GB1567024A - Antibiotic a-22082 and process for production thereof - Google Patents

Description

(54) ANTIBIOTIC A-22082 AND PROCESS FOR l'KODUCTION THEREOF (71) We, ELI LILLY AND COMPANY, a corporation of the State of Indiana, United States of America, having a principal place of business at 307 East McCarty Street, City of Indianapolis, State of Indiana, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to a new polypeptide antibiotic, designated A-22()82, which is produced by culturing a hitherto undescribed strain of the organism Aspergillus nidulans RRL 8112.

It is an object of this invention to provide a novel antibiotic mixture comprising antibiotic A-22()82. The antibiotic mixtures encompassed by the invention also contain other minor factors. It is also an object of this invention to provide a novel process to produce an antibiotic mixture comprising antibiotic A-22()82 and other minor factors, to separate out the antibiotic mixture comprising antibiotic A-22082 and other minor factors and to isolate antibiotic A-22082. The antibiotic mixture can be produced by culturing a novel strain of Asperglltçs nidulans NRRL 8112 under submerged aerobic fermentation conditions until a substantial level of antibiotic activity is produced. The antibiotic mixture can be separated from the fermentation medium by extraction with polar organic solvents. The antibiotic mixture may be further purified, and antibiotic A-22082 can be isolated by the use of a variety of techniques such as chromatography.

The present invention provides a novel antibiotic mixture comprising antibiotic A-22082 and other minor factors.

The present invention also provides a novel antibiotic A-22082 which is a white amorphous solid; which is soluble in methanol, ethanol. dimethylformamide, dimethyl sulfoxide, ethyl acetate and in aqueous solutions having a pH greater than 7.0; but which is insoluble in diethyl ether and petroleum ether; and which has: a) an approximate molecular weight of 1100, as determined by mass spectrometry and titration; b) an approximate elemental composition of 56.52 wt. percent carbon; 7.29 percent hydrogen, 8.68 percent nitrogen, and 27.09 percent oxygen; c) an approximate empirical formula of CSI-.5H79-8.7N70 d) the following specific rotations: [a] D25 - 44" (c0.5, CH3OH) [a] 2565 - 156 (c0.5, CH3OH) e) an infrared absorption spectrum in KBr disc with the following observable characteristic absorption maxima: 2.97 (strong), 3.30 (weak). 3.36 (shoulder), 3.39 (medium), 3.47 (weak), 5.97 (strong), 6.06 (strong), 6.45 (medium), 6.53 (medium), 6.83 (medium), 7.78 (weak), 8.00 (weak), 9.07 (weak) and 11.66 (weak) microns; f) ultraviolet absorption spectra in both neutral and acidic methanol with absorption maxima at 225 nm (E 18,000), 275 nm (E 3,000) and 284 nm (shoulder E 2,500) and absorption maxima in basic methanol at 245 nm (E 16,000) and 290 nm (E 3,000); g) a 13C nuclear magnetic resonance spectrum in perdeuteromethanol with the following characteristics: 6 176.1, 174.3, 173.4, 172.7, 172.4, 169.8, 158.4, 132.8, 130.9, 129.6, 129.0, 116.2, 77.0, 75.7, 74.4, 71.3, 70.9, 69.6, 68.3, 62.4, 58.7, 56.9, 56.1, 52.9, 39.0, 38.5, 36.8, 35.2, 33.9, 32.9, 32.6, 30.7. 30.4, 30.2, 28.2, 27.0, 26.5, 23.6, 20.1, 19.6, 14.4, and 11.3 ppm; h) a titratable group with a pKa value of 12.7 in 66% aqueous dimethylformamide; i) after hydrolysis, an amino-acid analysis which indicates the presence of threonine, hydroxyproline, and three other amino acids; j) an Rf value of 0.35 on silica-gel thin-layer chromatography using a benzene:methanol (7:3) solvent system and Candida albicans bioautography for detection; k) the following Rf values in the paper chromatographic systems indicated below, using Candida albicans bioautography for detection: Rf Value Solvent System 0.76 Butanol saturated with water 0.69 Butanol saturated with water plus 2% p-toluenesulfonic acid 0.75 Methanol:0.1 N HC1 (3:1) 0.17 Butanol:ethanol:water (13.5:15:150) 0.78 Methanol: 0.05 M sodium citrate at pH 5.7 (7:3); paper buffered with 0.05 M sodium citrate at pH 5.7 The present invention also provides a process for production of an antibiotic mixture comprising A-22082 as herein described comprising: a) cultivation of Aspergillus nidulans NRRL 8112 in a culture medium containing assimilable sources of carbohydrate. nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced by said organism in said culture medium; and b) optionally, the separation of antibiotic mixture A-22082 from the culture medium; and c) optionally, the isolation of antibiotic A-22082.

