GB1565821A - Antibiotic and process for production thereof - Google Patents

Description

PATENT SPECIFICATION ( 11) 1 565 821

( 21) Application No 39954/76 ( 22) Filed 27 Sep 1976 ( 19)( o ( 31) Convention Application No 619107 ( 32) Filed 2 Oct 1975 in s ( 33) United States of America (US)

\ O ( 44) Complete Specification Published 23 Apr 1980

In ( 51) INT CL 3 C 12 P 1/02 A 61 K 35/70 ( 52) Index at Acceptance C 2 A 1 B 1 Cl A 1 C 1 D 1 2 G 3 A 1 ( 72) Inventors: MARVIN MARTIN HOEHN KARL HEINZ MICHEL ( 54) ANTIBIOTIC AND PROCESS FOR PRODUCTION 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: 5

This invention relates to a novel antibiotic A-30912 mixture comprising at least 7 individual factors A, B, C, D, E, F and G The antibiotic A-30912 mixture is produced by culturing a novel strain of the organism Aspergillus rugulosus NRRL 8113.

The term "antibiotic mixture" as used in this specification refers to a mixture of co-produced individual antibiotic factors As will be recognized by those familiar with 10 antibiotic production by fermentation, the ratio of individual factors produced in an antibiotic mixture wil vary, depending on the fermentation conditions used.

The individual antibiotic factors of the present invention are designated antibiotic A-30912 factors A, B, C, D, E, F, and G.

It is the object of this invention to provide the novel antibiotic A30912 mixture and 15 antibiotic A-30912 factors B, C, D, E, F and G.

It is also the object of this invention to provide processes for production and separation of antibiotic A-30912 mixture comprising factors A, B, C, D, E, F and G and the isolation of factors A, B, C, D, E, F and G.

The present invention provides a novel antibiotic A-30912 mixture comprising factors A, 20 B, C, D, E, F and G.

The present invention also provides the novel process for production of antibiotic A-30912 mixture comprising factors A, B, C, D, E, F and G comprising:

a) cultivation of Aspergillus rugulous NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged 25 aerobic fermentation conditions until a substantial amount of antibiotic activity is produced; and b) optionally, the separation of antibiotic A-30912 mixture from the cutlure medium; and c) optionally, the isolation of antibiotic A-30912 factors A, B, C, D, E, F or G from the 30 antibiotic A-30912 mixture.

The antibiotic A-30912 mixture is extracted from the fermentation medium with polar organic solvents.

The known compound sterigmatocystin is also produced by Aspergillus rugulosus NRRL 8113 Sterigmatocystin is extracted either separately with a nonpolar organic solvent or 35 together with the antibiotic A-30912 mixture with polar organic solvents In the latter case, the antibiotic A-30912 mixture is separated from sterigmatocystin by concentrating the extracting solvent, adding the concentrate to an excess of nonpolar organic solvent such as diethyl ether, and separating the A-30912 antibiotic mixture as a precipitate Sterigmatocystin is separated in the filtrate The antibiotic A-30912 mixture is further purified by column 40 chromatography.

The antibiotic A-30912 mixture and the individual A-30912 factors are antifungal agents.

Infrared absorption spectra of the following A-30912 factors in K Br disc are presented in the accompanying drawings:

Figure 1 antibiotic A-30912 factor A 45 - 1 565 821 Figure 1 antibiotic A-30912 factor D Figure 3 antibiotic A-30912 factor B Figure 4 antibiotic A-30912 factor C Antibiotic A-30912 factor A A-30912 factor A is also similar to the polypeptide antibiotic Echinocandin B recently 5 reported by F Benz et al, Helv Chim Acta 57, 2459-2477 ( 1974).

Antibiotic A-30912 factor A is a white amorphous solid Elemental analysis of A-30912 factor A gave the following percentage composition:

Carbon, 56 52 wt %; hydrogen, 7 29 %; nitrogen, 8 68 %; oxygen, 27 09 % 10 The approximate empirical formula proposed for antibiotic A-30912 factor A is C 51-53 H 79-83 N 7017-19 Within this approximate range, the elemental analysis of A-30912 factor A corresponds especially well with an empirical formula of C 52 H 81 N 7018 H 20 (Calcd: C, 56 24; H, 7 54; N, 8 84; 0, 27 39).

Antibiotic A-30912 factor A has an approximate molecular weight of 1100, as determined 15 by mass spectrometry and titration.

The infrared absorption spectrum of antibiotic A-30912 factor A in K Br disc is shown in figure 1 of the accompanying drawings The following characteristic absorption maxima are observed: 2 97 (strong), 3 39 (medium), 3 47 (weak), 5 99 (strong), 6 10 (strong), 6 49 (medium), 6 56 (medium), 6 90, 8 00 (weak), 9 13 (weak), and 11 77 (week) microns 20 The ultraviolet absorption spectra of antibiotic A-30912 factor A in both neutral and acidic methanol exhibit absorption maxima at 225 nm (e 18,000), 275 nm (E 3,000) and 284 nm (shoulder E 2,500) The ultraviolet spectrum of factor A in basic methanol showns absorption maxima at 245 nm (E 16,000) and 290 nm (E 3,000) and also end absorption.

