EP0000991B1

EP0000991B1 - Antibiotics and pharmaceutical compositions containing them

Info

Publication number: EP0000991B1
Application number: EP78300252A
Authority: EP
Inventors: Walter Daniel Celmer; Walter Patrick Cullen; Liang Hsiung Huang; Mark Tilden Jefferson; Charles Edward Moppett; Riichiro Shibakawa; Junsuke Tone
Original assignee: Pfizer Inc
Current assignee: Pfizer Inc
Priority date: 1977-08-18
Filing date: 1978-08-16
Publication date: 1981-11-04
Also published as: EP0000991A1; DK144337C; IE781652L; DE2861282D1; DK144337B; JPS5512438B2; JPS5444602A; IE47180B1; IT7826811A0; US4148883A; IT1098192B; DK364178A

Description

This invention relates to new antibiotics produced by a new species of Nocardia, designated Nocardia argentinensis Huang sp. nov., when subjected to aerobic submerged fermentation.
The search for new antibiotics produced by soil micro-organisms has encompassed the screening of various genera of bacteria, and fungi including many species within each genus and many strains within each species.
Among the micro-organisms that have received somewhat less attention than others are those that belong to the genus Nocardia. This genus has the narrow hyphae of the Actinomycetales and is characterised by fragmentary substrate mycelium. The generic identity may be further supported by a cell wall of type IV as described by H. A. Lechevalier and M. P. Lechevalier, "A Critical Evaluation of the Genera of Aerobic Actinomycetes", pages 393-405, in "The Actinomycetales" (1970), edited by H. Prauser and published by Fischer, Jena. This genus is further characterised by a whole-cell sugar pattern of type A as described by M. P. Lechevalier, "Identification of Aerobic Actinomycetes of Clinical Importance," J. Lab. Clin. Med., 71(6), 934-944 (1968).
This invention provides new antibiotics, particularly those designated Compounds 47,444, 47,985 and 48,039, which are produced under submerged aerobic fermentation conditions by Nocardia argentinensis Huang sp. nov. ATCC 31306.
The micro-organism useful for the preparation of the antibiotics of this invention was isolated from a soil sample from Argentina.
This culture (Pfizer F.D. 25952), designated Nocardia argentinensis Huang sp. nov. was deposited in the American Type Culture Collection Rockville, as the type culture on 5th July, 1977, under their accession number ATCC 31306.
The culture was planted from a 5-day-old-slant into liquid ATCC No. 172 medium (American Type Culture Catalogue, 10th Edition, p. 235, 1972) and grown for 3 days at 28°C on a shaker. It was then homogenised for 30 seconds in a blender, centrifuged for 20 minutes, washed three times with sterile distilled water and planted on media commonly used for identification of members of the Actinomycetales.
The inoculated media were incubated at 28°C and records of results were made after suitable incubation time with most final results recorded at a period of 13 days. The colours were described in common terminology, but exact colour was determined by comparison with colour chips from the Colour Harmony Manual, fourth edition. About 20 grams of washed, autoclaved mycelium of the culture were used for cell wall analyses.
Identification media used for the characterisation of the culture and references for their composition are as follows:

1. Tryptone Yeast Extract Broth (ISP 1 medium, Difco).
2. Yeast Extract-Malt Extract Agar (ISP 2 medium, Difco).
3. Oatmeal Agar (ISP 3, medium, Difco).
4. Inorganic Salts-Starch Agar (ISP 4 medium, Difco).
5. Glycerol-Asparagine Agar (ISP 5 medium, Difco).
6. Peptone-Yeast Extract Iron Agar (ISP 6 medium- Ditco).
7. Tyrosine Agar (ISP 7 medium, Difco).
8. Gelatin-R. E. Gordon and J. M. Mihm, J. Bact. 73:15-27, 1957.
9. Starch-Ibid.
10. Organic Nitrate Broth-Ibid.
11. Dextrose Nitrate Broth-S. A. Waksman, The Actinomycetes, Vol. 2, medium no. 1, p. 328,1961, with 3 g dextrose substituted for 30 g sucrose and agar omitted.
12. Potato Carrot Agar-M. P. Lechevalier, J. Lab. and Clinical Med. 71:934-944, 1968 but use only 30 g potatoes, 2.5 g carrots and 20 g agar.
13. 2% Tap Water Agar.
14. Czapek-Sucrose Agar-S. A. Waksman, The Actinomycetes, Vol. 2 medium no. 1, p. 328, 1961.
15. Glucose Asparagine Agar-Ibid, medium no. 2, p. 328.
16. Glucose Yeast Extract Agar-Ibid, medium no. 29, p, 331.
17. Emerson's Agar-Ibid, medium no. 28, p. 331.
18. Nutrient Agar-Ibid, medium no. 14, p. 330.
19. Potato Dextrose Agar-J. N. Couch, J. Elisha Mitchell Soc. 79:53-70, 1963.
20. Gordon and Smith-Tyrosine Agar-R. E. Gordon and M. M. Smith J. Bact. 69:147-150, 1955.
21. Casein Agar-Ibid.
22. Calcium Malate Agar-S. A. Waksman, Bact. Rev. 21:1-29, 1957.
23. Skim Milk-Difco.
24. Cellulose utilisation
- (a) H. L. Jensen, Proc. Linn. Soc. N.S.W. 55:231-248, 1930
- (b) M. Levine and H. W. Schoenlein, A Compilation of Culture Media, medium 2511, 1930.
25. Carbohydrates-G. M. Leudemann and B. C. Brodsky, Anti-microbial Agents and Chemotherapy, 1964:47, 1965.
26. Temperature Range-ATCC medium 172 in ATCC Culture Collection Catalogue, 12th edition, p. 329, 1976.

The culture, Nocardia argentinensis Huang sp. nov., was described as follows as on the various culture media:

Yeast Extract-Malt Extract Agar - Growth good, white but pale orange (4 ga) near ends of streak, smooth to wrinkled, with white aerial mycelium; reverse pale orange (3 ga); no soluble pigment.
Oatmeal Agar - Growth moderate to good, white, thin, smooth, with white aerial mycelium; reverse colourless; no soluble pigment.
Inorganic Salts-Starch Agar - Growth poor to moderate, white, thin, smooth, with scant white aerial mycelium; reverse colourless; no soluble pigment. _N
Glycerol-Asparagine Agar - Growth moderate, white, thin, smooth, with white aerial mycelium; reverse pale orange (3 ea); no soluble pigment.
Gelatin - Growth good, white, slightly raised, smooth, with white aerial mycelium; reverse yellowish orange (3 ia); no soluble pigment.
Starch - Growth good, white, slightly raised, smooth, with white aerial mycelium; reverse pale yellowish (3 ga); no soluble pigment.
Potato Carrot Agar - Growth moderate, white with pale orange tint (near 3 ca), thin, smooth; reverse pale orange (3 ea to 3 ga); no soluble pigment.
Tap Water Agar - Growth poor, white, thin, smooth, with scant white aerial mycelium; reverse colourless to very pale orange (3 ca); no soluble pigment.
Czapek-Sucrose Agar - Growth poor to moderate, white, thin, smooth, with white aerial mycelium; reverse pale orange (3 ca); no soluble pigment..
Glucose Asparagine Agar - Growth good, white with small orange dots (4 ea), slightly raised and roughened, with white aerial mycelium; reverse yellowish orange (3 ga); no soluble pigment. Glucose-Yeast Extract Agar - Growth good, white but orange (4 ga) at ends of streak, raised, wrinkled; reverse orange (4 la); pale yellowish soluble pigment.
Emerson's Agar - Growth good, orange (4 ea to 4 ia), raised, wrinkled with short white aerial mycelium; reverse orange (4 la); no soluble pigment.
Nutrient Agar - Growth moderate, white, thin, smooth, with white aerial mycelium; reverse pale orange (3 ea); no soluble pigment.
Potato Dextrose Agar - Growth good, white with yellowish orange dots (3 ga to 3 ia), raised, slightly roughened with white aerial mycelium; reverse yellowish orange (3 ia); no soluble pigment.
Gordon and Smith' Tyrosine Agar - Growth moderate to good, white with very pale orange tint, thin, smooth with many small raised dots, aerial mycelium white; reverse like surface; soluble pigment brown (6 pn).
Casein Agar - Growth good, white, slightly raised, smooth but slightly wrinkled at the edge of streak, aerial mycelium white; reverse colourless; soluble pigment yellowish brown (3 Ic).
Calcium Malate Agar - Growth poor to moderate, white, thin, smooth, but granular in some areas, with white aerial mycelium; reverse very pale orange (3 ca); no soluble pigment.
Biochemical Properties - Gram-positive; non-acid-fast, no melanin; hydrogen sulfide not produced; gelatin liquefied; starch not hydrolysed; nitrate reduced to nitrite; no disintegration on both Jensen's cellulose and Levine and Schoenlein's cellulose; clearing but no coagulation on milk; casein digestion positive; tyrosine digestion positive; no digestion of calcium malate; no decomposition of xanthine; decomposition of hypoxanthine positive. Carbohydrate utilisation: glucose, inositol, glycerol, mannose, ribose, and trehalose utilised; arabinose, fructose, raffinose, sucrose, xylose and melibiose doubtfully- utilised; mannitol, rhamnose, adonitol, cellobiose, dulcitol, galactose, lactose, melezitose, salicin, sorbitol, sorbose and starch not utilised.
Morphological Properties - Morphological observations were made 8 hours, 24 hours, 5 days, 18 days, and 24 days after incubation of the culture on Czapek-Sucrose agar: Microcolonies consisting of an extensively branched substrate mycelium which begin to fragment into bacillary and coccoid cells after 24 hours incubation; fragmented cells rod-shaped or globose, smooth, measuring 0.8-1.0 µm or 1-2 (-3) x 0.6-0.9 ,um; aerial mycelium sparse, short, often zig-zagged may fragment into bacillary and coccoid cells as the culture ages.

Temperature Relations -

The culture was gram-positive, non-acid-fast, and was characterised by the fragmentation of substrate mycelium after 24 hours of incubation, the white aerial mycelium and the pale orange substrate mycelium. The cell wall had meso-diaminopimelic acid, arabinose, galactose, and mycolate of the nooardomycolate type characteristic of the species of Nocardia. It could not be identified with any known species of Nocardia and hereby described as new under the name Nocardia argentinensis Huang sp. nov. The specific epithet refers to Argentina where the soil sample yielding the culture was collected.
Cultivation of Nocardia argentinensis preferably takes place in aqueous nutrient media at a temperature of 24-36°C and under submerged aerobic conditions with agitation. Nutrient media which are useful for such purposes include a source of assimilable carbon such as sugars, starches and glycerol; a source of organic nitrogen such as casein, enzymatic digest of casein, soybean meal, cotton seed meal, peanut meal, wheat gluten, soy flour, meat meal and fish meal. A source of growth substances such as grain solubles and yeast extract as well as salts such as sodium chloride and calcium carbonate and trace elements such as iron, zinc, cobalt and manganese may also be utilised with advantageous results.
If excessive foaming is encountered during fermentation, antifoam agents such as vegetable oils or silicones may be added to the fermentation medium. Aeration of the medium in tanks for submerged growth is preferably maintained at the rate of from 1/2 to 2 volumes of free air per volume of broth per minute. Agitation may be maintained by means of agitators generally familiar to those in the fermentation industry. Aseptic conditions must, of course, be maintained through the transfer of the organism and throughout its growth.
Inoculum for the preparation of the antibiotic may be obtained by employing growth from a slant of the culture. The growth may be used to inoculate either shake flask or inoculum tanks or the inoculum tanks may be seeded from the shake flasks. Growth in shaken flasks will generally have reached its maximum in 2 to 4 days whereas inoculum in submerged inoculum tanks will usually be at the most favourable period in 1.5-3 days. Substantial antibiotic activity is obtained in the final fermenter stage in approximately 2 to 5 days.
The process of antibiotic production is conveniently followed during fermentation by biological assay of the broth employing a sensitive strain of Staphylococcus aureus or Micrococcus luteus. Standard plate assay technique is employed in which the zone of inhibition surrounding a filter paper disc saturated with broth is used as a measure of antibiotic potency.
Thin-layer chromatography employing silica gel is a useful tool for analysing the antibiotics produced by Nocardia argentinensis in fermentation media and the composition of crude and purified materials extracted from fermentation broths. Silica gel plates are employed with a developing system of chloroform:acetone (3:1 v/v). These antibiotics may be visualised by exposure to 254 nm light or bio- overlay with a thin layer of agar seeded with a sensitive strain of Staphylococcus aureus or Micrococcus luteus.
The antibiotics may be separated and recovered by extracting the whole, unfiltered fermentation broth with an organic solvent such as chloroform, ethyl acetate, methylisobutyl ketone or butanol at a pH range of 4.0 to 10.0. The solvent is concentrated to a thin syrup, defatted with heptane and chromatographed in chloroform on silica gel.
A method of separation and recovery of antibiotics 47,444, 47,985 and 48,039 is as follows: Whole fermentation broth is extracted with about 1/3 volume of methylisobutyl ketone followed by concentration in vacuo. The oily extract is triturated several times with heptane. The viscous concentrate is dispersed on silica gel in the presence of heptane and then added to a sintered glass filter coated with silica gel. The silica gel is washed successively with heptane, chloroform, varying ratios of chloroform:ethyl acetate and finally ethyl acetate. All steps in the purification sequence are monitored by thin-layer chromatography.
The appropriate column cuts are pooled, preferably treated with activated charcoal and rechromatographed on silica gel eluting with chloroform:ethyl acetate (1:1 v/v).
The present invention includes within its scope the dilute forms and crude concentrates of the mixture of antibiotics and the purified antibiotic Compound 47,444. The minor antibiotics Compound 47,985 and Compound 48,039 are present in such small amounts that it has not proved possible to isolate them in a state of homogeneity at the present time. All of these products are useful in combating micro-organisms, especially strains of Staphylococcus aureus that are resistant to other antibiotics.
Table I illustrates the antibacterial spectrum of Compound 47,444. These tests were run by preparing tubes of nutrient broth with gradually increasing concentrations of the pure antibiotic and then seeding the broths with the particular organism specified. The minimal inhibitory concentration indicated in Table I is the minimal concentration of the antibiotic (in micrograms/ml) at which the micro-organism failed to grow. The tests were conducted under standardised conditions as described in Proc. Soc. Exp. Biol. Et Med., 122, 1107 (1966).
In vivo protection afforded by Compound 47,444 against mice experimentally infected with Staphylococcus aureus 01 A005 is shown in Table II.
The compound was administered 30 minutes before infection and the figures quoted are the percentages of mice surviving 96 hours after infection, based on replicated studies using 10 mice per study.
Antibiotic Compound 47,444 can be administered via the oral or parenteral routes for the treatment in animals, including humans, of staphylococcal and other antibiotic-sensitive infections. In general, the antibiotic is most desirably administered in daily oral doses of 0.5 to 1 gram or parenteral injections of 100 to 500 mg, depending on the type and severity of the infection and weight of the subject being treated.
Antibiotic Compound 47,444 may be administered alone or in combination with pharmaceutically acceptable carriers, and such administration can be carried out in both single and multiple doses.
For purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and dicalcium phosphate may be employed along with various disintegrants such as starch, alginic acid and certain complex silicates together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and gum acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules, preferred materials include lactose as well as high molecuiar weight pulyethylene glycols. When aqueous suspension and/or elixirs are desired for oral administration, the essential active ingredient therein may be combined with various sweetening or flavouring agents, colouring matter or dyes, and if desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerol and various combinations thereof.
For purposes of parenteral administration, solutions of Compound 47,444 or solutions of a mixture of Compounds 47,444, 47,985 and 48,039 in sesame or peanut oil or in aqueous propylene glycol may be employed.

EXAMPLE I

A sterile aqueous medium having the following composition was prepared:
Cells from a slant culture of Nocardia argentinensis ATCC 31306 were transferred to each of a number of 300 ml shake flasks each containing 40 ml of the above medium and shaken at 28°C for three to four days.
A sterile aqueous medium having the following composition was prepared:
Fermentors containing two litres of the above described sterile medium were seeded with 2-4% v/v of grown inoculum. The temperature was maintained at 30°C. The broth was stirred at 1700 r.p.m. and aerated at the rate of about one volume of air per volume of broth per minute. When substantial antibiotic activity was obtained (based on antibiotic disc assay), ca. 2-5 days, the filtered or whole fermentation broth was twice extracted with 1/3 to 1/2 volume of methylisobutyl ketone. The solvent was separated from the aqueous phase and concentrated in vacuo to a viscous oil.

EXAMPLE II

The fermentation process of Example I may be repeated employing the following fermentation medium:

EXAMPLE III

EXAMPLE IV

The fermentation process of Example I was repeated. About 0.1 % v/v of the grown inoculum was used to inoculate a 7570 litre fermentor containing 4542 litres of the production medium of Example I. The fermentation was conducted at a temperature of 28°C and an aeration rate of one volume of air per volume of broth per minute. After substantial antibiotic activity was obtained (approximately 48 to 72 hours), 4163 litres of the whole fermentation broth, pH 8.4, was extracted with approximately 1324 litres of methylisobutyl ketone. Concentration of the solvent extract in vacuo gave rise to an oily extract (1,190 grams) containing antibiotic Compounds 47,444, 47,985 and 48,039. Trituratin with heptane (1 x 5.0 litres, 1 x 2.0 litres, 1 x 1.0 litres) then led to a viscous concentrate (241 grams) containing 95% of the antibiotic activity present in the starting extract of 1,190 grams.
The concentrate (241 grams) was dispersed on 500 grams of silica gel 60 (E. Merck, Darmstadt, Germany) in the presence of a litre of heptane and then added to a 2.0 litre sintered glass filter coated with 250 grams of silica gel 60. The silica gel was washed successively with a litre of heptane, 9 litres of chloroform, a litre of chloroform:ethyl acetate (9:1), a litre of chloroform:ethyl acetate (4:1), a litre of chloroform:ethyl acetate (7:3), a litre of chloroform:ethyl acetate (3:2), a litre of chloroform:ethyl acetate (1:1), a litre of chloroform:ethyl acetate (2.5:7.5) and 2.5 litres of ethyl acetate. All steps in the purification sequence were monitored by thin-layer chromatography. The greater part of the antibiotic activity was found in the last one litre of chloroform through the one litre of chloroform:ethyl acetate (1:1). These eluates were combined and evaporated in vacuo to an off-white foam (41 grams). The latter was dissolved in 400 ml of ethyl acetate and stirred for 30 minutes with 41 grams of Darco G60. Filtration followed by concentration in vacuo led to 36 grams of white solid.
The minor, less polar antibiotic Compound 47,985 was found in the heptane and chloroform eluates (first two litres) whereas the minor, more polar antibiotic Compound 48,039 was found in the chloroform:ethyl acetate (3:2-2.5:7.5) eluates.
The material from charcoal treatment (36 grams in chloroform solution) was then added to the top of a silica gel 60 column (5.08 cm x 96 cms) and developed with chloroform containing increasing amounts of ethyl acetate, using 20 ml cuts, from 100% chloroform to 50:50 chloroform:ethyl acetate. Appropriate cuts from this column were combined to afford substantially pure Compound 47,444 (25.8 grams). An analytical sample of antibiotic Compound 47,444 was derived by further chromatography of an aliquot of the 25.8 grams sample (column dimensions 2.54 cm x 92 cms) on silica gel PF₂₅₄ (E. Merck, Darmstadt, Germany) eluting with 50:50 chloroform:ethyl acetate. All attempts to crystallise antibiotic Compound 47,444 were unsuccessful. It was obtained as an amorphous white solid.
The minor antibiotic Compounds 47,985 and 48,039 were present in such small amounts that it has not proved possible to isolate them in a state of homogeneity at this time.
Compound 47,444 (sample dried overnight in vacuo 45-50°C0.

Elemental analysis

C 64.40% H 7.61% N 2.56% O 25.43% (by difference)

Molecular Formula

C₂₈H₃₇NO₈ (M⁺515), high resolution mass spectrum. The C¹³ nmr spectrum displays resonances consistent with the presence of 28 carbon atoms. Optical Rotation [α]_D = +49° (c = 1.0, methanol)

Ultraviolet Absorption Maxima

^MeOH 265 nm E ^1% 320 1 cm λ max Characteristic Infra-red Bands (KBr disc) in microns as shown in Figure 1:
2.90, 3.40, 5.76, 5.87, 6.40, 6.85, 7.05 7.63, 8.53, 9.0, 10.58, 11.34 and 13.35.

Solubilities

Soluble in methanol, ethanol, chloroform, methylene chloride, acetone, methylisobutyl ketone and ethyl acetate. Insoluble in heptane and water.

Claims

1. An antibiotic or mixture of antibiotics characterised in that it is produced by cultivating Nocardia argentinensis Huang sp. nov. ATCC 31306 under submerged aerobic conditions in an aqueous nutrient medium containing an assimilable source of carbon and nitrogen until substantial antibiotic activity is obtained and separating the antibiotic or mixture of antibiotics therefrom.

2. An antibiotic as claimed in claim 1 designated Compound 47,444 which is soluble in methanol, ethanol, chloroform, methylene chloride, acetone, methylisobutyl ketone and ethyl acetate; insoluble in heptane and water; has an absorption maximum in methanol in the ultraviolet light region of the spectrum at 265 nm with an

value of 320; has a molecular formula of C₂₈H₃₇NO₈; has an optical rotation of [α]_D = +49° at a concentration of 1% in methanol; and when pelleted in KBr, exhibits characteristic absorption of the infra-red region at the following wavelengths in microns: 2.90, 3.40, 5.76, 5.87, 6.40, 6.85, 7.05, 7.63, 8.53, 9.0, 10.58, 11.34 and 13.35.

3. A pharmaceutical composition suitable for combating bacterial infections comprising a pharmaceutically acceptable carrier and a therapeutically-effective amount of Compound 47,444.