The elemental analvsis of antibiotic A-22082 corresponds especially well with an empirical formula of Cs2H8lN7Ol8-H2O. (Calcd.: C, 56.24; H, 7.54: N, 8.84; 0, 27.39).

The infrared absorption spectrum of antibiotic A220X2 in KBr disc is presented in the accompanying drawing.

Antibiotic A-22082 is similar to but has a different spectrum of activity from the polypeptide antibiotic Echinocandin B reported by F. Benz et al., Helv. Chim. Acta 57, 2459-2477 (1974).

An organism useful for the preparation of antibiotic A-22082 is classified as a strain of Aspergillus nidulans (Eidam) Wint., which is in the Aspergillus nidulans form group. This classification is based on the descriptions of K.B. Raper and D.I. Fennel in "The Genus Aspergillus," The Williams and Wilkins Company, Baltimore. Md., 1965.

Color names were assigned according to the ISCC-NBS method (K.L. Kelly and D.B.

Judd, "The ISCC-NBS Method of Designating Color and a Dictionary of Color Names," U.S. Dept. of Commerce, Circ. 553, Washington, D.C., 1955). The Maerz and Paul color blocks are described by A. Maerz and M.R. Paul in "Dictionary of Color," McGraw-Hill Book Company, New York, N.Y., 1950.

Cultures were grown at 25"C unless otherwise specified.

CULTURAL CHARACTERISTICS OF A. NIDULANS NRRL 8112 Czapek's solution agar When grown at 25"C. for two weeks, the culture attains a diameter of 4.0 cm. The colony is rugulose and has a strongly crenate margin consisting of deeply submerged, brownish yellow hyphae. A colorless exudate, becoming pinkish with age. sometimes occurs. Loosely woven, nearly globose mycelial tufts occur randomly over the surface and in a submarginal line. As these tufts mature, they become more tightly woven and are interlaced with, and eventually enveloped in, hülle cells. Early growth ranges from white to buff. becoming pale yellow green (ISCC-NBS 12 and Maerz and Paul 10-A-1). In two weeks, growth becomes pale orange yellow (ISCC-NBS 73 and Maerz and Paul 10-B-3). After three weeks, widely scattered conidial heads, sometimes occurring in patches, are from white to light yellow initially, then becoming dark grayish yellow (ISCC-NBS 91 and Maerz and Paul 13-11-1).

No soluble pigment is evolved. The slightly buckled colony reverse ranges from white to light brown (ISCC-NBS 57 and Maerz and Paul 5-A-10) and darkens with age to dark purplish shades.

Conidial heads are at first radiate, but become short and columnar with age. Young globose heads may be up to 70u in diameter, but average 601l. Columnar heads are from 75 > to 125 long and from 301l to 65u wide and average 95 95,u x 47 .

Conidiophores, vesicles, and phialides are smooth-walled and pale brown. Conidiophores range in length from 38 > to 56 but may attain 85F. They are up to 5It wide.

Vesicles are subglobose to hemispherical and may be terminally flattened. They are generally from 8 > to 11,u in diameter, but average 9.6 .

Sterigmata are biseriate, and secondary sterigmata are frequently in pairs. Primary sterigmata are nearly cuneiform. They range in length from 3.2 to 6.3 and average 4.41l.

At their widest point they are 2.8F. Secondary sterigmata are flask-shaped and elongate.

They are 3.4 at their widest point and taper to 1 wide. They range in length from 4.7u to 7*9 > and average 6.2 > .