The 13 C nuclear magnetic resonance spectrum of antibiotic A-30912 factor A in 25 perdeuteromethanol shows the following characteristics; S 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.

Antibiotic A-30912 factor A has the following specific rotations: 30 lcal N 2 44 (c 0 5, CH 3 OH) le 1255 lcal 36 156 (c 0 5, CH 3 OH) Electrometric titration of antibiotic A-30912 factor A in 66 % aqueous dimethylformamide indicated the presence of a titratable group with a p Ka value of 12 8 (initial p H 6 9).

Amino-acid analysis of antibiotic A-30912 factor A indicated the presence, after hydrolysis, of threonine, hydroxyproline and three other as-yetunidentified amino acids.

Antibiotic A-30912 factor A is soluble in a variety of organic solvents such as methanol, 40 ethanol, dimethylformamide, dimethyl syulfoxide, and ethyl acetate: but is insoluble in nonpolar organic solvents such as diethyl ether and petroleum ether Antibiotic A-30912 factor A is also soluble in aqueous solutions, especially those having a p H greater than 7 0.

A-30912 Factor D Antibiotic A-30912 factor D is a white amorphous solid Elemental analysis of A-30912 45 factor D gave the following percentage composition: carbon, 56 37 wt percent; hydrogen, 8.17 percent; nitrogen, 8 54 percent; oxygen (by difference), 26 92 percent.

Antibiotic A-30912 factor D has an approximate molecular weight of 1100, based upon amino-acid analysis and its close structural relationship to antibiotic A30912 factor A.

The infrared absorption spectrum of antibiotic A-30912 factor D in K Br disc is shown in 50 figure 2 of the accompanying drawings The following characteristic absorption maxima are observed: 2 98 (strong), 3 31 (weak), 3 36 (shoulder), 3 40 (medium), 3 48 (weak), 5 76 weak), 6 01 (strong), 6 10 (shoulder), 6 49 (medium), 6 57 (medium), 6 90 (medium), 7 81 weak), 8 07 (weak), and 9 16 (weak) microns.

The ultraviolet absorption (UV) spectra of antibiotic A 30912 factor D in neutral and 55 acidic methanol exhibit absorption maxima at 225 nm (E 18,000) and 275 nm (e 2,500) The UV spectrum of A-30912 factor D in basic methanol exhibits absorption maxima at 240 nm (E 11,000) and 290 nm (e 3,000).

Antibiotic A-30912 factor D has the following specific rotation:

60 la Dl 50 (c 0 34, CH 3 OH).

Amino-acid analysis of antibiotic A-30912 factor D after hydrolysis, indicated the presence of threonine, hydroxyproline, histidine and three other amino acids One of the unidentified antibiotic A-30912-factor-D amino acids is identical to one of the unidentified 65 q% J 5 1 565 821 " antibiotic A-30912-factor-A amino acids.

Antibiotic A-30912 factor D is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate; but is insoluble in non-polar organic solvents such as diethyl ether and petroleum ether Antibiotic A-30912 5 factor D is soluble in aqueous solutions, especially those having a p H greater than 7 0.

A-30912 Factor B Antibiotic A-30912 factor B is a white amorphous solid Elemental analysis of A-30912 factor B gave the following approximate percentage composition: carbon, 57 36 wt 10 percent; hydrogen, 5 92 percent; nitrogen, 8 75 percent; oxygen, 26 19 percent.

The infrared absorption spectrum of A-30912 factor B in K Br disc is shown in figure 3 of the accompanying drawings The following characteristic absorption maxima are observed:

2.99, 3 41, 3 49, 6 06, 6 15, 6 54, 6 61, 6 94, 7 62, 8 07, 9 26, and 9 39 microns.

The ultraviolet absorption spectra of A-30912 factor B in both neutral and acidic 15 methanol exhibit absorption maxima at 223 nm (shoulder, S 16,000) amd 278 nm (E 2,400).

The ultraviolet spectrum of antibiotic A-30912 factor B in basic methanol shown absorption maxima at 242 nm (e 13,900) and 292 nm (e 1,800).

A-30912 factor B has the following approximate specific rotations:

la 121 20 lalD -47 (c 0 5, CH 3 OH) lal 5-170 (c 0 5, CH 3 OH) Electrometric titration of A-30912 factor B in 66 % (w/v) aqueous dimethylformamide 25 indicated the presence of a titratable group with a p Ka value of about 13 0 (initial p H 7 91).

Amino-acid analysis of A-30912 factor B indicated the presence, after standard acid hydrolysis, of threonine, hydroxyproline and several as-yet-unidentified amino acids.

A-30912 factor B is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate; but is insoluble in nonpolar 30 organic solvents such as diethyl ether and petroleum ether A-30912 factor B is also soluble in aqueous solutions, especially those having a p H greater than 7 0.

A-30912 Factor C Antibiotic A-30912 factor C is a white amorphous solid Elemental analysis of A-30912 35 factor C gave the following approximate percentage composition: carbon, 56 76 wt.

percent; hydrogen, 7 88 percent; nitrogen, 1061 percent; oxygen, 25 09 percent.

