EP0000991B1 - Antibiotiques et compositions pharmaceutiques les contenant - Google Patents

Antibiotiques et compositions pharmaceutiques les contenant Download PDF

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Publication number
EP0000991B1
EP0000991B1 EP78300252A EP78300252A EP0000991B1 EP 0000991 B1 EP0000991 B1 EP 0000991B1 EP 78300252 A EP78300252 A EP 78300252A EP 78300252 A EP78300252 A EP 78300252A EP 0000991 B1 EP0000991 B1 EP 0000991B1
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EP
European Patent Office
Prior art keywords
antibiotic
agar
antibiotics
white
chloroform
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Expired
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EP78300252A
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German (de)
English (en)
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EP0000991A1 (fr
Inventor
Walter Daniel Celmer
Walter Patrick Cullen
Liang Hsiung Huang
Mark Tilden Jefferson
Charles Edward Moppett
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
    • A61P31/04—Antibacterial agents

Definitions

  • 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.
  • 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.
  • micro-organism useful for the preparation of the antibiotics of this invention was isolated from a soil sample from Argentina.
  • 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.
  • 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.
  • 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 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 I The fermentation process of Example I may be repeated employing the following fermentation medium:
  • Example I The fermentation process of Example I may be repeated employing the following fermentation medium:
  • Example I 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.
  • silica gel 60 E. Merck, Darmstadt, Germany
  • 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 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 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.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Communicable Diseases (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Oncology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Compounds Of Unknown Constitution (AREA)

Claims (3)

1. Antibiotique ou mélange d'antibiotiques, caractérisé en ce qu'il est produit par culture de Nocardia argentinensis Huang sp. nov. ATCC 31306 dans des conditions aérobies en immersion dans un milieu nutritif aqueux contenant une source assimilable de carbone et d'azote jusqu'à ce qu'une activité antibiotique importante ait été obtenue, et séparation de l'antibiotique ou du mélange d'antibiotiques.
2. Antibiotique suivant la revendiction 1, appelé composé 47 444, qui est soluble dans le méthanol, l'éthanol, le chloroforme, le chlorure de méthylène, l'acétone, la méthylisobutylcétone et l'acétate d'éthyle; insoluble dans t'heptane et l'eau; qui a un maximum d'absorption dans le méthanol dans la région de la lumière ultraviolette du spectre à 265 nm avec une valeur E 1 % de 320; qui a 1 cm pour formule moléculaire C28H37NO8; qui a une rotation optique [α]D = +49° à une concentration de 1 % dans le méthanol; et qui, incorporé à une pastille de KBr, présente une absorption caractéristique dans la région infrarouge pour les longueurs d'ondes suivantes exprimées en 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 et 13,35.
3. Composition pharmaceutique apte à combattre des infections bactériennes, comprenant un support acceptable du point de vue pharmaceutique et une quantité thérapeutiquement efficace du composé 47 444.
EP78300252A 1977-08-18 1978-08-16 Antibiotiques et compositions pharmaceutiques les contenant Expired EP0000991B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US825563 1977-08-18
US05/825,563 US4148883A (en) 1977-08-18 1977-08-18 Antibiotics produced by new species of nocardia

Publications (2)

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EP0000991A1 EP0000991A1 (fr) 1979-03-07
EP0000991B1 true EP0000991B1 (fr) 1981-11-04

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EP78300252A Expired EP0000991B1 (fr) 1977-08-18 1978-08-16 Antibiotiques et compositions pharmaceutiques les contenant

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US (1) US4148883A (fr)
EP (1) EP0000991B1 (fr)
JP (1) JPS5444602A (fr)
DE (1) DE2861282D1 (fr)
DK (1) DK144337C (fr)
IE (1) IE47180B1 (fr)
IT (1) IT1098192B (fr)

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4224314A (en) * 1979-03-02 1980-09-23 Pfizer Inc. Antibiotics produced by species of Nocardia
US4293651A (en) * 1979-10-02 1981-10-06 The Upjohn Company Process for producing antibiotic using saccharopolyspora
US4360683A (en) * 1980-08-06 1982-11-23 The Upjohn Company Antibiotic nodusmicin derivatives
US4461903A (en) * 1980-08-06 1984-07-24 The Upjohn Company Antibiotic nodusmicin derivatives
US4363922A (en) * 1980-08-06 1982-12-14 The Upjohn Company Esters of antibiotic nodusmicin
US4588817A (en) * 1980-08-06 1986-05-13 The Upjohn Company Antibiotic nodusmicins
US4351769A (en) * 1980-08-25 1982-09-28 The Upjohn Company Antibiotic composition of matter
US4448970A (en) * 1981-02-19 1984-05-15 The Upjohn Company Nargenicin derivatives
US4436747A (en) 1982-10-21 1984-03-13 Pfizer Inc. Nargenicin C1
US4605624A (en) * 1982-10-21 1986-08-12 Pfizer Inc. Nocardia species capable of producing nargenicin C1
PE5591A1 (es) * 1988-12-19 1991-02-15 Lilly Co Eli Un nuevo grupo de compuestos de macrolida
US5202242A (en) * 1991-11-08 1993-04-13 Dowelanco A83543 compounds and processes for production thereof
US5539089A (en) * 1991-11-08 1996-07-23 Dowelanco A83543 aglycones and pseudoglycones
US5227295A (en) * 1991-11-08 1993-07-13 Dowelanco Process for isolating A83543 and its components
US5591606A (en) * 1992-11-06 1997-01-07 Dowelanco Process for the production of A83543 compounds with Saccharopolyspora spinosa
WO1994020518A1 (fr) * 1993-03-12 1994-09-15 Dowelanco Nouveaux composes a83543 et procede de production desdits composes
US6001981A (en) * 1996-06-13 1999-12-14 Dow Agrosciences Llc Synthetic modification of Spinosyn compounds
WO2016061772A1 (fr) 2014-10-22 2016-04-28 Merck Sharp & Dohme Corp. Composés nargénicine et leurs utilisations de comme agents antibactériens
DE202022102100U1 (de) 2022-04-20 2022-04-27 Prabhakar Ramesh Bhandari Synergistische antibiotische pharmazeutische Zusammensetzung zur Behandlung von Staphylococcus aureus-Infektionen

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4038383A (en) * 1975-01-17 1977-07-26 Pfizer Inc. Mixture of antibiotics produced by a species of actinoplanes

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JPS5512438B2 (fr) 1980-04-02
US4148883A (en) 1979-04-10
DK364178A (da) 1979-02-19
IE781652L (en) 1979-02-18
EP0000991A1 (fr) 1979-03-07
IT1098192B (it) 1985-09-07
IT7826811A0 (it) 1978-08-17
IE47180B1 (en) 1984-01-11
JPS5444602A (en) 1979-04-09
DE2861282D1 (en) 1982-01-14
DK144337B (da) 1982-02-22
DK144337C (da) 1982-08-23

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