Conidia are rugulose, globose, yellow to green en masse, and range from 1.6 to 4.6 in diameter, averaging 2.5 .

The ascogenous state is encrusted in hiille cells on the colony surface but may also be found at levels beneath the surface. Hülle cells are hyaline and may become pinkish to brownish as cleistothecia develop. The hulle cells are globose to subglobose or oval to elongate and, in general, are from 14 to 18 in diameter. Cleistothecia are globose,thickwalled, and are hyaline when young and dark purple when fully mature. They range from 185 > to 320,u in diameter and average 200y.

Asci are hyaline, globose to oval, and may disintegrate before cleistothecia mature.

Globose asci are 12.6 in diameter. Oval asci average 14.111 x 11.1 and range from 12.6 to 17.4 long and fron 9.5 to 12.6y wide.

Ascospores are smooth walled, orange, and adorned with two delicately pleated, parallel equatorial crests which are unbroken and 0.81l wide. If the crest is equatorial, it is through the long axis of a lenticular ascospore which is from 4.3 to 5.1 long and from 2.7 to 3.5 wide and averages 4.5 to 3.21l. If the crest is peripheral, the ascopore appears globose, and the body has a diameter equal to the length of the lenticular view.

Malt extract agar When grown at 250C., the culture grows rapidly, achieving a diameter of 5 cm. in ten days and up to 7 cm. in three to four weeks. Colonies are velutinous, yellow green. By the seventh day small white clusters of hülle cells occur randomly over the surface and in a submarginal band. The hülle cell clusters become dull yellow with age. The crenate colony periphery consists of deeply submerged yellowish brown hyphae. In general, the surface is moderate olive green (ISCC-NBS 125 and Maerz and Paul 24-L-1), and the colony reverse is moderate olive (ISCC-NBS 107 and Maerz and Paul 15-L-4).

The conidial and the ascogenous states are similar to those of Czapek's solution agar with several exceptions. Vesicles are smaller, ranging from 6.3 to 11.01l in diameter and averaging 8.7 . Globose asci are 11.01l in diameter. and asci are from 11.1 to 12.6y long and from 9.5 to 10.3,u wide, averaging 11.3 x 10.1F. Ascospores average 4.0 x 3.2 .

Certain characteristics of the antibiotic A-22082-producing strain of Aspergillus nidulans NRRL 8112 differ from the characteristics of the organism described by Raper and Fennel, supra. The A-22082-producing strain produces smaller conidiophores, conidia, and hulle cells, but slightly larger conidial heads and secondary sterigmata. The A-22082-producing strain produces an exudate, whereas the organism described by Raper and Fennel does not.

All other characteristics conform to those of the published description and confirm the identification of the A-22082-producing oganism as a new strain of Aspergillus nidulans (Eidam) Wint.

The Aspergillus nidulans culture useful for the production of the novel antibiotic mixture comprising antibiotic A-22082 and other minor factors has been deposited and made a part of the stock culture collection of the Northern Regional Research Laboratory, U.S. Dept.

of Agriculture, Agricultural Research Service, Peoria. Illinois 61604. The Aspergillus nidulans culture is available to the public under the number NRRL 8112.

The known compound sterigmatocystin is produced by Aspergillus nidulans NRRL 8112.

During the recovery process, sterigmatocystin is extracted together with the antibiotic mixtures by extraction of the fermentation media with polar organic solvents. The antibiotic mixtures are separated from sterigmatocystin by precipitration techniques.

Separation of the antibiotic mixture comprising antibiotic A-22082 and other minor factors from the other components of the fermentation media used adsorption on a silica-gel column and elution with acetonitrile:water (97:3). Antibiotic A-22082 was isolated from the other minor factors by chromatographic separation on a silica-gel column with elution by acetonitrile:water (97:3).

The culture medium employed to grow A. nidulans NRRL 8112 can be any one of a number of media. For economy in production, optimal yield, and ease of product isolation, however, certain culture media are preferred. Thus, for example, a preferred carbohydrate source in large-scale fermentation is sucrose, although glucose, maltose and glycerol may be employed. Preferred nitrogen sources are enzyme-hydrolyzed casein and corn steep liquor.

