EP0000258B1 - Polycyclic ether antibiotics, process for producing them and animal feed compositions containing them - Google Patents

Polycyclic ether antibiotics, process for producing them and animal feed compositions containing them Download PDF

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Publication number
EP0000258B1
EP0000258B1 EP78300057A EP78300057A EP0000258B1 EP 0000258 B1 EP0000258 B1 EP 0000258B1 EP 78300057 A EP78300057 A EP 78300057A EP 78300057 A EP78300057 A EP 78300057A EP 0000258 B1 EP0000258 B1 EP 0000258B1
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EP
European Patent Office
Prior art keywords
antibiotic
mixture
antibiotics
ethyl acetate
medium
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
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EP78300057A
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German (de)
English (en)
French (fr)
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EP0000258A1 (en
Inventor
Walter Daniel Celmer
Walter Patrick Cullen
Charles Edward Moppett
John Richard Oscarson
Liang Hsiung Huang
Riichiro Shibakawa
Junsuke Tone
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Pfizer Inc
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Pfizer Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
    • C07G11/00—Antibiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/826—Actinomyces

Definitions

  • This invention is concerned with new members of the acidic polycyclic ether group of antibiotics, a class of compounds characterized biologically by their effect on cation transport in mitochondria.
  • This family of antibiotics includes monensin (J. Amer. Chem. Soc., 89:5737m 1967); nigericin (Biochem. Biophys. Res. Comm., 33:29, 1968); grisorixin (J. Chem. Soc. Chem. Commun., 1421, 1970); dianemycin (J. Antibiotics, 22:161, 1969); salinomycin (J. Antibiotics, 27:814, 1974); X-537A (J. Chem. Soc. Chem. Commun., 967, 1972); X-206 (J. Chem. Soc. Chem. Commun., 927, 1971); and A204A (J. Amer. Chem. Soc., 95:3399, 1973).
  • polycyclic ether antibiotics listed above are active against Gram-positive bacteria, fungi and protozoa, and also exhibit potent anticoccidial activity.
  • Eimeria tenella, E. necatrix, E. brunetti, E. acervulina, E. maxima and E. mivati produce damage either directly through destruction of epithelial cells of the digestive tract or indirectly through production of toxins.
  • Three other species of protozoa belonging to the same genus, E. mitis, E. hagani and E. praecox, are considered to be relatively innocuous, but are capable of reducing weight gain, lowering feed efficiency and adversely affecting egg production.
  • the polycyclic ether antibiotics possess a high degree of effectiveness against all species of Eimeria. These antibiotics can, therefore, be regarded as "broad spectrum” coccidiostats.
  • This invention is concerned with new polycyclic ether antibiotics produced by a new species of actinomycete under submerged aerobic conditions in aqueous nutrient media.
  • Antibiotic compounds 47,433 and 47,434 or mixtures of antibiotic compounds 47,433 and 47,434 and their cationic salts are active against a variety of micro-organisms, effective in controlling coccidiosis in poultry and act to improve feed utilisation efficiency in ruminants.
  • the antibiotic producing micro-organism used in the present invention isolated from a soil sample in Japan, was found on examination to have the morphological features of an actinomycete such as narrow hyphae and sparse aerial mycelium. No spores were found on the media tested with the exception of tyrosine agar on which hyphal swellings were produced on substrate mycelium.
  • the culture was planted from an agar slant into liquid ATCC 172 medium (American Type Culture Catalogue, 10th Edition p. 235 1972) grown for 4 days at 28°C on a rotary shaker and planted from the resultant growth to fresh liquid ATCC 172 medium. After 7 days of incubation at 28°C on a shaker, it was centrifuged, washed twice with sterile distilled water and then planted on media commonly used for identification of members of the actinomycetes.
  • liquid ATCC 172 medium American Type Culture Catalogue, 10th Edition p. 235 1972
  • the culture (Pfizer F.D. 25934) was described as follows on the various media:
  • Cultivation of the actinomycete culture preferably takes place in aqueous nutrient media at a temperature of 28-36°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, magnesium, zinc, cobalt, and manganese may also be utilised with advantageous results.
  • 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 about 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 flasks 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 3 to 5 days whereas inoculum in submerged inoculum tanks will usually be at the most favourable period in 3 to 4 days.
  • Substantial antibiotic activity is obtained in the final fermenter stage in approximately 3 to 5 days.
  • the antibiotic levels range from 50 to 500 mg per litre.
  • the process of antibiotic production is conveniently followed during fermentation by biological assay of the broth employing a sensitive strain of Staphylococcus aureus or Bacillus subtilis.
  • Standard plate assay technique is employed in which the zone of inhibition surrounding a filter paper disc saturated with the broth is used as a measure of antibiotic potency.
  • Thin-layer chromatography employing silica gel is a useful tool for analyzing the antibiotics produced in fermentation media and the composition of crude and purified materials extracted from the fermentation broths.
  • the Analtech silica gel GF chromatograms are developed with ethyl acetate.
  • the antibiotics, compound 47,433 (major, least polar) and compound 47,434 (minor, more polar) are visualised by spraying with 3% vanillin in ethanolic sulfuric acid (97:3 v/v). They show up as pinkish red spots on a white background on warming on a steam bath or a hot plate.
  • Bio-overlay with agar seeded with a sensitive strain of Staphylococcus aureus or Bacillus subtilis is a further procedure for direction of these antibiotics.
  • 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.
  • an organic solvent such as chloroform, ethyl acetate, methylisobutyl ketone or butanol at a pH range of 4.0 to 10.0.
  • a major portion of the antibiotic activity is contained in the mycelium and may be extracted therefrom by slurrying the separated mycelium with a water-soluble solvent such as methanol. The solvent is concentrated to a thin syrup.
  • a method of separation and recovery of antibiotics 47,433 and 47,434 is as follows: Separated wet mycelium from fermentation broth is extracted several times with methanol. The methanol is evaporated in vacuo to provide an aqueous extract which is extracted several times with chloroform. The chloroform extracts are combined and evaporated under vacuum to a viscous oil which is dissolved in heptane. Silica gel is added to the solution and the resultant slurry is evaporated to dryness on a rotary evaporator. The silica gel is placed on a large sintered glass funnel and washed with heptane, chloroform, ethyl acetate and acetone.
  • the desired antibiotics are contained almost exclusively in the ethyl acetate fraction. This fraction is evaporated to dryness, redissolved in ethyl acetate and stirred with water. The pH is adjusted to 9.0 with 1.0 N sodium hydroxide. The ethyl acetate phase is separated, dried over anhydrous sodium sulfate and evaporated under vacuum. The residue is taken up in a small volume of methanol at which time crystallisation occurs.
  • the crystalline material may be further purified by column chromatography employing silica gel developed with ethyl acetate-heptane (30:70). Appropriate column cuts containing compound 47,433 are combined and evaporated to dryness. The residue is dissolved in ethyl acetate and the pH adjusted to 5.0 while stirring with water. The ethyl acetate phase is separated and added to 5% disodium phosphate buffer and the pH adjusted to 9.0 with 1.0 N sodium hydroxide. The ethyl acetate phase is separated and dried over anhydrous sodium sulfate. The residue is taken up in acetone whereupon crystallisation occurs.
  • Antibiotic compounds 47,433 and 47,434 exhibit inhibitory action against the growth of a number of Gram-positive microorganisms. These compounds are their cationic salts exhibit excellent activity against coccidiosis infections in poultry. When incorporated into the diet of chickens at dose levels of 2.5 to 100 ppm, these compounds are effective in controlling infections due to Eimeria tenella, E. acervulina, E. maxima, E. brunetti and E. necatrix.
  • the "average degree of infection” indicates the average lesion score at each dose level, while the “ratio” (established by dividing the lesion score of each treated group by the lesion score of the infected control) indicates the effective reduction of the degree of infection by the antibiotic compound at each dose level.
  • antibiotic compound 47,434 or mixtures of antibiotic compound 47,433 and antibiotic compound 47,434.
  • An increase in the propionic acid content in the rumen fluid indicates that a desirable response in overall ruminant performance has been brought about by the growth promotant in the feed composition.
  • the change in propionic acid content is expressed as percent of the propionic acid content found in the control rumen fluid. Long term in vivo feeding studies are used to show a reliable correlation between propionic acid increase in the rumen fluid and improved animal performance.
  • Rumen fluid is collected from a fistulated cow which is fed on a commercial fattening ration plus hay.
  • the rumen fluid is immediately filtered through cheese cloth, and 10 ml added to a 50 ml conical flask containing 400 mg of standard substrate (68% corn starch + 17% cellulose + 15% extracted soybean meal), 10 ml of a pH 6.8 buffer and the test compound.
  • the flasks are gassed with oxygen free nitrogen for about two minutes and incubated in a shaking water bath at 39°C for about 16 hours. All tests are conducted in triplicate.
  • Antibiotic compounds 47,433 and 47,434 and mixtures of antibiotic compounds 47,433 and 47,434 may be incorporated in feed compositions as the free acid, sodium salt, potassium salt or mixtures thereof.
  • Crude antibiotic mixtures of compounds 47,433 and 47,434 or the dried fermentation medium containing the two antibiotics may be incorporated in feed compositions for ruminants or monogastric animals at the desired potency concentrations for improving feed utilisation, or incorporated into the diet of chickens at the desired dose levels for controlling coccidiosis infections in poultry.
  • a sterile aqueous medium having the following composition was prepared:
  • the fermentation was conducted at 28-36°C with stirring at 1700 revolutions per minute and aeration at 1.5 to 2 volumes of air per volume of broth per minute until substantial activity was obtained (48-120 hours).
  • the whole broth, without pH adjustment, was twice extracted with 1/3 to 1/2 volume of methyl isobutyl ketone.
  • the separated solvent extracts were combined and concentrated under vacuum to a thin syrup.
  • Example 1 The inoculum medium of Example 1 was distributed in 700 ml amounts in 4 to 8 shake flasks and inoculated with cells of Actinomycete sp. ATCC 31286. After incubation at 28°C on a rotary shaker for 3 to 8 days, a 3 to 5% v/v inoculum was introduced into a 190 litre fermenter containing 95 litres of the following sterile medium:
  • the fermentation was conducted for a period of 5 days at 30°C with an aeration rate of one volume of air per volume of medium per minute.
  • the separated mycelium from 95 litres of broth was extracted three times with 19 litres (each time) of methanol.
  • the combined methanolic extracts were evaporated under vacuum to provide an aqueous extract of about 12 litres which was extracted 4 times with 4 litres (each time) of chloroform.
  • the chloroform extracts were combined and evaporated under vacuum to yield 51 grams of a. viscous yellow oil.
  • the oil was dissolved in 500 ml of heptane. Column grade silica gel 60 (E. Merck, Darmstadt, Germany), about 500 grams, was added to the solution and the resultant slurry was evaporated to dryness on a rotary evaporator.
  • the silica gel was then placed on a large sintered glass funnel and washed successively with two litres each of heptane, chloroform, ethyl acetate and acetone.
  • the desired antibiotics were shown by thin-layer chromatography to be contained almost exclusively in the ethyl acetate fraction. This fraction was evaporated to dryness (24 grams) and the other fractions were discarded.
  • the material was dissolved in 125 ml of ethyl acetate and stirred with 125 ml of water. The pH was raised to 9.0 with 1.0 N sodium hydroxide. The ethyl acetate phase was dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was taken up in a small volume of methanol at which time crystallisation occurred. The crystals were removed by filtration and washed with methanol (5.1 grams).
  • the crude crystalline material was further purified by column chromatography on a 2.54 x 100 cm column packed with silica gel 60 in heptane.
  • a portion of the crude crystalline material (2.5 grams) was applied to the column in solution in ethyl acetate-heptane (30:70) and the column developed with the same solvent system at a rate of 10 ml/minute with column cuts taken every two minutes.
  • the column cuts were monitored by thin-layer chromatography.
  • the column was washed with heptane and the remaining 2.6 grams of crude crystalline material processed in the same manner.
  • the residue was dissolved in 100 ml of ethyl acetate and the pH adjusted to 5.0 with 85% phosphoric acid while stirring with 100 ml of water.
  • the ethyl acetate phase was added to 100 ml of 5% disodium phosphate buffer and the pH adjusted to 9.0 with 1 N sodium hydroxide.
  • the ethyl acetate phase was dried with anhydrous sodium sulfate and evaporated to dryness.
  • the residue was taken up in acetone whereupon crystallisation occurred. Crystals were collected by filtration and dried under high vacuum at room temperature to yield 2.7 grams of compound 47,433 as the sodium salt.
  • the sodium salt of compound 47,433 is soluble in chloroform, ethyl acetate and methylisobutyl ketone; it is insoluble in water.
  • the free acid was derived by washing an ethyl acetate solution of the sodium salt of Compound 47,433 with a pH 5.0 aqueous phase (water adjusted to pH 5.0 with 85% phosphoric acid). The solvent layer was concentrated in vacuo and crystallised from heptane as the free acid.
  • the free acid m.p. 89-99°C, is soluble in methanol, acetone, chloroform, methylisobutyl ketone and ethyl acetate; it is insoluble in water.
  • the potassium salt of Compound 47,433 was obtained by washing an ethyl acetate solution of the free acid with aqueous dipotassium hydrogen phosphate adjusted to pH 9.0 with 1.0 N potassium hydroxide. It was crystallised from heptane.
  • the potassium salt, m.p. 202-205°C is soluble in chloroform, ethyl acetate and methylisobutyl ketone; it is insoluble in water.
  • the silver salt of Compound 47,433 was prepared by the addition of silver nitrate in aqueous methanol to an aqueous methanolic solution of the sodium salt. Removal of the methanol under vacuum led to the separation of the silver salt.
  • the salt is soluble in chloroform, ethyl acetate and methylisobutyl ketone; it is insoluble in water.
  • the sodium salt of Compound 47,434 is soluble in chloroform, ethyl acetate and methylisobutyl ketone; it is insoluble in water.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Fodder In General (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Feed For Specific Animals (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Compounds Of Unknown Constitution (AREA)
EP78300057A 1977-06-24 1978-06-20 Polycyclic ether antibiotics, process for producing them and animal feed compositions containing them Expired EP0000258B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US05/809,620 US4148882A (en) 1977-06-24 1977-06-24 Polycyclic ether antibiotics produced by new species of actinomycete
US809620 1991-12-17

Publications (2)

Publication Number Publication Date
EP0000258A1 EP0000258A1 (en) 1979-01-10
EP0000258B1 true EP0000258B1 (en) 1981-03-11

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EP78300057A Expired EP0000258B1 (en) 1977-06-24 1978-06-20 Polycyclic ether antibiotics, process for producing them and animal feed compositions containing them

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US (1) US4148882A (it)
EP (1) EP0000258B1 (it)
JP (3) JPS5412302A (it)
CA (1) CA1131145A (it)
DE (1) DE2860512D1 (it)
DK (1) DK283578A (it)
IE (1) IE47005B1 (it)
IT (1) IT1097275B (it)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4533553A (en) * 1981-07-20 1985-08-06 Pfizer Inc. Polycyclic ether antibiotic
US4546084A (en) * 1982-07-26 1985-10-08 Bristol-Myers Company Biologically pure culture of Actinomadura Sp.
GR78648B (it) * 1982-07-26 1984-09-27 Bristol Myers Co
US4683204A (en) * 1984-10-09 1987-07-28 Eli Lilly And Company Process for producing antibiotic A80190
US4683201A (en) * 1984-10-09 1987-07-28 Eli Lilly And Company Antibiotic A80190-producing Actinomadura oligospora and process
US4582822A (en) * 1984-10-09 1986-04-15 Eli Lilly And Company Antibiotic A80190, pharmaceutical compositions containing same and method of use
US4859599A (en) * 1984-10-11 1989-08-22 The Dow Chemical Company Antibiotic A26201-1 and antibiotic A26201-2 produced by a novel strain of actinoplanes
US4613503A (en) * 1984-10-11 1986-09-23 The Dow Chemical Company Antibiotic A26201-1 and antibiotic A26201-2 produced by a novel strain of actinoplanes
US4725621A (en) * 1986-03-03 1988-02-16 Warner-Lambert Company CL-1957E antibiotic compound and its production
WO1993012800A1 (en) * 1987-11-20 1993-07-08 Dirlam John P An acidic polycyclic ether useful as an anticoccidial agent and as a growth promotant
HU203789B (en) * 1988-02-08 1991-09-30 Pfizer Process for producing polycyclic acidic ether-type antibiotica of anticoccidial and growth-stimulating activity
WO1989012105A1 (en) * 1988-06-09 1989-12-14 Pfizer Inc. Acidic polycyclic ether antibiotic having anticoccidial and growth promotant activity
US5298524A (en) * 1988-06-09 1994-03-29 Pfizer Inc. Acidic polycyclic ether antibiotic having an anticoccidial and growth promotant activity
US5034224A (en) * 1989-05-30 1991-07-23 American Cyanamid Company Method and composition for treating protozoal infections
HUT59932A (en) * 1989-06-01 1992-07-28 Pfizer Microbiological process for producing acidic, polycyclic ether-type antibiotica
US5242814A (en) * 1989-10-10 1993-09-07 Eli Lilly And Company Polyether antibiotic
US5043353A (en) * 1989-10-10 1991-08-27 Eli Lilly And Company A80789 polyether antibiotic
US5891727A (en) * 1990-04-16 1999-04-06 Pfizer Inc. Acidic polycyclic ether useful as an anticoccidial agent and as a growth promotant

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3985872A (en) * 1974-11-15 1976-10-12 Eli Lilly And Company Dihydro-A204
CH615459A5 (en) * 1975-03-11 1980-01-31 Sandoz Ag Process for the preparation of a metabolite

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Publication number Publication date
CA1131145A (en) 1982-09-07
JPS57154187A (en) 1982-09-22
DE2860512D1 (en) 1981-04-09
JPS5412302A (en) 1979-01-30
JPS57154188A (en) 1982-09-22
EP0000258A1 (en) 1979-01-10
JPS5735915B2 (it) 1982-07-31
IE47005B1 (en) 1983-11-30
DK283578A (da) 1978-12-25
IT1097275B (it) 1985-08-31
IT7824912A0 (it) 1978-06-23
IE781254L (en) 1978-12-24
US4148882A (en) 1979-04-10

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