kaerber method (D.J.

Finney Statistical Methods in Biological Assay pp.524-530 Charles Griffin and Co.Ltd. London 1952).

The LD^-'s for the various routes are in table IX. □u In general, death occurred within a few minutes after treatment; 2/4 animals treated i.p. with 20 mg/kg died on day 2. All the surviving animals showed slight sedation during the first ten hours of observation. Gross necroscopy did not reveal any alterations. . ‘ - ‘40 Table IX ANTIBIOTIC GE1655 COMPLEX ACUTE TOXICITY IN THE MOUSE LD50 mg/kg Route i.v. i.p. p.o. >500 Transcutaneous toxicity Antibiotic GE1655 complex was solubilized in p DMSO and pH 7 buffered methylcellulose (Methocel ,Dow Chemical Co.) (10:90).

CD3 mice (weight 20-22 g, Charles River ) of both sexes were used(4 mice per dose level). The animals were anaesthetized with 3 ml/kg of an aqueous solution containing 0.05 mg/ml of fentanyl and 2.5 mg/ml of p droperidol (Leptofen Carlo Erba) plus 5 mg/kg of chlordiazepoxide (Valium Roche) i.p.; the back of each mouse was abraded with emery paper and then treated topically with a single dose of 10 or 5 mg of antibiotic GE1655 complex.

No symptoms of toxicity were observed. - ‘41 In view of the broad spectrum of activity, the antibiotics of the present invention are adaptable to being utilized in various applications of antifungal compositions. In such use, compounds may be admixed with a biologically inert carrier, optionally with the aid of a surface active dispersing agent, the nature of which would vary depending on whether the use is for the control of pathogens infecting man or animals, or for control of fungi in agriculture such as in soil or plant parts.

A further object of this invention is to provide pharmaceutical dosage forms particularly useful for the topical administration of antibiotic GE1655 complex and its factors in treatment of vaginal infections. According to this invention topical dosage forms are provided including vaginal tablets, pessaries, creams, ointments, gels, suppositories, lotions, foams, powders, suspensions, drug delivery systems and the like which permit delivery and release of the active substance into the infection sites.

The pharmaceutical dosage forms contain antibiotic GE1655 complex or one of its factors and one or more excipients such as, for example: starch, lactose, glucose, talc, cellulose for solid dosage forms; methylcellulose, modified vegetable oils, mineral oils, polyalkylene glycols, fatty acids and alcohols and the like for semi-solid dosage forms; water, alkanols, glycerol, lanolin, polyethylene glycols, mineral oil, pharmaceutical acceptable organic solvents (e.g. DMSO, methyl-decyl-sulfoxide) and the like for liquid or semi-liquid dosage forms. The dosage forms may optionally contain other active ingredients or ingredients which preserve and favour the antimicrobial action of antibiotic GE1655 complex l F - '42 and its factors in the infection sites (e.g. antiseptics, emulsifiers, surfactants, and the like).

Useful indications for the preparation of suitable topical dosage forms can be found in: Remington’s Pharmaceutical Sciences, 17th Edition, 1985 (Mack Publishing Company, Easton, Pennsylvania).

The amount of active substance in the finished dosage forms depends on the minimal inhibitory concentration of the antibiotics of the invention against the infection causative agents and their particular type of formulation.

The dosage may obviously be adjusted according to the severity of the infection and the type of patients. Experimental tests for determining the sensitivity of the microorganisms isolated from the patient may also offer useful indications to select the appropriate dosage. In general terms, the effective dosage ranges between 10 and 600 mg, preferably 100 and 400 mg, for each vaginal application one to three times daily. The course of treatment may last from 3 to 10 days or longer, if required.

Liquid or semi-liquid dosage forms such as creams, lotions, ointments, foams, and suspensions, generally contain from 0.05 to 5 % by weight of antibiotic.

If necessary, this range may be broadened without any substantial modification of the characteristics of the respective dosage form. Solid intravaginal unit dosages such as vaginal tablets and suppositories can be manufactured in different dosages. For instance, they may contain from 10 to 600 mg of the antibiotics of the invention.

Typical drug delivery systems incorporating the antibiotics of the invention are formulated, for , ' Γ - -43 instance, with biodegradble polymers for controlled release such as those described at pages 106-119 of the book: Drug Delivery Systems. Fundamentals and Techniques - Edited by P. Johnson and J.G. Loyd-Jones, 1987, Ellis Horwood Ltd. Chichister, England.