Nutrient inorganic salts can be incorporated in the culture media. These include the customary soluble salts capable of yielding sodium, magnesium, calcium, ammonium, chloride, carbonate: sulfate and nitrate ions. Essential trace elements necessary for the growth and developmwent of the organism should also be included in the culture medium.

Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of organism.

It may be necessary to add small amounts (i.e. 0.2 ml./l.) of an antifoam agent such as polypropylene glycol to large-scale fermentation media if foaming becomes a problem.

For production of a substantial quantity of antibiotic A-22()82, submerged aerobic fermentation in tanks is preferred. Small quantities of antibiotic A-220X2 may be obtained by shake-flask culture. Because of the time lag in antibiotic production commonly associated with inoculation of large tanks with the spore form of the organism, it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inoculating a small volume of culture medium with the spore form or mycelial fragments of the organism to obtain a fresh. actively growing culture of the organism. The vegetative inoculum is then transferred to a larger tank. The medium used for the growth of the vegetative inoculum can be the same as that used for larger fermentations, but other media can also be employed.

A. nidulans can be grown at temperatures between 20"C. and 4() C. Optimum A-22082 production appears to occur at temperatures of about 25-30"C.

As is customary in aerobic submerged culture processes. the culture medium is aerated by sparging with sterile air while being mechanically agitated by conventional turbine impellers in a fully baffled vessel. Efficient antibiotic production is obtained when the dissolved oxygen content of the medium is maintained at, or above, 35 percent of air saturation.

The production of antibiotic A-22082 can be followed during the fermentation by testing samples of alcoholic extracts of the whole broth for antibiotic activity against an organism known to be sensitive to the antibiotic. One assay organism useful in testing for the presence of antibiotic A-22082 is Candida albicans. The bioassay is conveniently performed by paper-disc assay on agar plates.

Generally, antibiotic activity is detectable on the third day of the fermentation.

Maximum production of antibiotic activity usually occurs between about the fourth and the sixth days with A. nidulans.

Antibiotic A-22082 is a useful antifungal agent. The antifungal activity of antibiotic A-22082 was demonstrated by in vitro tests. Antifungal activity was measured by the conventional disc-diffusion method (6-mm pads were dipped in solutions containing antibiotic A-22082; pads were placed on agar plates seeded with test organism). Table II summarizes the minimal inhibitory concentration (MIC) per disc at which antibiotic A-22082 inhibited representative organisms.

TABLE II Test organism MIC (mcg./disc) Candida albicans 0.625 Trichophyton mentagrophytes 0.078 Candida tropicalis 3.12 In vitro disc-diffusion tests also indicate that antibiotic A-22()82 inhibits Geotrichum candidum and four species of Microsporum.

The antifungal activity of antibiotic A-22082 was further demonstrated by in vivo tests.

When two doses of antibiotic A-22082 were administered to Candida albicans-infected mice, protection against C. albicans was provided. A measure of the protection afforded is the EDs(, value [the effective dose in mg./kg. which protects 50 percent of the mice; see W.

Wick et al., J. Bacteriol. 81, 233-235 (19610]. The END30 values for antibiotic A-22082 against Candida albicans in mice were 30 mg./kg (intraperitoneal administration) and 50 mg./kg. (subcutaneous administration).

There were no signs of acute toxicity when antibiotic A-22082 was administered intraperitoneally (ip) or subcutaneously (sc) to mice at 100 mg./kg. twice per day for three days (a total of 600 mg./kg.). There were also no signs of acute toxicity when antibiotic A-22082 was administered ip to mice at 200 mg./kg. three times over 24 hours (a total of 600 mg./kg.).

When used as an antifungal agent, antibiotic A-22082 is administered parentally and is commonly administered together with a pharmaceutically-acceptable carrier or diluent.

The dosage of antibiotic A-22082 will depend upon a variety of factors, such as the nature and severity of the particular infection involved.

In order to illustrate more fully the operation of this invention, the following examples are provided.