The infrared absorption spectrum of A-30912 factor C in K Br disc is shown in figure 4 of the accompanying drawings The following characteristic absorption maxima are observed:

2.98, 3 39, 3 43, 3 51, 6 01, 6 12, 6 47, 6 90, 7 04, 7 22, 7 38, 8 00 8 30, and 9 13 microns 40 The ultraviolet absorption spectra of A-30912 factor C in both neutral and acidic methanol exhibit absorption maxima at 223 nm (shoulder, e 7,300) and 275 nm)e 1,350).

The ultraviolet spectrum of antibiotic A 30912 factor C in basic methanol shows absorption maxima at 240 nm (c 12,400) and 290 nm (E 5,200).

A-30912 factor C has the following approximate specific rotations: 45 l 45 lalD -33 (c 0 5, CH 3 OH) lal 365-119 (c 0 5, CH 3 OH).

Electrometric titration of A-30912 factor C in 66 % aqueous dimethylformamide 5 indicated the presence of a titratable group with a p Ka value of about 13 08 (initial p H 7.93).

Amino-acid analysis of A -30912 factor C indicated the presence, after standard acid hydrolysis, of threonine, hydroxyproline and several as-yet unidentified amino acids 55 A-30912 factor C is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate; but is insoluble in nonpolar organic solvents such as diethyl ether and petroleum ether A-30912 factor C is also soluble in aqueous solutions, especialy those having a p H greater than 7 0.

The seven individual factors of the antibiotic A-30912 mixture can be separated and 60 identified by the use of thin-layer chromotography (TLC) Silica gel is a preferred adsorbent; and benzene:methanol ( 7:3, V:V) is a preferred solvent system.

The Rf values of antibiotic A-30912 factors A-G, using silica gel (Merck, Darmstadt) TLC, the benzene:methanol ( 7:3) solvent system, and Candida albicans bioautography are given in Table I.

cl A -a 1 565 821 4 TABLE I

Antibiotic A-30912 Factor Rf Value A 0 35 5 B 0 45 C 0 54 D O 59 E 0 27 10 F 0 18 G 0 13 The Rf values of antibiotic A-30912 factor A in various paperchromatographic systems, again using Candida albicans as a detectionorganism, are given in Table II 15 TABLE II

A-30912 Factor A Rf Value Solvent system 20 0.76 Butanol saturated with water 0.69 Butanol saturated with water plus 2 % p-toluenesulfonic acid 25 0.75 Methanol:0 1 N HC 1 ( 3:1) 0.17 Butanol:ethanol:water ( 13 5:15:150) 30 0.78 Methanol:0 05 M sodium citrate at p H 5 7 ( 7:3); paper buffered with 0 05 M sodium citrate at p H 5 7 35 The organism useful for the preparation of the antibiotic A-30912 mixture was isolated from a soil sample from the ruins of Pempeii, Italy The A-30912 producing organism is classified as a strain of Aspergillus rugulosus Thom and Raper, which is in the Aspergillus nidulans form group This classification is based on the description of K B Raper and D I Fennel 40 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 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, 45 "McGraw-Hill Book company, New York, N Y, 1950.

Cultures were grown at 25 C unless otherwise specified.

Culture Characterostics of A rugulosus NRR 8113 Czapek's Solution Agar.

The culture grows slowly, attaining 1 5 to 2 0 cm in diameter in 15 days at 25 C The colony surface is convex and velutinous, becoming wrinkled with age near the center and 50 then umbonate The mycelium periphery is a 2-mm wide band of deeply submerged colorless hyphae and is sinuate A pinkish-brown exudate forms on the marginal aerial hyphae In from 7 to 14 days a pale purple soluble pigment is produced in the agar surrounding the colony The pigment diffuses throughout the colony by 15 days.

After 5 days the colony surface ranges from white to buff, and the colony reverse is 55 brownish orange centrally and brownish to brownish purple in the peripheral regions In 10 days the colony is moderate yellowish pink (ISCC-NBS 29 and Maerz and Paul 11-A-7).

After 14 days the colony is light grayish red (ISCC-NBS 28 and Maera and Paul 4-G-7) The marginal area becomes verruculose and is strong yellow (ISCC-NBS 84 and Maerz and Paul 10-L-5) due to conidiation Scattered dull yellowish clusters of hille cells occur randomly 60 over the surface and along the margin of the colony With age, the strong yellow patches and marginal area become yellowish green After 3 weeks, an orange-purple tone is observed in the new aerial components of the margin Initially, the colony reverse is slightly concave As it matures, the colony flattens to the agar surface, and the reverse becomes slightly wrinkled By 10 days the reverse is light brown (ISCC-NBS 57 and Maerz and Paul 65 A 1 565 821 5-A-10) In 15 days it is grayish red (ISCC-NBS 10 and Maerz and Paul 6-J3).

The conidiogenous state is sparse; and conidiophores are scattered over the surface, sometimes occurring as patches or in a submarginal band Conidial heads are at first loosely radiate and globose; with aging, they may develop as short columnar forms which are more compact Globose heads are from 70 I to 80 t in diameter and average 50 g Columnar heads 5 may be up to 140 lt long and 70,u wide.