For non-medical application, the products of the present invention, either singly or as mixture may be employed in compositions in an inert carrier which includes finely divided dry or liquid diluents, extenders, fillers, conditioners and excipients, including various clays, diatomaceous aerth, talc, and the like, or water and various organic liquids such as lower alkanols, for example ethanol and isopropanol, or kerosene, toluene, and other petroleum distillate fractions or mixtures thereof.

In practicing the invention, an antifungal amount of the compositions may be applied directly to areas where fungal control is desired.

The following examples further illustrate the invention and have not to be interpreted as limiting it in any way. - '44 Examples Example 1 : Production of antibiotic GE1655 A culture of Streptomvces hyqroscopicus ATCC 55085 is grown on oatmeal agar slant for one week at 28’-30°C and then used to inoculate a 500 ml Erlenmeyer flask containing 100 ml of seed medium having the following composition : Yeast extract 2 g/1 Soybean meal 8 g/1 Dextrose 20 g/1 NaCl 1 g/1 CaCO3 4 g/1 Distilled water q.s. 1000 ml (adjusted to pH 7.3 before sterilization) The inoculated flask is incubated at 28’C for 60 hours on a rotary shaker (200 rpm) and then transferred into a 10 1 jar fermentor containing 4 1 of the seed medium. This new culture is grown for 48 hours at 28’-30’C with stirring (about 900 rpm) and aeration (about one standard liter of air per volume per minute).

The obtained broth is then used to inoculate a 70 1 jar fermentor containing 50 1 of production medium : Starch 2% Peptone 0.25% Hydrolyzed casein 0.25% ' f - '45 Yeast extract Beef extract Soybean meal Calcium carbonate (brought to pH 7.4 0.3% 0.2% 0.2% 0.1% before sterilization) Fermentation stirring 500 aereation 0.5 temperature 28 conditions: rpm v/v/min. (volume/volume/minute) C and incubated for about 72 hours.

Antibiotic production is monitored by well-agar assay using Candida albicans ATCC 10231 grown in minimum Davis medium. The inhibition zones are evaluated after incubation overnight at 28°C.

Example 2: Recovery of antibiotic GE1655 complex After 72 hours of fermentation the above obtained broth is harvested and filtered in the presence of a filter aid (Hyflo).

Antibiotic GE1655 is recovered from the filtrate.

The filtrate is adjusted at about pH 7 and 1.5 liter of resin S112 (Dow Chemical Co.) are added and stirred for 3 hours. After this time the resin is collected by filtration on a Bukner funnel, washed with 1 liter of water for 2 times. The resin is eluted with 2 liters of a buffer having the following composition : - '46 10 NaH-PO (sodium dihydrogen phosphate monohydrate) 2.5 g/1 Methanol Tetrahydrofuran with a ratio of 1:1:1 (v/v) adjusted at pH 7.5 with NaOH IN.

The eluate is concentrated under reduced pressure to give an aqueous residue which is extracted, first with 350 ml of ethyl acetate for 2 times and then with 400 ml of n-butanol for 2 times.

The ethyl acetate extract is discarded, the butanolic extract is concentrated under reduced pressure and the crude antibiotic GE1655 complex is precipitated by addition of petroleum ether from the concentrated organic phase.

The precipitated antibiotic is collected by filtration and dried giving 10.9 g of crude antibiotic GE1655 complex.

Example 3: Purification of antibiotic GE1655 complex Part of the crude antibiotic GE1655 complex obtained from the process illustated in the example 2, is purified by chromatography on Sephadex LH 20 (Pharmacia Fine Chemicals, Ab) according to the following procedure: 200 mg are dissolved in methanol and applied to a chromatography column containing 100 g of resin LH 20 prepared in methanol. The column is developed with methanol and 40 fractions of 7 ml each are collected.

Fractions are analized by HPLC and . 1 1 - '47 microbiologically against Candida albicans and pooled according to their antibiotic content.

The pooled fractions containing pure antibiotic GE1655 complex are concentrated under reduced pressure and precipitated by adding petroleum ether to give 41 mg of antibiotic GE1655 complex.

Example 4 : Isolation of antibiotics GE1655 factor A, factor B, and factor C Part of the crude GE1655 complex is purified by preparative HPLC according to the following procedure: A portion of this crude antibiotic (20 mg) is dissolved in acetonitrile/water 1:1 (v/v) and injected into a HPLC chromatographic system equipped with a silanized silica gel column (Nucleosil C18, 5 micrometer, 250 x 40 mm, Stagroma, CH-8304 Wallisellen). Elution is made with a linear gradient of a mixture of solution A and B from 5% to 10% of B in A, in 23 min, at a flow rate of 20 ml/min.

Solution A is a mixture of 9 mM ammonium formiate and acetonitrile 60:40 (v/v), adjusted to pH 6.5. Solution B is a mixture of 9 mM ammonium formiate, acetonitrile and tetrahydrofuran, 20:60:20 (v/v), adjusted at pH 6.5.

The column is connected to a Shimadzu SPD-6 AV U.V. detector at 254 nm.

The fractions of 8 chromatographic runs, having homogeneous antibiotic content, are pooled and concentrated to remove acetonitrile and tetrahydrofuran, thus obtaining separated residual solutions containing, respectively, antibiotic GE1655 » - ‘48 factor A, antibiotic GE1655 factor B, and antibiotic GE1655 factor C.

The 3 different residues are lyophilized to give 15 mg of antibiotic GE1655 factor A (retention time 11.4 min.), 19 mg of antibiotic GE1655 factor B (retention time 10.5 min.), and 5 mg of antibiotic GE1655 factor C (retention time 10.3 min.). - '49 10 Brief description of the accompanying drawings Fiqure 1 shows the U.V. spectrum of GE1655 complex in different solvents: dotted line indicates the U.V. spectrum of GE1655 complex in a methanol solution indicates the U.V. spectrum of GE1655 complex in HCl 0.1 M indicates the U.V. spectrum of GE1655 complex in phosphate buffer pH 7.4 indicates the U.V. spectrum of GE1655 complex in KOH 0.1 M.

Fiqure 2 shows the I.R. spectrum of GE1655 complex in KBR pellet.

Figura 3 shows the 1H NMR spectrum (500 MHz) of GE1655 complex in DMSO-dg.

Figura 4 shows the U.V. spectrum of GE1655 factor B in different solvents: indicates the U.V. spectrum of GE1655 factor B in a methanol solution indicates the U.V. spectrum of GE1655 factor B in phosphate buffer pH 7.4.

Claims (19)

Claims

1. An antibiotic substance selected from antibiotic GE1655 complex, antibiotic GE1655 factor A, antibiotic GE1655 factor B, antibiotic GE1655 factor C, a mixture of said factors in any proportion and a pharmaceutically acceptable salt thereof.

2. Antibiotic GE1655 complex having the following characteristics, in the non-salt form: A) ultraviolet absorption spectrum, which is shown in Figure 1 of the accompanying drawings, and exhibits the following absorption maxima: Lambda max (nm) 0.1 M HCl 230 240 268 (shoulder) 0.1 Μ KOH 240 268 Phosphate buffer 235 (shoulder) pH 7.4 240 268 Methanol 240 260 (shoulder) B) Infrared absorption spectrum in KBr pellet which is shown in Figure 2 of the accompanying drawings 1 ' f - -51 and exhibits the following absorption maxima (cm ) : 3398; 2926; 2854; 1705; 1640; 1458; 1419; 1383; 1299; 1279; 1263; 1230; 1141; 1069; 1005; 970; 918; 856; 837; 662; 604; 559. C) ^H-NMR spectrum which is shown in Figure 3 and exhibits the following groups of signals ( in ppm) at 500 MHz recorded in DMSO-dg (hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00 ppm): 7.55 (br £ 3); 7. .16 (m); 6. 00-5. .75 (m) ; 5. 95-5.80 (m) ; 5.38 (m) ; 5.08 (m) t 4.78 (m) ; 4.02 (m); 3.90 (m) ; 3.83 (m) ; 3.67 (m) r 3.55 (m) ; 3.09 (m) ; 2.95 (t) ; 2.73 (s) ! 2.6 8( s) ; ; >.5 (DMSOdg ) ; 2.38 (m) ; 2.00- -1.90 (m) 1.82 (m) ; 1.74 (m) ; 1.59 (s) ; 1.02 (d); 0.9 3 (d) ; 0.9 0-0.75 (d) . D) A positive ion FAB spectrum, obtained on a Kratos MS-50 double focusing mass spectrometer, using 8 *kV accelerating voltage and a saddle field atom -5 gun with Xe gas (2 X 10 torr pressure indicated on the source ion gauge) at 6 kV voltage and 1 mA current in a glycerol matrix acidified with 0.1 N CH^COOH in positive ionization mode, showing three protonated molecular ions M + 1 at m/z 1082, 1068, and 1054 E) Retention-times (R^) of 10.3, 10.56, 11.12 min. when analyzed by reverse phase HPLC under the following conditions: column: Bakerbond R Octyl (C8) (reverse phase silanized silica gel; 5 micrometer) 4.6 X 250 mm - -52 precolumn: Brownlee Labs RP 8 (octasilane silica gel; 5 micrometer) eluent A: CH 3 CN - 6.6mM ammonium citrate 20:80(v/v) eluent B: CH 3 CN - tetrahydrofuran - isopropyl alcohol - 6.6 mM ammonium citrate 35:25:10;30(v/v) elution mode: linear gradient from 40% to 60% of eluent B in eluent A in 20 min. flow rate: 1.8 ml/min U.V. detector: 254 nm.

3. Antibiotic GE1655 factor A having the following characteristics: 20 A) ultraviolet absorption spectrum which exhibits the following absorption maxima: Lambda max (nm) Phosphate buffer 240 pH 7.4 268 Methanol 240 260 IE 913058 ( · - - ‘53 - B) 1 H-NMR spectrum which exhibits the following groups of signals (in ppm) at 500 MHz recorded in DMSO-d^ (hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00 ppm)): 5 7.45 (br s); 7.17 (m); 6.30-6.15 (m); 5.95-5.80 (m) ; 5.39 (m) ; 5.08 (m) ; 4.72 (m) ; 4.02 (m) ; 3.83 (m) ; 3.67 (m); 3.3 (H 2 0); 3.09 (m); 2.73 (s) ; 2.50 (DMSOdJ ; 2.38 (m); 2.00-1.90 (m); 1.73 (m) ; D 1.59 (s) ; 1.02 (d); 0.95 (d); 0.90-0.75 (d) 10 C) Positive ion FAB spectrum , obtained on a Kratos MS-50 double focusing mass spectrometer, using 8 kV accelerating voltage and a saddle field atom . -5 gun with Xe gas (2 X 10 torr pressure 15 indicated on the source ion gauge) at 6 kV voltage and 1 mA current in a glycerol matrix acidified with 0.1N CH COOH in positive J + ionization mode, showing the M+H peak at m/z 1082 20 D) 25 Retention-time (R fc ) of 11.12 min. when analyzed by reverse phase HPLC under the following conditions: p column: Bakerbond Octyl (C8) (reverse phase silanized silica gel; 5 micrometer) 4.6 X 250 mm precolumn: Brownlee Labs RP 8 (octasilane silica gel; 5 micrometer) eluent A: CH 3 CN 20:80(v/v) 6.6mM ammonium citrate - 54 10 15 A > eluent B: CH 3 CN - tetrahydrofuran - isopropyl alcohol - 6.6 mM ammonium citrate 35:25:10:30(v/v) elution mode: linear gradient from 40% to 60% of eluent B in eluent A in 20 min. flow rate: 1.8 ml/min U.V. detector: 254 nm.

4. Antibiotic GE1655 factor B having the following characteristics: Ultraviolet absorption spectrum, which is shown in Figure 4 of the accompanying drawings, and exhibits the following absorption maxima: Lambda max (nm) Phosphate buffer 240 pH 7.4 268 Methanol B) 240 260 1 H-NMR spectrum which exhibits the following groups of signals (in ppm) at 500 MHz recorded in DMSO-d, (hexadeuterodimethylsulfoxide) using TMS D as the internal standard (0.00 ppm)): 7.57 (br s); 7.16 (m); 6.30-6.15 (m); 5.95-5.80 (m); 5.39 (m); 5.08 (m); 4.72 (m); 4.02 (m) ; 3.90 (m) 3.83 (m) ; 3.67 (m); 3.3 (H 2 0); 3.06 (m); - '55 2.70 (s); 2.50 (DMSOdg); 2.37 (m) ; 2.00-1.90 (m) ; 1.73 (m) ; 1.59 (s) ; 1.02 (d) ; 0.94 (d) ; 0.90-0.75 (d) C) Positive ion FAB spectrum, obtained on a Kratos MS-50 double focusing mass spectrometer, using 8 kV accelerating voltage and a saddle field atom -5 gun with Xe gas (2 X 10 torr pressure indicated on the source ion gauge) at 6 kV voltage and 1 mA current in a glycerol matrix acidified with 0.1N CH 3 COOH in positive ionization mode. FAB-MS analysis showing the M+H + peak at m/z 1068 D) Retention-time (R t ) of 10.56 min when analyzed by reverse phase HPLC under the following conditions: p column: Bakerbond Octyl (C8) (reverse phase silanized silica gel; 5 micrometer) 4.6 x 250 mm precolumn: Brownlee Labs RP 8 (octasilane silica gel; 5 micrometer) eluent A: CH 3 CN - 6.6mM ammonium citrate 20:80(v/v) eluent B: CH 3 CN - tetrahydrofuran - isopropyl alcohol - 6.6 mM ammonium citrate 35:25:10:30(v/v) elution mode: linear gradient from 40% to 60% of eluent B in eluent A in 20 min. flow rate: 1.8 ml/min - 56 10 U.V. detector: 254 nm.

5. Antibiotic GE1655 factor C having the following characteristics: A) Positive ion FAB spectrum, obtained on a Kratos MS-50 double focusing mass spectrometer, using 8 kV accelerating voltage and a saddle field atom -5 gun with Xe gas (2 X 10 torr pressure indicated on the source ion gauge) at 6 kV voltage and 1 mA current in a glycerol matrix acidified with 0.1 N CHCOOH in positive . . . . + ionization mode showing the M+H peak at m/z 1054 B) Retention-time (R t ) of 10.3 min when analyzed by reverse phase HPLC under the following conditions: p column: Bakerbond Octyl (C8) (reverse phase silanized silica gel; 5 micrometer) 4.6 X 250 mm precolumn: Brownlee Labs RP 8 (octasilane silica gel; 5 micrometer) eluent A: CHgCN - 6.6mM ammonium citrate 20:80(v/v) eluent B: CH^CN - tetrahydrofuran - isopropyl alcohol - 6.6 mM ammonium citrate 35:25:10:30(v/v) elution mode: linear gradient from 40% to 60% of eluent B in eluent A in 20 min. flow rate: 1.8 ml/min - 57 U.V. detector: 254 nm.

6. A process for preparing antibiotics of claim 1 which comprises cultivating Streptomvces hvgroscopicus under submerged aerobic conditions in 5 the presence of assimilable sources of carbon, nitrogen, and inorganic salts, recovering and isolating the produced antibiotic GE1655 complex therefrom, purifying it and optionally separating it into factors A, B, and C.

7. A process according to claim 6 in which the GE1655 producing strain is Streptomvces hvgroscopicus ATCC 55085 or a mutant or variant thereof.

8. A process according to claim 6 in which the cultivation of the GE1655 producing strain is carried out at a temperature of 24 °C to 35°C, preferably 28°C, for about 2 days to about 5 days.

9. A process according to claim 6 wherein the factors A, B, and C are separated by means of reverse phase HPLC technique.

10. A pharmaceutical composition which contains a compound of claim 1 in admixture with a pharmaceutical acceptable carrier.

11. A compound of claim 1 for use as a medicine.

12. Use of a compound of claim 1 for preparing a medicament useful for controlling fungal infections.

13. Use of a compound of claim 1 for control of fungi in soil or plant parts. - '58 io

14. Use as claimed in claim 12 in which the fungus causing the infection is a Candida albicans.

15. Streptomyces hvgroscopicus ATCC 55085.

16. An antibiotic substance substantially as described herein by way of Example.

17. A process for preparing antibiotics as defined in claim 1 substantially as described herein by way of Example.

18. An antibiotic whenever prepared by a process as claimed in any of claims 6 to 9 or 17.

19. A pharmaceutical composition comprising an antibiotic substance as claimed in claim 18.