EXAMPLE 1 A. Shake-flask fermentation of antibiotic mixture comprising antibiotic A-22082 and other minor factors using A. nidulaizs NRRL 8112 A culture of Aspergillus nidulans NRRL 8112 was prepared and maintained on an agar slant having the following composition: Ingredient Amount Tomato paste 2 percent Baby oatmeal 2 percent Agar 2 percent Deionized water 94 percent The slant was inoculated with Aspergillus nidulans NRRL 8112, and the inoculated slant was incubated at 25"C. for about 7 days. The mature slant culture was covered with beef serum and scraped with a sterile loop to loosen the spores. The resulting suspension was lyophilized into six pellets.

One lyophilized pellet thus prepared was used to inoculate 50 ml. of a vegetative medium having the following composition: Ingredient Amount (percent) Glucose 1.0 Glycerol 1.0 Cottonseed meal 2.5 CaCO3 0. l Water 95.4 The inoculated vegetative medium was incubated in a 250-ml. Erlenmeyer flask at 25"C.

for 48 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.

B. Tank fermentation of antibiotic mixture comprising antibiotic A-22082 and other minor factors using A. nidulans NRRL 8112 In order to provide a larger volume of inoculum, 10 ml. of the above-described incubated vegetative medium was used to inoculate 200 ml. of a second-stage vegetative growth medium having the same composition as that of the vegetative medium. This second-stage medium was incubated in a 2-liter wide-mouth Erlenmeyer flask at 25"C. for 48 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.

Incubated second-stage vegetative medium (800 ml.) prepared as above-described, was used to inoculate 100 liters of sterile production medium having the following composition: Ingredient Amount (percent) Sucrose 2.0 Maltose 1.0 Malt extract 1.0 Molasses 0.5 Corn steep liquor 0.5 Enzymatic hydrolysate of casein* 0.5 Water 94 5 *N-Z-Case, Sheffield Chemical Co., Norwich, N.Y.

The pH of the medium was 7.1 after sterilization by autoclaving at 12U C. for 30 minutes at 15-20 pounds pressure. The inoculated production medium was allowed to ferment in a 165-liter fermentation tank for five days at a temperature of 25"C. The fermentation medium was aerated with sterile air at the rate of 0.4 V/V/M and was stirred with conventional agitators at 250 RPM.

EXAMPLE 2 Separaton of the antibiotic mixture comprising antibiotic A-22082 and others minor factors Whole fermentation broth (200 1.), obtained as described in Example I . was filtered using a filter aid (Hyflo Super-cel, a diatomaceous earth, Johns-Manville Products Corp.). The separated mycelia were extracted with methanol (100 1.). The methanol extract was concentrated under vacuum to a volume of about 50 1. The concentrated methanol extract was acidified to pH 3.5-4.0 by the addition of hydrochloric acid. The resulting solution was extracted twice with 1/2 volumes of chloroform. The chloroform extracts were combined and concentrated to a volume of about one liter.

A portion of this chloroform concentrate (250 ml.) was added to acetonitrile (100 ml.).

The resulting solution was filtered, and the filtrate was concentrated under vacuum to a volume of about 150 ml. This concentrated filtrate was applied to a silica-gel column (5.5 x 54 cm.: Woelm silica gel). The column was eluted with acetonitrile:water (97.3) at a flow rate of 5 ml. per minute, collecting fractions having a volume of approximately 10 ml.

Elution was monitored by silica-gel thin-layer chromatography, using a benzene:methanol (7:3) solvent system. The presence of the antibiotic mixture comprising antibiotic A-22082 and other minor factors was detected by bioautography using Candida albicans. Column fractions 136-190 contained the antibiotic mixture. These fractions were combined and evaporated under vacuum to give 453 mg. of the antibiotic mixture.

EXAMPLE 3 Isolation of antibiotic A-22082 A portion of the antibiotic mixture comprising antibiotic A-22082 and other minor factors (200 mg.), obtained as described in Example 2, was chromatographed on a silica-gel column (1.5 x 6.0 cm.; Woelm silica gel). The column was eluted with acetonitrile:water (95:5) at a flow rate of one ml. per min.. collecting 5-ml. fractions. The fractions were monitored as described in Example 2. Fractions 1-3 which contained antibiotic A-22082 were combined and concentrated under vacuum to an oil. This oil was again chromatographed on a silica-gel column (1.5 x 12 cm.; Woelm silica gel), eluting with acetonitrile:water (97:3).