Conidia are globose to subglobose, rugulose, and greenish gold en masse They range from 2 8 g to 3 9 lt in diameter and average 3 2 t.

Sterigmata are biseriate and colorless Primary sterigmata range in length from 4 7 l 1 to 11 O 01 and average 7 O i At their widest point they are 2 4 p and taper to 1 6 g Secondary 10 sterigmata may occur singly or in pairs, arising from the primaries, and are flask shaped At their widest point they measure 3 O t and taper apically to 9 41 t where they become tubular.

The tubular apex is 1 21 i long The overall length ranges from 5 5,u to 12 6 It and averages 9.21 i.

Vesicles are globose to subglobose or hemispherical and may be apically flattenened, 15 becoming brownish with age They range from 7 4 R to 11 2 in diameter and average 9 4 lt.

Conidiophores are smooth, relatively thick-walled, and are at first hyaline then evolve to a light cinnamon grown They are slightly wider at the vesicle and may taper slightly near the foot cell The average width is 5 9 lt Conidiophores range from 47 71 l to 166 6 t and average 106 lt in length They arise from submerged hyphae or laterally from aerial hyphal 20 filaments.

The ascogenous state appears in up to 20 days The small yellowish clusters of hille cells which occur on the surface may be found at any level in the mycelium They consist of hille cells which envelope one or more cleistothecia Hiille cells are globose to subglobose or oval to elongate, are thick-walled and hyaline Globose huille cells range from 18 it to 24 lx in 25 diameter and average 21 81/.

Cleistothecia are globose to subglobose, thick-walled, relatively tough and fibrous At first relatively colorless, they become reddish purple and darken with age They measure from 165 l, to 4701 t in diameter and average 275 lx.

Malt extract agar 30 Colonies grown at 25 C expand rapidly, attaining 4-5 cm in 10-12 days At first a grayish white, colonies become moderate olive green (ISCC-NBS 90 and Maerz and Paul 23-E-6) in 4 days The sinuate to weakly lobate periphery consists of tightly packed, short, white aerial hyphae Small yellowish clusters of hille cells dot the margin and are randomly scattered over the felt-like agar surface After 20 days these hiille cell clusters tend to encrust much of 35 the surface The colony reverse is grayish yellow (ISCC-NBS 90 and Maerz and Paul 11-B-1).

The ascogenous state appears in 15 days The small yellowish clusters of hille cells which occur on the surface may be found at any level in the mycelium They consist of hfille cells which envelope one or more cleistothercia Hiille cells may encrust large areas over the 40 conidial heads Hulle cells are globose to subglobose or oval to elongate, are thick-walled and hyaline Globose hille cells range from 18 lt to 24 lt in diameter and average 21 8,.

Cleistothecia are globose to subglobose and are dark reddish brown They range from 3891 t to 700 x in diameter and average 506 lt.

Asci are fragile, hyaline, and subglobose to oval Subglobose asci are from 8 7 t to 11 9 lt 45 in diameter and average 10 3 it Oval asci are from 10 3 ii-14,21 i x 8 7 ll-10 3 l and average 12.1 l I x 9 1 t.

Ascospores are red-prange, rugulose, with two parallel, delicately pleated equatorial crests which are up to 0 8 t wide and unbroken The ascospore appears lenticular through the long axis When the crest is peripheral, the ascospore is globose In the globose view it is 50 from 4 9 g to 6 2 lt in diameter and averages 5 4 l.

Two characteristics of the antibiotic A-30912 producing strain of Aspergillus rugulosus differ from the characteristics of A rugulosus described by Raper and Fennel, supra The A-30912-producing strain has larger conidial heads and ascospores.

The Aspergillus rugulosus culture useful for the production of the antibiotic A-30912 55 mixture has been deposited and made a part of the stock culture collection of the Northern Regional Research Laboratory, U S Department of Agriculture, Agricultural Research Service, Peoria, Illinois 61604, from which it is available to the public under the number NRRL 8113.

Any one of a number of culture media can be employed to grow Aspergillus rugulosus 60 NRRL 8113 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 glucose, although molasses, starch, lactose, sucrose, maltose and glycerol may be employed Preferred nitrogen sources are enzyme-hydrolyzed casein and soluble meat peptone, although distiller's grains and monosodium glutamate 65 1 565 821 may be used 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 development of the organism should also be included in the culture medium Such trace elements commonly occur as 5 impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organism.

It may be necessary to add small amounts (i e 0 2 ml /1) 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 the antibiotic A-30912 mixture, submerged 10 aerobic fermentation in tanks is preferred Small quantities of the antibiotic A-30912 mixture 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 vegative inoculum The vegative inoculum is prepared by inoculating a small volume of culture medium with the spore form or mycelial fragments of 15 the organism to obtain a fresh, actively growing culture of the organism The vegetative innoculum 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 The antibiotic A-30912-producing organism can be grown at 20 temperatures between 200 and 430 C; the organism grows well at temperatures of about '-300 C Optimum production of the antibiotic A-30912 mixture appears to occur at a temperature of about 250 C.

As is customary in aerobic submerged culture processes, sterile air is blown through the culture medium For efficient antibiotic production, the volume of air employed in tank production is preferably above 0 4 volume of air per volume of culture medium per minute (V/V/M).

Production of the antibiotic A-30912 mixture 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 A-30912 antibiotics One assay organism useful in testing for the presence of the antibiotic A-30912 mixture is Candida albicans The bioassay 30 is conveniently performed by paper-disc assay on seeded agar plates.

Generally, antibiotic activity can be detected on the second day of fermentation.

Maximum production of antibiotic activity usually occurs between the third and the sixth days.

The antibiotic A-30912 factors are antifungal agents Illustrative of the antifungal activity 3 of the antibiotic A-30912 factors are in vitro tests with antibiotic A30912 factors A and D.

These tests are summarized in Table III Antifungal activity was measured by the conventional disc-diffusion method ( 6-mm pads were dipped in solutions containing test compound; pads were placed on agar plates seeded with the test organism) Results are given as the minimal inhibitory concentration (MIC) per disc at which the test compound 4 inhibited the test organism.

TABLE III

Test Organism MIC (mcg/disc) Antibiotic Antibiotic A-30912 A-30912 Factor A Factor D Candida albicans 0 625 0 5 Trichophyton mentagrophytes 0 078 h 1 0.07 1 565 821 A-30912 factor A is very active in in in vitro disc-diffusion tests against dermatophytes.

The results of these tests are summarized in Table IV.

TABLE IV

TABLE IV

A-30912 Factor A VS dermatophytes Dermatophyte Trichophyton mentagrophytes Trichophyton gallinae Trichophyton megininim Trichophyton quinckeanum Trichophyton rubrum Trichophyton schoenceinii Trichophyton terrestre Trichophyton tonsurans No of Isolates MIC(mcg/disc 1.25 039 > 1.25 0.0195 > 1.25 <.0098 0.0195 0.0195 > 1.25 0 156 Microsporium gypseum Microsporium audouinii Microsporium canis 0.156 0 038 1.25 0 156 1.25 0 0098 Microsporium cookei Nannizzia incurvata Phalaphere jean salemi Epidermatophyton floccosum Geotrichum candidum 1.25 0 0195 0.312 > 1.25 1.25 > 1.25 0 156 Keratinomyces ajellio 0.156 The antifungal activity of the antibiotic A-30912 factors was further demonstrated by in vivo tests The in vivo tests were carried out in the following manner: Three doses of test compound are given to Candida albicans infected mice at 0, 4, and 24 hours post-infection.

The protection which the test compound provides is measured as an ED 50 value lthe effective dose in mg/kg which protects 50 percent of the mice; see W Wick et al, H, 1 565 821 Bacteriol, 81 233-235 ( 1961)l The ED 50 values for antibiotic A-30912 factor A against Candida albicans in mice were 30 mg/kg (intraperitoneal administration) and 50 mg/kg (subcutaneous administration) The ED 50 value for antibiotic A-30912 factor D against Candida albicans in mice was 33 mg/kg (subcutaneous administration).

There were no signs of acute toxicity when antibiotic A-39012 factor A was administered 5 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-30912 factor A was administered ip to mice at 200 mg/kg three times per day (a total dose of 600 mg/kg).

There were no signs of acute toxicity when antibiotic A-30912 factor D was administered 10 sc to mice three times per day at 14 mg/kg (a total dose of 42 mg/kg).

When used as antifungal agents, the antibiotic A-30912 factors are administered parenterally and are commonly administered together with a pharmaceutically-acceptable carrier or diluent The dosage of antibiotic A-30912 factor will depend upon a variety of conditions, such as the nature and severity of the particular infection involved 15 In order to illustrate more fully the operation of this invention, the following examples are provided.

EXAMPLE I 20

A Shake-flask Fermentation 20 A culture of Aspergillus rugulosus NRRL 8113 was prepared and maintained on an 18 x ml agar slant having the following composition:

Amount Ingredient (percent)by wt) 25 Dextrin 1,0000 Enzymatic hydrolystate of casein 0 2000 Yeast extract 0 1000 Beef extract 0 1000 30 KCI 0 0200 Mg SO 4 7 H 20 0 200 Fe SO 4,7 H 20 O 004 Water 98 5596 35 N-Z-Amine A, Sheffield Chemical Co,

Norwich, N Y.

The slant was inoculated with Aspergillus rugurlosus NRRL 8113, and the inoculated slant was incubated at 25 C for about 7 days The mature slant culture was covered with 40 beef serum and scraped with a sterile loop to loosen the spores One-half of the resulting suspension was used to inoculate 50 ml of a vegetative medium having the following composition:

Amount 45 Ingredient (Percent by wt) Sucrose 2 5 Molasses 3 6 Corn steep liquor 0 6 50 Enzymatic hydrolystate of cassein 1 0 K 2 HPO 4 0 2 Water 92 1 N-Z-Case, Sheffield Chemical Co, 55

Norwich, N Y.

The inoculated vegetative medium was incubated in a 250-ml wide-mouth Erlenmeyer flask at 25 C for 24 hours on a shaker rotating through an arc two inches in diameter at 250 RPM 60 This incubated vegetative medium may be used directly to inoculate the second-stage vegetative medium Alternatively and preferably, it can be stored for later use by maintaining the culture in the vapor phase of liquid nitrogen The culture is prepared for such storage in multiple small vials as follows: In each vial is placed 2 ml of incubated vegetative medium and 2 ml of a glycerol-lactose solution having the following composition: 65 R Q Cl 7 1 565 821Ingredient Amount Glycerol 20 %(w/w) Lactose 10 % Deionized water 70 % 5 The prepared suspensions are stored in the vapor phase of liquid nitrogen.

A stored suspension ( 1 ml) thus prepared was used to inoculate 50 ml of a first-stage vegetative medium having the same composition earlier described for the vegetative medium The inoculated first-stage vegetative medium was incubated in a 250-ml 10 wide-mouth Erlenmeyer flask at 25 C for 22 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.

B Tank Fermentation In order to provide a larger volume of inoculum, 10 ml of the abovedescribed incubated first-stage vegetative medium was used to inoculate 400 ml of a secondstage vegetative 15 growth medium having the same composition as that of the vegetative medium The second-stage medium was incubated in a 2-liter wide-mouth Erlenmeyer flask at 25 C for 25 hours on a shaker rotating through an arc to inches in diameter at 250 RPM.

Incubated second-stage vegetative medium ( 800 ml), prepared as abovedescribed, was used to inoculate 100 liters of sterile production medium having the following composition: 20 Ingredient Amount Glucose 25 g/liter Starch 10 g/liter 25 Peptone 10 g/liter Blackstrap molasses 5 g/liter Enzymatic hydrolysate of casein 4 g/liter Mg SO 4 7 H 20 0 5 g/liter Czapek's mineral stock 2 0 g/liter 30 Ca CO 3 2 0 g/liter Deionized water q s 1 liter W.P No 159, Inolex Biomedical Corp, Glenwood, III 35 N-Z Amine A, Sheffield Chemical Co,

Norwich, N Y.

Czapek's mineral stock has the following 40 composition; Ingredient Amount Fe SO 47 H 2 O (dissolved in 45 2 ml conc HC 1) 2 g KCI 100 g Mg SO 4-7 H 20 100 g Deionized water q S to 1 liter 50 The p H of the medium was 6 8 after sterilization by autoclaving at 121 C for 30 min at about 16-18 pounds per sq in pressure The inoculated production medium was allowed to ferment in a 165-liter fermentation tank at a temperature of 25 C for four days The fermentation medium was aerated with sterile air at the rate of 0 5 V/V/IM The fermentation medium was stirred with conventional agitators at 300 RPM 55 EXAMPLE 2

Separation of the Antibiotic A-30912 Mixture Whole fermentation broth ( 200 1), obtained by the method described in Example 1, was stirred thoroughly with methanol ( 200 1) for one hour and then was filtered, using a filter 60 aid (Hyflo Super-cel, a diatomaceous earch, Johns-Manville Products Corp) The p H of the filtrate was adjusted to p H 4 0 by the addition of 5 N HC 1 The acidified filtrate was extracted twice with equal volumes of chloroform The chloroform extracts were combined and concentrated under vacuum to a volume of about four liters This concentrate was added to about 60 liters of diethyl ether to precipitate the A-30912 mixture The precipitate 65 1 565 821 i V was separated by filtration and dried to give 38 g of the antibiotic A30912 mixture as a gray powder The filtrate was concentrated under vacuum to give an oil; this oil was dissolved in methanol ( 500 ml) to precipitate additional antibiotic A-30912 mixture This precipitate was also separated by filtration and dried to give and addition 3 5 g of the antibiotic A-30912 5 mixture.

EXAMPLE 3

Isolation of Antibiotic A-30912 Factor A antibiotic A-30912 mixture ( 20 g), obtained as described in Example 2, was placed on a silica-gel column ( 4 x 107-cm), Woelm) in acetonitrile:water ( 95:5) The column was eluted 10 with acetonitrile:water ( 95:5) at a flow rate of 1 to 2 ml per minute, collecting fractions having a volume of approximately 20 ml Fractions were checked by thinlayer silica-gel chromatography, using the acetonitrile:water ( 95:5) solvent system and Candida albicans bioautography.

Fractions 74 through 125 were combined and concentrated The concentrated solution 15 crystallized upon standing to give an additional 124 mg of sterigmatocystin Fractions 212 through 273 were combined and concentrated under vacuum to give an oil This oil was dissolved in a small volume of methanol The methanol solution was added to diethyl ether ( 15 volumes) The precipitate which formed was separated and dried to give 23 mg of antibiotic A-30912 factor D Fractions 274 through 437 contained antibiotic A-30912 factors 20 A, B, C and D Fractions 482 through 900 contained antibiotic A-30912 factors A, E, F and G Fractions 438 through 481 were combined and concentrated under vacuum to give an oil.

This oil was dissolved in a small volume of methanol; and the methanol solution was added to diethyl ether ( 15 volumes) The precipitate which formed was separated and dried to give 25 2 17 g of antibiotic A-30912 factor A.

EXAMPLE 4.

Isolation of Antibiotic A-30912 Factor D A partially purified antibiotic A-30912 mixture containing antibiotic A30912 factors A, B, C, and D was obtained as described in Example 3 for fractions 274-437 This material 30 ( 750 mg) was chromatographed on a silica-gel column ( 2 2 x 51 cm Woelm silica gel), collecting fractions having a volume of approximately 15 ml and eluting with the following solvents.

Fractions Solvents 35 1-25 acetonitrile 26-62 acetonitrile + 1 % water 63-700 acetonitrile + 1 5 % water 40 The column fractions were monitored by silica-gel thin-layer chromatography, using acetonitrile:water ( 95:5) and benzene:methanol ( 7:3) solvent systems and Candida albicansbioautography Fractions 535-685, which contained antibiotic A-30912 factor D, were combined and concentrated under vacuum to give an oil This oil was dissolved in a small amount of methanol and added to diethyl ether ( 15 volumes) The precipitate which formed 45 was separated by filtration and dried to give 69 mg of antibiotic A-30912 factor D.

EXAMPLE 5

Isolation of A-30912 factors B and C Partially purified A-30912 antibiotic complex containing A-30912 factors A, B, C, and D 50 was obtained as described in Example 3 for fractions 212-437 This material ( 18 g) was dissolved in a minimal volume of acetonitrile:

water ( 4:1) and chromatographed on an aluminum oxide column ( 3 8 x 56 cm, Woelm), collecting fractions having a volume of approximately 20 ml the column was eluted with the following solvents: 55 Fractions Solvent 1-300 acetonitrile:water ( 4:1) 301-509 acetonitrile:water ( 7:3) The column fractions were monitored by silica-gel thin-layer chromatography as described in Example 4 On the basis of these results, fractions were combined and concentrated to oils; the oily residues were dissolved into small volumes of methanol the methanol solutions were precipitated with 10-15 volumes of diethyl ether The manner in which the fractions were combined, the weight of material obtained, and the factor content 65 in 1 n T 1 1 565 821 of the combined fractions are summarized below.

Fraction Weight (g) Factors 6-28 0 23 5 6.28 5 80 A-30912 C, D 34-114 2 90 A-30912 B 115-164 1 20 A-30912 A, B 165-509 1 90 A-30912 A 10 10 Insoluble material obtained before ether precipitation.

In order to obtain purified A-30912 factor C, a portion of fractions 6-28 ( 2 g) was dissolved in methanol, adsorbed onto a sufficient quantity of silica gel (grade 62), dried, 15 and added on top of a silica-gel column ( 1 9 x 80 cm, grade 62), packed in acetonitrile The column was eluted with acetonitrile at a flow rate of 2 ml/minute, collecting factions having a volume of about 10 ml At fraction 117, the solvent was changed to acetonitrile:water ( 99:1) The column fractions were again monitored by thin-layer chromatography On the basis of the TLC results, fractions were combined and concentrated to give oily residues; 20 the oily residues were dissolved into a small volumes of methanol; the methanol solutions were precipitated with 10-15 volumes of diethyl ether The factor content and weight of the fractions of interest are summarized below:

Fractions Weight (g) Factors 25 341-479 0 250 D 480-540 0 015 D 541-899 0 391 C, D 30 900-1675 0 340 C

Claims (14)

WHAT WE CLAIM IS

1 An antibiotic A-30912 mixture comprising factors A, B, C, D, E, F and G, which factors are herein described.

2 Antibiotic A-30912 factor B which is a white amorphous solid; which is soluble in 35 methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate and in aqueous solutions having a p H greater than 7 0; but which is insoluble in diethyl ether and petroleum ether; and which has:

a) an approximate elemental composition of 57 36 percent carbon, 5 92 percent 40 hydrogen, 8 75 percent nitrogen, and 26 19 percent oxygen; b) an infrared absorption spectrum in K Br disc with the following observable characteristic absorption maxima: 2 99, 3 41, 3 49, 6 06, 6 15, 6 54, 6 61, 6 94, 7 62, 8 07, 9.26, and 9 39 microns; c) ultraviolet absorption spectra in both neutral and acidic methanol with absorption maxima at 223 (shoulder, e 16,000) and 278 (e 2,400) and in basic methanol with absorption 45 maxima at 242 nm (E 13,900) anmd 292 (e 2,800); d) the following approximate specific rotations:

lalD -47 (c 0 5, CH 3 OH) 50 lal 365-170 (c 0 5, CH 30 o H); e) a titratable group with a p Ka value of about 13 0 in 66 % aqueous dimethylformamide; 55 f) an Rf value of 0 45 on silica-gel thin-layer chromatography using a benzene:methanol ( 7:3) solvent system and Candida albicans bioautography for detection; and g) after standard acid hydrolysis, an amino-acid analysis which indicates the presence of threonine, hydroxvproline, and several as-vet-unidentified amino acids.

3 Antibiotic A-30912 factor C which is a white amorphous solid which is soluble in 60 methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate and in aqueous solutions having a p H greater than 7 0; but which is insoluble in diethyl ether and petroleum ether; and which has:

a) an approximate elemental composition of 56 76 percent carbon, 7 88 percent hydrogen, 10 61 percent nitrogen, and 25 09 percent oxygen; 65 I I 1 n 1 565 821 b) an infrared absorption spectrum in K Br disc with the following observable characteristic absorption maxima: 2 98, 3 39, 3 43, 3 51, 6 01, 6 12, 6 47, 6 90, 7 04, 7 22, 7.38, 8 00, 8 30, and 9 13 microns; c) ultraviolet absorption spectra in both neutral and acidic methanol with absorption maxima at 223 nm (shoulder, E 7,300) and 275 nm (E 1,350) and in basic methanol with absorption maxima at 240 nm (e 12,400) and 290 nm (e 5,200); d) the following approximate specific rotations:

llalt 5 -33 (c 0 5, CH 3 OH) 10 D 1 lal 255119 (c 0 5, CH 3 OH); e) A titratable group with a p Ka value of about 13 08 (initial p H 7 93) in 66 % aqueous 15 dimethylformamide; f) an Rf value of O 54 on silica-gel thin-layer chromatography using a benzene:methanol ( 7:3) solvent system and Candida albicans bioautography for detection; and g) after standard acid hydrolysis, an amino-acid analysis which indicates the presence of threonine, hydroxyproline, and several as-yet-unidentified amino acids.

4 Antibiotic A-30912 factor D which is a white amorphous solid; which is soluble in 20 methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate and in aqueous solutions having a p H greater than 7 0; but which is insoluble in diethyl ether and petroleum ether; and which has:

a) an approximate molecular weight of 11000; b) an approximate elemental composition of 56 37 wt percent carbon, 8 17 percent 25 hydrogen, 8 54 percent nitrogen, and 26 92 percent oxygen (by difference); c) the following specific rotation:

lal 3 -50 (c 0 34, CH 3 OH); 30 d) an infrared absorption spectrum in K Br disc with the following observable characteristic absorption maxima: 2 98 (strong), 3 31 (weak), 3 36 (shoulder), 3 40 (medium), 3 48 (weak),

5 76 (weak), 6 01 (strong), 6 10 (shoulder), 6 49 (medium), 6 57 (medium), 6 90 (medium), 7 81 (weak), 8 07 (weak), and 9 16 (weak) microns; 3) ultraviolet absorption spectra in both neutral and acidic methanol with absorption maxima at 225 (e 18,000) and 275 nm (e 2,500) and in basic methanol with absorption maxima at 240 nm (E 11,000 and 290 nm (E 3,000); f) an amino-acid analysis, after hydrolysis, which indicates the presence of threonine, hydroxyproline, histidine, and three other amino acids; and g) an Rf value of 0 59 on silica-gel thin-layer chromatography using a benzene:

methanol ( 7 3) solvent system and Candida albicans bioautography for detection.

Antibiotic A-30912 factor E having a Rf value of 0 27 by thin-layer chromatography on silica gel using a benzene:methanol ( 7:3) solvent system.

6 Antibiotic A-30912 factor F having a Rf value of O 18 by thin-layer chromatography 45 on silica gel using a benzene:methanol ( 7:3) solvent system.

7 Antibiotic A-30912 factor G, having a Rf value of O 13 by thin-layer chromatography on silica gel using a benzene:methanol ( 7:3) solvent system.

8 A process for production of the antibiotic A-30912 mixture comprising factors A, B, C, D, E F and G as herein described comprising:

a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing 5 assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced: and b) optionally, the separation of antibiotic A-30912 mixture from the culture medium

9 A process as claimed in Claim 8 further comprising the steps of isolating the 55 isolating the 5 antibiotic A-30912 factors A, B, C, D E, F or G from the antibiotic A30912 mixture.

The antibiotic A-30912 mixture comprising factors A, B, C, D, E, F and G substantially as hereinbefore described with particular reference to Example 1 or 2.

11 The process of producing the antibiotic A-30912 mixture and antibiotic A-30912 factors A, B, C D E, F and G substantially as hereinbefore described with particular 60 reference to any one of Examples 1 to 5.

12 Antibiotic A-30912 mixture, or any one of Factors B, C, D, E, F or G, whenever prepared by a process according to claim 8 or 11.

13 A pharmaceutical formulation comprising Antibiotic A-30912 mixture, or any one of Factors B, C, D, E,:F or G, associated with a pharmeceutically acceptable carrier 65 13 1 565 821 13 therefor.

14 Aspergillus rugulosus NRRL 8113 in a synthetic culture medium containing assimilable sources of carbohydrates, nitrogen, and inorganic salts.

K W H McVEY, 5 Chartered Patent Agent, Erl Wood Manor, Windlesham, Surrey, England Agent for the Applicants.

Printed for Her Majesty's Stationery Office, by Croydon Printing Company Limited, Croydon, Surrey, 1980.

Published by The Patent Office, 25 Southampton Buildings, London, WC 2 A l AY,from which copies may be obtained.