Fractions 15-20 which contained A-22082 were combined and concentrated under vacuum to an oil. This oil was dissolved in methanol (2 ml.); the methanol solution was added to diethyl ether (20 ml.). The precipitate which formed was separated by filtration and dried to give 35 mg. of antibiotic A-22082.

WHAT WE CLAIM IS: 1. An antibiotic mixture comprising antibiotic A-22082 as herein described 2. Antibiotic A-22082 which is a white amorphous solid: which is soluble in methanol, ethanol, dimethyl-formamide, dimethyl sulfoxide, ethyl acetate and in aqueous solutions having a pH greater than 7.0; but which is insoluble in diethyl ether and petroleum ether; and which has: a) an approximate molecular weight of 1100. as determined by mass spectrometry and titration; b) an approximate elemental composition of 55.62 percent carbon; 7.29 percent hydrogen, 8.68 percent nitrogen, and 27.09 percent oxygen; c) an approximate empirical formula of C5, 53H7983N7O,7 l9; d) the following specific rotations:

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (8)

**WARNING** start of CLMS field may overlap end of DESC **. Ingredient Amount (percent) Sucrose 2.0 Maltose 1.0 Malt extract 1.0 Molasses 0.5 Corn steep liquor 0.5 Enzymatic hydrolysate of casein* 0.5 Water 94 5 *N-Z-Case, Sheffield Chemical Co., Norwich, N.Y. The pH of the medium was 7.1 after sterilization by autoclaving at 12U C. for 30 minutes at 15-20 pounds pressure. The inoculated production medium was allowed to ferment in a 165-liter fermentation tank for five days at a temperature of 25"C. The fermentation medium was aerated with sterile air at the rate of 0.4 V/V/M and was stirred with conventional agitators at 250 RPM. EXAMPLE 2 Separaton of the antibiotic mixture comprising antibiotic A-22082 and others minor factors Whole fermentation broth (200 1.), obtained as described in Example I . was filtered using a filter aid (Hyflo Super-cel, a diatomaceous earth, Johns-Manville Products Corp.). The separated mycelia were extracted with methanol (100 1.). The methanol extract was concentrated under vacuum to a volume of about 50 1. The concentrated methanol extract was acidified to pH 3.5-4.0 by the addition of hydrochloric acid. The resulting solution was extracted twice with 1/2 volumes of chloroform. The chloroform extracts were combined and concentrated to a volume of about one liter. A portion of this chloroform concentrate (250 ml.) was added to acetonitrile (100 ml.). The resulting solution was filtered, and the filtrate was concentrated under vacuum to a volume of about 150 ml. This concentrated filtrate was applied to a silica-gel column (5.5 x 54 cm.: Woelm silica gel). The column was eluted with acetonitrile:water (97.3) at a flow rate of 5 ml. per minute, collecting fractions having a volume of approximately 10 ml. Elution was monitored by silica-gel thin-layer chromatography, using a benzene:methanol (7:3) solvent system. The presence of the antibiotic mixture comprising antibiotic A-22082 and other minor factors was detected by bioautography using Candida albicans. Column fractions 136-190 contained the antibiotic mixture. These fractions were combined and evaporated under vacuum to give 453 mg. of the antibiotic mixture. EXAMPLE 3 Isolation of antibiotic A-22082 A portion of the antibiotic mixture comprising antibiotic A-22082 and other minor factors (200 mg.), obtained as described in Example 2, was chromatographed on a silica-gel column (1.5 x 6.0 cm.; Woelm silica gel). The column was eluted with acetonitrile:water (95:5) at a flow rate of one ml. per min.. collecting 5-ml. fractions. The fractions were monitored as described in Example 2. Fractions 1-3 which contained antibiotic A-22082 were combined and concentrated under vacuum to an oil. This oil was again chromatographed on a silica-gel column (1.5 x 12 cm.; Woelm silica gel), eluting with acetonitrile:water (97:3). Fractions 15-20 which contained A-22082 were combined and concentrated under vacuum to an oil. This oil was dissolved in methanol (2 ml.); the methanol solution was added to diethyl ether (20 ml.). The precipitate which formed was separated by filtration and dried to give 35 mg. of antibiotic A-22082. WHAT WE CLAIM IS:

1. An antibiotic mixture comprising antibiotic A-22082 as herein described

2. Antibiotic A-22082 which is a white amorphous solid: which is soluble in methanol, ethanol, dimethyl-formamide, dimethyl sulfoxide, ethyl acetate and in aqueous solutions having a pH greater than 7.0; but which is insoluble in diethyl ether and petroleum ether; and which has: a) an approximate molecular weight of 1100. as determined by mass spectrometry and titration; b) an approximate elemental composition of 55.62 percent carbon; 7.29 percent hydrogen, 8.68 percent nitrogen, and 27.09 percent oxygen; c) an approximate empirical formula of C5, 53H7983N7O,7 l9; d) the following specific rotations:

[a]XyS - 44 44C (c0.5, CH3OH) [α]365 -156 (c0.5, CH30H) e) an infrared absorption spectrum in KBr disc with the following observable characteristic absorption maxima: 2.97 (strong),

3.30 (weak), 3.36 (shoulder), 3.39 (medium), 3.47 (weak), 5.97 (strong), 6.06 (strong), 6.45 (medium), 6.53 (medium), (z.83 (medium), 7.7X weak), 8.00 (weak), 9.07 (weak) and 11.66 (weak) microns; f) ultraviolet absorption spectra in both neutral and icidic methanol with absorption maxima at 225 nm (E 18,()()()), 275 nm (e 3,()()()) and 284 nm (shoulder # 2,5()()) and absorption maxima in basic methanol at 245 nm (e 16,000) and 290 nm (e 3,000); g) a 13C nuclear magnetic resonance spectrum in perdeuteromethanol with the following characteristics: # 176.1, 174.3, 173.4, 172.7, 172.4, 169.X, 158.4. 132.8, 130.9, 129.6, 129.0, 116.2,77.0, 75.7, 74.4, 71.3, 70.9, 69.6, 68.3, 62.4, 58.7, 56.9, 56.1, 52.9, 39.0, 38.5, 36.8, 35.2, 33.9, 32.9. 32.6, 30.7, 30.4, 3().2, 28.2, 27.(), 26.5, 23.6, 20.1, 19.6, 14.4, and 11.3 ppm; h) a titratable group with a pK, value of 12.7 in 66% aqueous dimethylformamide; i) after hydrolysis, an amino-acid analysis which indicates the presence of threonine, hydroxyproline, and three other amino acids; j) an Rf value of 0.35 on silica-gel thin-layer chromatography using a benzene:methanol (7:3) solvent system and C'andida albicans bioautography for detection; k) the following Rf values in the paper chromatographic systems indicated below, using Candida albicans bioautography for detection: R- value Solvent system ().76 Butanol saturated with water 0.69 Butanol saturated with water plus 2% wt./vol p-toluenesulfonic icid 0.75 Methanol:().1 N HCI (3:1) 0.17 Butanol:ethanol:water (13.5:15:150) 0.78 Methanol:0.()5 M sodium citrate at pH 5.7 (7:3); paper buffered with 0.05 M sodium citrate at pH 5.7 3. A process for production of an antibiotic mixture comprising antibiotic A-22082 as herein described comprising: a) cultivation of Aspergillus nidulans NRRL 8112 in a culture medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced by said organism in said culture medium; and b) optionally. the separation of antibiotic mixture A-22082 from the culture medium; and c) optionally, the isolation of antibiotic A-22082.

4. Antibiotic A-22082 substantially as hereinbefore described with particular reference to any one of Examples 1, 2 or 3.

5. The process of producing antibiotic A-22082 substantially as hereinbefore described with particular reference to any one of Examples 1, 2 and 3.

6. Antibiotic A-22082 whenever prepared by a process according to claim 3 or 5.

7. A pharmaceutical formulation comprising A-22082 associated with a pharmaceutically-acceptable carrier therefor.

8. Aspergillus nidulans NRRL 8112 in a synthetic culture medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts.