EP0232373A1 - Antibiotiques a base de tetracyclines, leur preparation, compositions antibiotiques les contenant et microorganismes utiles pour leur preparatioin - Google Patents

Antibiotiques a base de tetracyclines, leur preparation, compositions antibiotiques les contenant et microorganismes utiles pour leur preparatioin

Info

Publication number
EP0232373A1
EP0232373A1 EP86905061A EP86905061A EP0232373A1 EP 0232373 A1 EP0232373 A1 EP 0232373A1 EP 86905061 A EP86905061 A EP 86905061A EP 86905061 A EP86905061 A EP 86905061A EP 0232373 A1 EP0232373 A1 EP 0232373A1
Authority
EP
European Patent Office
Prior art keywords
antibiotic
chloro
atcc
var
brunnea
Prior art date
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.)
Withdrawn
Application number
EP86905061A
Other languages
German (de)
English (en)
Inventor
Elizabeth Bargmann Smith
Hanan Karaman Munayyer
Michael Joseph Ryan
George Henry Miller
Mahesh Gordhandas Patel
Ann Camille Horan
Joseph Anthony Marquez
Richard Willis Vaughan
Manohar Gopal Kalyanpor
Jay Allan Waitz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck Sharp and Dohme LLC
Original Assignee
Schering Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from US06/764,275 external-priority patent/US4752605A/en
Application filed by Schering Corp filed Critical Schering Corp
Publication of EP0232373A1 publication Critical patent/EP0232373A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P29/00—Preparation of compounds containing a naphthacene ring system, e.g. tetracycline
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/03—Actinomadura

Definitions

  • This invention relates to new tetracycline antibiotics, 7-chloro-8-methoxytetracycline and its 4ahydroxy and 2'-N-methyl derivatives, isolated from and produced by fermentation of Actinomadura brunnea var. antibiotica var. nov. ATCC 53108, Actinomadura brunnea var. antibiotica ATCC 53180, Actinomadura brunnea ATCC 39216, and Dactylosporangium vescum ATCC 39499, under controlled conditions using biologically pure cultures of these new microorganisms.
  • the present invention relates to biologically pure cultures of various microorganisms, in particular the microorganisms Actinomadura brunnea (a novel species) having the identifying characteristics of ATCC 39216, Actinomadura brunnea var. antibiotica var. nov. having the identifying characteristics of ATCC 53108, Actinomadura brunnea var. antibiotica var. nov. having the identifying characteristics of ATCC 53180, and Dactylosporangium vescum (a novel species) having the identifying characteristics of ATCC 39499; as well as mutants and variants of said microorganisms.
  • Each of said cultures is capable of producing a characteristic antibiotic or antibiotic complex in a recoverable guantity upon fermentation under aerobic conditions in an agueous medium containing assimilable sources of nitrogen and carbon.
  • Each of these antibiotics or antibiotic complexes is a further feature of the present invention.
  • strains of microorganisms produce antibiotics or antibiotic complexes as follows:
  • A. brunnea ATCC 39216 produces an antibiotic complex 81-47 containing the novel antibiotic 7-chloro-8methoxy-2'-N-methyltetracycline;
  • A. brunnea var. antibiotica var. nov. ATCC 53108 produces an antibiotic complex ES-119 containing the above-mentioned novel antibiotic 7-chloro-8-methoxy2'-N-methyl-tetracycline and also the novel antibiotic 7chloro-8-methoxytetracycline, which can be isolated substantially pure;
  • A. brunnea var. antibiotica var. nov. ATCC 53180 produces an antibiotic complex Tet. 7 containing the novel antibiotic 7-chloro-8-methoxytetracycline substantially free of 7-chloro-8-methoxy-2'-Nmethyltetracycline;
  • D. vescum ATCC 39499 produces the novel antibiotic 7273 complex comprising the novel antibiotic 7-chloro-4a-hydroxy-8-methoxytetracycline.
  • R is a hydrogen atom or a hydroxy group and R 1 is a hydrogen atom or a methyl group, with the proviso that at least one of R and R 1 is a hydrogen atom.
  • the invention also comprises pharmaceutically acceptable salts of tetracyclines of formula I.
  • the antibiotics and antibiotic complexes of this invention are produced by cultivating a strain of a microorganism hereinabove described in a pH- and temperature-controlled aqueous nutrient medium containing assimilable sources of nitrogen and carbon under aerobic conditions, until a composition of matter having substantial antibiotic activity and containing the antibiotic or complex of this invention is produced.
  • the present invention also provides a pharmaceutical composition comprising an antibiotically effective amount of a tetracycline of the formula I above or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.
  • the present invention provides a method of eliciting an antibiotic effect in a host, e.g., a mammal, having a susceptible infection, which comprises administering to said host an antibiotically effective amount of a tetracycline of the formula I above or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition defined above.
  • Figure 1 is the infrared spectrum of 7-chloro8-methoxytetracycline
  • Figure 2 is the H NMR spectrum of 7-chloro-8methoxytetracycline
  • Figure 3 is the infrared spectrum of 7-chloro8-methoxy-2'-N-methyltetracycline in KBr;
  • Figure 4 is the 1 H NMR spectrum of 7-chloro-8methoxy-2'-N-methyltetracycline in a mixture of acetonedg and methanol-d 4 ;
  • Figure 5 is the infrared spectirum of 7-chloro4a-hydroxy-8-methoxytetracycline in KBr;
  • Figure 6 is the 1 H NMR spectrum of 7-chloro-4ahydroxy-8-methoxytetracycline in dimethyl sulfoxide-d 6 ;
  • Figure 7 is the fully-decoupled proton 13 C-NMR spectrum of 7-chloro-4a-hydroxy-8-methoxytetracycline in dimethyl sulfoxide-d 6 ;
  • Figure 8 is the chemical ionization mass spectrum of 7-chloro-4a-hydroxy-8-methoxytetracyclipe.
  • One antibiotic complex of this invention is produced when the elaborating organism of the novel species Actinomadura brunnea, in particular a strain having the identifying characteristics of ATCC 39216, is grown in an appropriate nutrient medium.
  • This antibiotic complex may be isolated from the fermentation broth by solvent extraction and filtration, employing the following procedure:
  • This antibiotic complex is made up of at least two active components, one of which has been isolated and characterized as the novel 8-substituted tetracycline, 7chloro-8-methoxy-2'-N-methyltetracycline.
  • the active antibiotics can be isolated from this antibiotic complex (as the HCl salt) by chromatography using, for example, a Sephadex G-25 gel column.
  • the eluate (dilute aqueous HCl) from the column was monitored by determining the activity of each fraction against S. aureus and E. coli.
  • the desired active fractions were combined and lyophilized to give a light yellow powder which was crystallized from methylene chloride:hexane to give the novel 7-chloro-8-methoxy-2'N-methyletracycline.
  • a - indicates peaks buried under DMSO peak, but observed when spectrum was run in D 2 O/Dioxan.
  • 7-Chloro-8-methoxy-2'-N- methyltetracycline had a GMM of 33 against 23 gramnegative tetracycline-susceptible organism compared to a GMM of 2.3 for tetracycline.
  • the 23 gram-negative organisms included nine strains of E. coli, eight of Klebsiella, four of Enterobacter and two of Salmonella.
  • 7-Chloro-8-methoxy-2'-N-methyltetracycline had a GMM of 0.86 against nine Methicillin-resistant Staphylococci, a GMM of 0.48 against 54 Methicillin-susceptible organisms, a GMM of 0.15 against 25 Streptococci (including Groups A, B, C, G, S. pneumoniae, S. viridans, S. faecium and S. faecalis), and a GMM of 0.41 against 10 strains of Bacteroides fragilis (tested in Mueller-Rinton agar with
  • Antibiotic ES-119 is produced when the elaborating microorganism, Actinomadura brunnea var. antibiotica var. nov. having the identifying characteristics of ATCC 53108 is grown in an appropriate medium.
  • Antibiotic ES-119 may be isolated from the fermentation broth by employing the following procedures:
  • step (b) On a small scale, the separation in step (b) is normally accomplished by solvent extraction of said solution using one volume of an organic solvent (e.g. nbutanol saturated with water) each time for each volume of said solution.
  • organic solvent e.g. nbutanol saturated with water
  • step (b) On a larger (preparative) scale, column chromatography on a neutral resin (e.g.
  • XAD-2, -4 or -16 a neutral polystyrene resin available from Rohm and Haas, Philadelphia, PAKeluting with aqueous alcohol mixtures (e.g., 25.%, 50% methanol in water and 100% methanol) and an alcohol containing dilute aqueous mineral acid (e.g.., methanol:0.02N HCl) is normally employed to remove the bioloqically inactive organics and produce an eluate containing the antibiotic ES-119.
  • aqueous alcohol mixtures e.g., 25.%, 50% methanol in water and 100% methanol
  • dilute aqueous mineral acid e.g.., methanol:0.02N HCl
  • Antibiotic Tet. 7 is produced when the elaborating microorganism, Actinomadura brunnea var. antibiotica var. nov. having the identifying characteristics of ATCC 53180 is grown in an appropriate medium.
  • Antibiotic Tet. 7 may be isolated from the fermentation broth by employing the procedures described hereinabove in reference to antibiotic ES-119.
  • the antibiotic ES-119 is made up of at least two active (tetracycline) components (in about 60:40 ratio), so no relevant physicochemical data can be obtained for the antibiotic ES-119.
  • the major (60%) active component isolated from antibiotic ES-119 was identified as 7-chloro-8-methoxy2'-N-methyltetracycline, described above, and the other active component isolated was characterized as the novel 7-chloro-8-methoxy-tetracycline.
  • the active antibiotics including 7-chloro-8methoxytetracycline can be separated from antibiotic ES119 by chromatography using, for example, high performance reverse-phase liquid chromatography on C 18 columns e.g. u-Bondapak C 18 , or Sephadex G-25 gel column chromatography. For large scale preparation, use of a Sephadex G-25 gel column chromatography is preferred.
  • the eluate from the chromatography column was monitored by disc testing the activity of each fraction against B. subtilis, ATCC 6633 and E. coli, OLA 290R5.
  • the desired active fractions were pooled and lyophilized to give 7chloro-8-methoxytet ⁇ racycline and 7-chloro-8-methoxy-2'-Nmethyltetracycline.
  • the antibiotic Tet. 7 contains almost exclusively 7-chloro-8-methoxytetracycline substantially free (i.e., less than about 1%) of 7-chloro-8-methoxy-2'N-methyltetracycline. Purification of antibiotic Tet. 7 and isolation of 7-chloro-8-methoxytetracycline is performed by using procedures described hereinabove in reference to antibiotic ES-119.
  • antibiotic ES-119 and antibiotic Tet. 7 and the common component of each, 7- ⁇ hloro-8-methoxytetracycline, were tested in vitro and found to be active against a variety of gram-positive and gram-negative bacteria.
  • 7-Chloro-8methoxytetracycline had a GMM of 4.2 against 23 gramnegative (tetracycline-susceptible) organisms compared to a GMM of 2.3 for tetracycline and 33.0 for 7-chloro-8methoxy-2'-N-methyl-tetracycline.
  • the twenty-three gramnegative organisms included 9 strains of E. coli; 8 strains of Klebsiella; 4 strains of. Enterobacter and 2 strains of Salmonella.
  • 7-Chloro-8-methoxytetracycline exhibited a GMM of 0.21 against 9 Methicillin-resistant Staphylococci, a GMM of 0.14 against 54 Methicillin- suspectible Staphylococci organisms, a GMM of 0.06 against 25 Streptococci (including Groups A, B, C, G; S. pneumoniae, S. viridans, S. faecium and S. faecalis), and a GMM of 0.16 against 10 strains of Bacteroides fragilis (tested in Mueller-Hinton agar with 5% sheep blood).
  • Antibiotic 7273 complex is produced when the elaborating organism of the novel species Dactylosporangium vescum, in particular a strain having the identifying characteristics of ATCC 39499, is grown in an appropriate nutrient medium.
  • Antibiotic 7273 complex may be isolated from the fermentation broth by solvent extraction and filtration, and by employing the following procedure:
  • the antibiotic 7273 complex is made up of at least two active components, one of which has been isolated and characterized as the novel 4a-,8-substituted chlortetracycline of this invention, 7-chloro-4a-hydroxy8-methoxytetracycline.
  • the active antibiotics including 7-chloro-4ahydroxy-8-methoxytetracycline can be isolated from the antibiotic 7273 complex (as the HCl salt) by chromatography using, for example, a Sephadex G-25 gel column.
  • the eluate (dilute aqueous HCl) from the column was monitored by determining the activity of each fraction against S. aureus and E. coli.
  • the desired active fractions were combined and lyophilized to. give 7-chloro4a-hydroxy-8-methoxytetracycline as a light yellow powder.
  • the antibiotic 7273 complex containing at least two biologically active components is active against a variety of gram-positive and gram-negative bacteria when tested in vitro.
  • 7-chloro4a-hydroxy-8-methoxytetracycline is active against a variety of gram-positive and gram-negative bacteria when tested in vitro.
  • 7-chloro-4a-hydroxy-8-methoxytetracycline showed activity against 91 gram-positive tetracycline-susceptible organisms with a Geometric Mean Minimum Inhibitory Concentration (GMM, mcg/mL) of 1.77 which is similar to the GMM for tetracycline (0.46).
  • GMM Geometric Mean Minimum Inhibitory Concentration
  • 7-Chloro-4a-hydroxy-8methoxytetracycline had a GMM of 7.8 against 23 gramnegative tetracycline-susceptible organisms compared to a GMM of 2.3 for tetracycline.
  • the 23 gram-negative organisms included nine strains of E. coli, eight of Klebsiella, four of Enterobacter and two of Salmonella.
  • 7-Chloro-4a-hydroxy-8-methoxytetracycline had a GMM of 5.4 against nine Methicillin-resistant Staphylococci, a GMM of 2.83 against 54 Methicillin-susceptible organisms, a GMM of 0.45 against 25 Streptococci (including Groups A, B, C, G; S. pneumoniae, S. viridans, S. faecium and S. faecalis), and a GMM of 0.57 against 10 strains of Bacteroides fragilis (tested in Mueller-Hinton agar with 5% sheep blood).
  • 7-Chloro-4a-hydroxy-8-methoxytetracycline had a GMM of 58.7 against 8 chlortetracycline-resistant (MIC>8) strains including 7 of Staphylococcus and 1 of Streptococcus compared to a GMM of 20.7 for chlortetracycline.
  • 7-Chloro-4a-hydroxy-8methoxytetracycline had a GMM of 3.0 against 22 chlortetracycline-susceptible strains including 8 of E. coli, 2 of Enterobacter, 9 of Klebsiella, 1 each of Salmonella, Serratia and Shigella, compared to a GMM of 3.3 for chlortetracycline.
  • 7-Chloro-4a-hydroxy-8methoxytetracycline had about the same potency as chlortetracycline against chlortetracycline-susceptible gram-negative strains but was about 8-fold less potent than chlortetracycline against chlortetracyclinesusceptible gram-positive strains.
  • MHB Mueller-Hinton Broth
  • A, B, D as identified in Tables IIA and IIIA
  • TC tetracycline
  • DOX doxycycline
  • MIN minocycline.
  • novel tetracycline antibiotics are substantially non-toxic at therapeutic doses; indeed, their low toxicit.y is similar to that of 7chlorotetracycline, as is shown in the following table where the compounds are again identified as in. Tables IIA and IIIA:
  • mice LD 50 s in mice, i.v., mg/kg:
  • the present invention comprises also a method of eliciting an antibacterial effect in a host, e.g., a warm-blooded mammal such as a human being having a susceptible bacterial infection which comprises admini-stering to said host an antibiotically effective amount of 7-chloro-8-methoxytetracycline, 7-chloro-4ahydroxy-8-methoxytetracycline or 7-chloro-8-methoxy-2'-Nmethyltetracycline, or of a pharmaceutically acceptable salt thereof.
  • eliciting is meant treating or preventing susceptible bacterial infection.
  • compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective quantity of 7-chloro-8-methoxytetracycline, 7chloro-4a-hydroxy-8-methoxytetracycline or 7-chloro-8methoxy-2'-N-methyltetracycline, or of a pharmaceutically acceptable salt thereof, and are also features of the invention.
  • the preferred pharmaceutically acceptable salts are the acid addition salts.
  • Pharmaceutically acceptable acid addition salts of 7-chloro-8-methoxytetracycline, 7chloro-4a-hydroxy-8-methoxytetracycline and 7-chloro-8methoxy-2'-N-methyltetracycline are those formed from strong acids containing pharmaceutically acceptable anions, such as -the hydrochloride, hydrobromide, hydrogen sulfate and tricfiloroacetate.
  • Acid addition salts may also be formed with carboxylic acids having 2-18 carbon atoms such as aliphatic, cycloaliphatic, aromatic and heterocyclic carboxylic acids, including dicarboxylic acids.
  • Exemplary of such acids are acetic, propionic, stearic, tartaric, maleic, cyclopropylcarboxylic, cyclopentylcarboxylic, adamantoic, furoic, nicotinic, thenoic, picolinic, benzoic, phenylacetic and the like.
  • the antibiotics of. this invention may be combined with any suitable pharmaceutical carrier and administered orally, parenterally or topically in a variety of formulations.
  • the antibiotic of this invention may be compounded in the form of tablets, capsules, elixirs or the like. Tablets and capsules may contain such excipients as starch or lactose; liquid oral forms may contain coloring or flavoring agents.
  • Topical preparations may be in the form of creams, hydrophobic and hydrophilic ointments, or aqueous, non-aqueous or emulsion-type lotions. Typical carriers for such formulations are water, oils, greases, polyesters and polyols.
  • Parenteral formulations, e.g., injectable dosage forms are usually liquids such as solutions or suspensions, and typical carriers are distilled water and saline solution.
  • the dose to be administered in any particular dosage form will be determined by the attending clinician after consideration of various factors, such as the age and condition of the animal species being treated, the susceptibility of the infecting organism to the antibiotic, and the stage and severity of the infection.
  • the oral dosage administered is from about 1.0 mg to about 25 mg per kilogram of body weight, preferably about 5 mg to about 10 mg per kilogram, per day, in single or divided doses.
  • the concentration of the novel antibiotic in topical formulations is from about 1% to about 5%, preferably about 1% to about 3%.
  • parenteral dosage administered to humans is from about 100 mg to about 2000 mg per day, in single or divided doses, with about 500 mg to about 1000 mg being preferred.
  • the microorganism used for the production of the antibiotic complex containing 7-chloro-8-methoxy'-2'N-methyltetracycline is a biologically pure culture of Actinomadura brunnea (ATCC 39216). Actinomadura brunnea is classified as a novel species.
  • a culture of this microorganism has been deposited with the Northern Utilization and Research Division, Agriculture Research Service, U.S. Department of Agriculture in Peoria, Illinois where it has been assigned accession number NRRL 15216. Subcultures of Actinomadura brunnea NRRL 15216 are available to the public without restriction from the aforementioned agency. A culture of this microorganism has been made a' part of the collection of the American Type Culture Collection (ATCC) in Rockville, Maryland where it has been assigned accession number ATCC 39216. Subcultures of Actinomadura brunnea ATCC 39216 are available to the public without restriction.
  • ATCC American Type Culture Collection
  • the microorganism was isolated from a sample of soil collected near Phoenix, Arizona. It has been characterized and found to have the microscopic, macroscopic, and whole cell hydrolysis properties of the genus Actinomadura. Description of the Producing Strains The taxonomic methods used herein for Tables IV through XVII are those cited by R. E. Gordon and V. Blanchard, "Some criteria for the recognition of Nocardia madura", J. Gen. Microbiol., 45, pp 355-364 (1966); by
  • the microorganism of this invention is categorized as a species of the genus Actinomadura. Of the named, deposited strains of this genus listed in Table VIII, the microorganism of this invention shares vegetative mycelia pigmentation with the species A. helvata, A. flava and A. melliaura.
  • the microorganism of this invention may be distinguished from the above listed species of Actinomadura by comparisons of the characteristics listed in Table VIII.
  • A. brunnea differs from A. helvata (1) in morphology of the spore-bearing hyphae: A. helvata forms short, coiled chains of spores as side branches along the length of the aerial mycelium; A. brunnea forms abundant long, branching aerial mycelia which fragment into long, straight to flexous chains of more than 20 elliptical spores; (2) in the utilization of mannitol: A . helvata utilizes mannitol, but A. brunnea does not; and (3) in antibiotic production: A. brunnea produces the novel tetracycline antibiotic 7-chloro-8-methoxy-2'-Nmethyltetracycline, but A. helvata exhibits no antibiotic activity.
  • A. brunnea differs from A. flava (1) in the formation of aerial mycelia: A. flava rarely forms aerial mycelia when compared to A. brunnea; and (2) in the utilization of mannitol: A. flava utilizes mannitol while A. brunnea does not.
  • A. brunnea differs from A. melliaura (1) in morphology: A. melliaura forms short to long spore chains, and the terminal end of the aerial mycelium forks into two sporophores bearing a straight chain of spores; (2) in the utilization of methyl- ⁇ -D-glucoside and mannitol by A. melliaura but not by A. brunnea; and (3) in the type of antibiotic production: A. melliaura produces a fused indole aminoglycoside while A. brunnea produces the novel tetracycline antibiotic 7-chloro-8methoxy-2'-N-methyltetracycline.
  • this microorganism On the basis of these morphological, physiological and culture characteristics, as well as the production of the novel 7-chloro-8-methoxy-2'-N-methyltetracycline this microorganism is considered to represent a distinct, new species of the genus Actinomadura. It is proposed that the microorganism be designated Actinomadura brunnea, Horan. and Brodsky sp. nov. The species name selected refers to the brown vegetative mycelial pigments formed.
  • An antibiotic complex of this invention is produced when the elaborating microorganism, Actinomadura brunnea, is grown in an aqueous nutrient medium under submerged aerobic conditions at a temperature of about 27°C to 40oC, preferably at from 27°C to 35°C, and at a pH of from about 6.5 to 8.0 with agitation until substantial antibiotic activity is imparted to the medium. Temperature studies indicate that the organism grows rapidly at 30°C. Therefore, the fermentation is preferably conducted employing a single temperature pattern of 30°C for the first 24 hours as well as for the period 24 to 96 hours. The fermentation is generally conducted to from 3 to 7 days, preferably for 4 days.
  • samples of the medium may be assayed every 24 hours for antibiotic content by bioassay of the whole broth against S. aureus ATCC 209P (pH 7.0) and E. coli ATCC 10536 (pH 8.0).
  • the growth of the organism (packed cell volume), pH and dissolved oxygen levels can be determined either intermittently or continuously.
  • Any suitable nutrient medium containing a source of carbon, for example an assimilable carbohydrate, and a source of nitrogen, for example an assimilable nitrogenous or proteinaceous material, may be used.
  • the medium employed for the fermentation may for example contain NZ-Amine A (an enzymatic hydrolysate of casein) and soluble starch as the major sources of nitrogen and carbon.
  • NZ-Amine A an enzymatic hydrolysate of casein
  • soluble starch as the major sources of nitrogen and carbon.
  • the fermentation is generally conducted by initially sterilizing the fermentation medium prior to the addition of the inoculum.
  • the pH of the fermentation medium is generally maintained at from 6.5 to 8.0, a pH of from 6.5 to 7.5 being preferred. Prior to sterilization the pH of the medium is usually adjusted to 6.7, and prior to inoculation the pH is usually adjusted to 7.5.
  • the fermentation is initiated by addition of the inoculum to the broth.
  • inoculum volume is 5% of total broth volume.
  • the inoculum is prepared by addition of a sample of the frozen whole broth to an appropriate medium.
  • a particularly preferred medium comprises beef extract, 0.3%; tryptone, 0.5; dextrose, 0.1%; potato starch, 2.4%; yeast extract, 0.5%; and calcium carbonate, 0.2%.
  • the pH of the inoculum medium is adjusted to 7.5 prior to sterilization.
  • the inoculum stage of the fermentation usually requires from 24 to 120 hours, preferably 1 to 2 days, and is generally conducted at about 30°C..
  • the microorganism used for the production of antibiotic ES-119 is a biologically pure culture of Actinomadura brunnea var. antibiotica var. nov.
  • a culture of this microorganism has been made a part of the collection of the American Type Culture Collection (ATCC) in Rockville, Maryland where it has been assigned accession number ATCC 53108. Subcultures of Actinomadura brunnea var. antibiotica ATCC 53108 are available to the public without restriction.
  • ATCC American Type Culture Collection
  • the microorganism used for the production of antibiotic ES-119 was produced by exposure of a spontaneous mutant of a' culture of Actinomadura brunnea ATCC 39216 to a mutagenic agent, e.g., N-methyl-Nnitroso-N'-nitro-guanidine. Representatives of the mutated population of Actinomadura brunnea were plated and allowed to grow. Two replicate agar plates (such as starch yeast agar plates) were prepared and the mutated populations were allowed to grow until single colonies were observed. Usually, after about four days single colonies were observed, and thereafter one of the replicate plates was directly overlaid with agar containing an appropriate gram-negative indicating organism, e.g., E.
  • the desired mutant colonies of Actinomadura brunnea var. antibiotica var. nov. ATCC 53108 were recognized and recovered from the unoverlaid replicate plate by comparison with a clear zone of inhibition they exhibited against the gramnegative indicator strain on the overlaid plate.
  • Antibiotic ES-119 produced from A. brunnea var. antibiotica ATCC 53108 comprises about a 60:40 mixture of 7-chloro-8-methoxy-2'-N-methyltetracycline and 7-chloro8-methoxytetracycline; the spontaneous mutant of the parent A. brunnea ATCC 39216 produced about a 80:20 mixture of the two compounds.
  • Actinomadura brunnea var. antibiotica var. nov. has been characterized and found to have the microscopic, macroscopic, and whole cell hydrolysis properties of the genus Actinomadura.
  • the microorganism used for the production of antibiotic Tet. 7 is a biologically pure culture ofActinomadura brunnea var. antibiotica var. nov.
  • a culture of this microorganism has been made a part of the collection of the American Type Culture Collection (ATCC) in Rockville, Maryland where it has been assigned accession number ATCC 53180. Subcultures of Actinomadura brunnea var. antibiotica ATCC 53180 are available to the public without restriction.
  • the microorganism used for production of antibiotic Tet. 7 was produced by exposure of a high level streptomycin-resistant mutant isolated from a culture of Actinomadura brunnea ATCC 39216 to a mutagenic agent, e.g., N-methyl-N-nitroso-N'-nitro-guanidine.
  • Actinomadura brunnea var antibiotica ATCC 53108 and ATCC 53180 occurs from 27° to 45 °C on yeast-dextrose agar. At 50°C slight growth occurs and the strains survive for 8 hours. No growth occurs at 10°C. Optimum growth is observed at about 35°C. a) Observations made after 14-21 days at 30°C.
  • the mutant strains (ATCC 53108 and ATCC 53180) and the parent microorganism (ATCC 39216) produce 7-chloro-8-methoxy-2'N-methyltetracycline.
  • these microorganisms On the basis of these morphological, physiological and culture characteristics, as well as the production of the novel 7-chloro-8-methoxy-tetracycline these microorganisms have the identifying characteristics of ATCC 53108 and 53180, and are induced mutational variants of A. brunnea ATCC 39216. Thus, these microorganisms, ATCC 53108 and ATCC 53180, are considered new varieties of A. brunnea ATCC 39216.
  • Antibiotic ES-119 and antibiotic Tet. 7 are produced when the elaborating microorganisms, Actinomadura brunnea var. antibiotica ATCC 53108 and Actinomadura brunnea var. antibiotica ATCC 53180 respectively, are grown in an aqueous nutrient medium under submerged aerobic conditions at a temperature of about 27°C to 45°C, preferably at from 27°C to 35°C, and at a pH of from about 6.5 to 8.0 with agitation until substantial antibiotic activity is imparted to the medium. Temperature studies indicate that the organism grows more rapidly at 35°C than at 30oC. However, antibiotic production is greater if the temperature is lowered to 30°C at the end of the exponential growth period at 35°.
  • fermentation may also be conducted by employing a two-temperature pattern of 35°C for the first 24 hours and 30oC for the period 24 to 96 hours.
  • the fermentation may be more conveniently conducted by employing a single temperature pattern of 30°C for the first 24 hours as well as for the period 24 to 96 hours.
  • the fermentation is generally conducted for from 3 to 7 days, preferably for 4 days.
  • samples of the medium may be assayed every 24 hours (starting at 18 hours) for antibiotic content by bioassay of the whole broth against S. aureus, ATCC 209P (PH 8.0) and E. coli, OLA 290R5 (pH 8.0).
  • the growth of the organisms packed cell volume
  • pH and dissolved oxygen levels may be determined either intermittently or continuously.
  • Any suitable nutrient medium containing a source of carbon, for example an assimilable carbohydrate, and a source of nitrogen, for example an assimilable nitrogenous or proteinaceous medium, may be used.
  • the medium employed for the inoculum stages of the fermentation may for example contain beef and yeast extracts, cerelose and soluble starch as the major sources of nitrogen and carbon.
  • Replacement of the beef yeast extract by NZ-Amine A (an enzymatic hydrolysate of casein) and adjusting the amounts of cerelose and soluble starch and addition of cobalt chloride produces a medium preferred for the fermentation stage especially for large scale fermentations.
  • the microorganism ATCC 53108 produces antibiotic ES-119 containing a 60:40 mixture of 7-chloro-8-methoxy-2'-Nmethyltetracycline and 7-chloro-8-methoxytetracycline, and ATCC 53180 produced antibiotic Tet.
  • the foregoing media are exemplary of the nutrients utilized by Actinomadura brunnea var. antibiotica ATCC 53108 and Actinomadura brunnea var antibiotica ATCC 53180 to produce antibiotic ES-119 and antibiotic Tet. 7, respectively.
  • a wfde range of nutrients obtained from a number of suppliers may be substituted for the foregoing, and that generally good growth and antibiotic production can be obtained, such nutrients being the functional equivalent of those set forth herein.
  • the fermentation is generally conducted by initially sterilizing the fermentation medium prior to the addition of the inoculum.
  • the pH of the fermentation medium is generally maintained at from 6.5 to 8.0, preferably 6.5 to 7.5. Prior to sterilization, the pH of the medium is usually adjusted to 6.7.
  • the fermentation is initiated by addition of the inoculum to the broth.
  • the inoculum volume is 5% of total broth.
  • the inoculum is prepared by addition of a sample of the frozen whole broth to an appropriate medium.
  • a particularly preferred medium for the 1st and 2nd inoculum stages for antibiotic ES-119 and antibiotic Tet. 7 comprises 3 g of beef extract, 5 g of tryptone, 5 g of yeast extract, 1 g of cerelose, 24 g of potato starch, 2 g of calcium carbonate and optionally 1 mL of AF-1 antifoam (Antifoam B available from Dow Corning Corp., Midland, MI 48641).
  • Fermentation is completed by addition of a portion (generally about 5 volume %) of the inoculum (usually the 2nd stage inoculum) to an appropriate medium.
  • a particularly preferred medium comprises (per liter): 5 g of yeast extract, 5 g of NZ-amine A; 20 g soluble starch, 10 g of cerelose, 1 ml of 0.001M Cobalt (II) chloride, 0.4 g of calcium carbonate and optionally 1 ml of a surfactant, e.g., AF-1 Antifoam.
  • the pH of the inoculum medium is adjusted to 7.5 prior to sterilization.
  • the inoculum stage of the fermentation usually reguires from 24 to 120 hours, preferably 1 to 2 days, and is generally conducted at about 30°C.
  • the fermentation stage usually reguires 90 to 165 hours, preferably 90 hours, and is generally conducted at 30°C, with agitation (e.g. 300-350 rpm) and under an air flow rate of, for example, 3.5 L/min.
  • the microorganism used for the production of antibiotic 7273 complex is a biologically pure culture of Dactylosporangium vescum (ATCC 39499). Dactylosporangium vescum is classified as a novel species.
  • a culture of this microorganism has been made a part of the collection of the American Type Culture Collection (ATCC) in Rockville, Maryland where it has been assigned accession number ATCC 39499. Subcultures of Dactylosporangium vescum ATCC 39499 are available to the public without restriction.
  • ATCC American Type Culture Collection
  • the microorganism was isolated from a sample of soil collected in the Kasie Valley of Zambia. It has been characterized and found to have the microscopic. macroscopic, and whole cell hydrolysis properties of the genus Dactylosporangium.
  • Dactylosporangium vescum ATCC 39499 A comparison of the characteristics of Dactylosporangium vescum ATCC 39499 with those of other species of Dactylosporangium is listed in Table XVII.
  • Whole cell analysis of the microorganism Dactylosporangium vescum ATCC 39499 found hydroxydiaminopimelic acid as the characteristic cell wall amino acid, and arabinose and xylose as the characteristic whole cell sugars.
  • Physiologic characteristics differentiating D . vescum ATCC 39499 from the described species of Dactylosporangium are presented in Table XVII. None of the species of Dactylosporangium share with 13. vescum the combination of tan to yellow vegetative mycelial pigments, abundant and rapid formation of sporangioles, formation of yellow diffusible pigments, utilization of glycerol and rhamnose, ability to grow in the presence of 3% NaCl and the production of the novel 7-chloro-4ahydroxy-8-methoxytetracycline.
  • Strain ATCC 39499 is therefore considered to be a distinct, new species of Dactylos-porangium designated D. vescum (Theimann, Pagani and Beretta) Horan and Brodsky sp. nov., and this strain is the type strain of the new species.
  • D. vescum is the type strain and, should another strain be found, the type strain would also be the type subspecies.
  • Antibiotic 7273 complex is produced when the elaborating microorganism, Dactylosporangium vescum, is grown in an aqueous nutrient medium under submerged aerobic conditions at a temperature of about 27°C to 40°C, preferably at from 27°C to 35°C, and at a pH of from about 6.5 to 8.0 with agitation until substantial antibiotic activity is imparted to the medium. Temperature studies indicate that the organism grows rapidly at 30°C. Therefore, the fermentation is preferably conducted employing a single temperature pattern of 30°C for the first 24 hours as well as for the period 24 to 96 hours. The fermentation is generally conducted for from 65 to 96 hours, preferably for 66 hours.
  • samples of the medium may be assayed every 24 hours (starting at 48 hrs.) for antibiotic content by bioassay of the whole broth against S. aureus ATCC 209P (pH 8.0) and E. coli ATCC 10536 (pH 8.0).
  • the growth of the organism (packed cell volume), pH and dissolved oxygen levels are determined either intermittently or continuously.
  • Any suitable nutrient medium containing a source of carbon, for example an assimilable carbohydrate, and a source of nitrogen, for example an assimilable nitrogenous or proteinaceous material, may be used.
  • the medium employed for the fermentation may for example contain NZ-Amine A (an enzymatic hydrolysate of casein) and soluble starch as the major sources of nitrogen and carbon.
  • NZ-Amine A an enzymatic hydrolysate of casein
  • soluble starch as the major sources of nitrogen and carbon.
  • the foregoing media are exemplary of the nutrients utilized by Dactylosporangium vescum to produce antibiotic 7273 complex.
  • Dactylosporangium vescum to produce antibiotic 7273 complex.
  • a wide range of nutrients obtained from a number of suppliers may be substituted for the foregoing, and that generally good growth and antibiotic production can be obtained, such nutrients being the functional equivalent of those set forth herein.
  • the fermentation is generally conducted by initially sterilizing the fermentation medium prior to the addition of the inoculum.
  • the pH of the fermentation medium is generally maintained at from 6.5 to 8.0, a pH of from 6.5 to 7.5 being preferred. Prior to sterilization the pH of the medium is usually adjusted to 6.7, and prior to inoculation the pH is usually adjusted to 7.0.
  • the fermentation is initiated by addition of the inoculum to the broth.
  • inoculum volume is 2.5% of total broth volume.
  • the inoculum is prepared by addition of a sample of the frozen .whole broth to an appropriate medium.
  • a particularly preferred medium comprises beef extract, 0.3%; tryptone, 0.5; cerelose, 0.1%; potato starch, 2.4%; yeast extract, 0.5%; and calcium carbonate, 0.2%.
  • the pH of the inoculum medium is adjusted to 7.5 prior to sterilization.
  • the inoculum stage of the fermentation usually requires from 24 to 120 hours, preferably 1 to 2 days, and is generally conducted at about 30°C with agitation. Agitation and a forced air flow, generally about 3.5 L/min., are employed during the fermentation.
  • Example 2 Separation of Antibiotic Complex Isolation of 7-chloro-8-methoxy-2'-N-methyltetracycline Dissolve a 100 mg portion of the crude antibiotic complex of Example lC in 10 mL of 0.02N HCl. Adsorb the solution so formed on a 2.54 cm x 63.5 cm gel column containing 300 mL of Sephadex G-25 filtration gel (medium; dry particles size 50-150 mm). (Sephadex G-25 is a cross-linked dextran available from Pharmacia Fine Chemicals, Inc. Piscataway, N.J.) Elute the column with 0.02N HCl at a flow rate of about 3.0 mL per minute. Monitor the activity of each fraction (10 mL) against S.
  • Sephadex G-25 is a cross-linked dextran available from Pharmacia Fine Chemicals, Inc. Piscataway, N.J.
  • aureus ATCC 209P (pH 7.0) and E. coli ATCC 10536 (pH 8.0) using a disc diffusion assay. Spot the active fraction on thin layer chromatography plates developed in a 2:2:1 (v/v/v) chloroform:methanol:pH 3.5 acetate buffer. Detect the antibiotic components by bioautography against both S. aureus and E. coli.
  • AF-1 Antifoam is Dow Corning Antifoam B available from Dow Corning Corp. Adjust the pH of the germination medium to 7.5. Sterilize the medium and after cooling add 0.5 mL of a frozen whole broth sample from a previously prepared inoculum of A. brunnea var antibiotica ATCC 53108 or of
  • step B Adjust the pH of the whole fermentation broth of step B to 2 with sulfuric acid and remove the insoluble mycelia by centrifugation. Extract 2 L of centrifugate at pH 2 twice with 2 L of water-saturated rbutanol. Combine the n-butanol solutions and remove the solvent by vacuum stripping at 40°C to give a residue. Dissolve the residue in 16 mL of water and pass the aqueous solution of antibiotic ES-119 through a 0.22 micron filter.
  • XAD-16 resin a neutral polystyrene resin available from Rohm & Haas, Philadelphia, PA
  • Example 3(C) Place the filtrate from Example 3(C) on a 0.78 cm x 30 cm HPLC column containing y Bondapak C-18 (an 18 carbon chain attached to a silica support available from Waters Associates, Inc., Framingham, MA 01701).
  • a mobile phase consisting of a linear, 15 min gradient from 100% buffer A (30:60:10 (v/v/v) methanol :water :0.2M phosphate/ phosphoric acid buffer, pH 2.5) to 100% buffer B (50:20:20:10 (v/v/v/v) methanol:acetonitrile:water : 0.2M phosphate/phosphoric acid buffer, pH 2.5) at a flow rate of 3.5 mL per min. Collect fractions every 20 sec.
  • Example 3(C) Monitor the antibiotic activity of each fraction by disc testing each, fraction against a gramnegative organism, e.g. E. coli, and a gram-positive organism, e.g. B. subtilis. Pool the fractions with activity against E. coli, and evaporate the solvent to provide a solid residue. Repeat the HPLC procedure to provide pure 7-chloro-8-methoxytetracycline having the physico-chemical data presented in Table II.
  • Example 3 Add 5 volume % of a frozen whole broth of A. brunnea var antibiotica ATCC 53108 or A. brunnea var antibiotica ATCC 53180 to 70 mL of the sterilized germination medium (pH 7.5) of Example 3 (A) (1) in a 300 mL Erlenmeyer shake flask. Incubate at 30°C for 48 hrs. with continual shaking at 300 rpm.
  • Samples can also be monitored by bioautography after chromatography on TLC plates. Based on the whole broth analysis, both the antibiotic ES-119 and antibiotic Tet. 7 have comparable activity which reaches a maximum in both fermentations at 90 hours.
  • the antibiotic ES-119 contains about 10 mg/L of 7-chloro-8methoxytetracycline and about 15 mg/L of 7-chloro-8methoxy-2'-N-methyltetracycline.
  • the antibiotic Tet. 7 contains somewhat less of 7-chloro-8-methoxytetracycline than found in antibiotic ES-119. No significant quantity of 7-chloro-8-methoxy-2'-N-methyltetracycline was detected (e.g., less than about 1%) by TLC against a known sample in antibiotic Tet. 7.
  • Per vial 7-chloro-8-methoxytetracycline, 7chloro-4a-hydroxy-8-methoxytetracy ⁇ line or 7-chloro-8methoxy-2'-N-methyltetracycline (hereinafter "drug") as a sterile powder.
  • Unit dosages may be 100 mg, 200 mg, 500 mg, 1 g and 2 g.
  • Priority Country US (81) Designated States: AT (European patent), BE (European tent), CH (European patent), DE (European patent), D

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Dérivés de 7-chlorotétracycline utilisés en tant que nouveaux antibiotiques pouvant être obtenus par cultures de souches d'Actinomadura brunnea et Dactylosporangium vescum. Ces dérivés sont actifs contre les aérobies gram-positives et gram-négatives.
EP86905061A 1985-08-08 1986-08-06 Antibiotiques a base de tetracyclines, leur preparation, compositions antibiotiques les contenant et microorganismes utiles pour leur preparatioin Withdrawn EP0232373A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US76374085A 1985-08-08 1985-08-08
US76374285A 1985-08-08 1985-08-08
US763740 1985-08-08
US763742 1985-08-08
US06/764,275 US4752605A (en) 1985-08-09 1985-08-09 7-chloro-4a-hydroxy-8-methoxytetracycline, antibiotic compositions containing them and a method of using
US764275 1985-08-09

Publications (1)

Publication Number Publication Date
EP0232373A1 true EP0232373A1 (fr) 1987-08-19

Family

ID=27419573

Family Applications (1)

Application Number Title Priority Date Filing Date
EP86905061A Withdrawn EP0232373A1 (fr) 1985-08-08 1986-08-06 Antibiotiques a base de tetracyclines, leur preparation, compositions antibiotiques les contenant et microorganismes utiles pour leur preparatioin

Country Status (3)

Country Link
EP (1) EP0232373A1 (fr)
DK (1) DK176787A (fr)
WO (1) WO1987000832A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5024839A (en) * 1987-12-24 1991-06-18 E. R. Squibb & Sons, Inc. Dactylocycline A and dactylocycline B

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL111166C (fr) * 1957-04-05
FR1553651A (fr) * 1962-11-05 1969-01-17
US3360559A (en) * 1964-11-30 1967-12-26 American Cyanamid Co Substituted 1, 3, 4, 10, 11, 12-hexahydroxynaphthacene-2-carboxamides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8700832A2 *

Also Published As

Publication number Publication date
DK176787A (da) 1987-06-09
WO1987000832A2 (fr) 1987-02-12
WO1987000832A3 (fr) 1987-06-18
DK176787D0 (da) 1987-04-07

Similar Documents

Publication Publication Date Title
US4559301A (en) Process for preparing macrocin derivatives
US5286631A (en) Process for producing the A10255 complex and corresponding microorganism
US4252898A (en) Antibiotics A6888C and A6888X
US4705688A (en) Antibiotic LL-E19020 α and β
EP0274873B1 (fr) Antibiotique A10255 complexe et facteurs, procédé, micro-organismes pour sa fabrication
JPH0774228B2 (ja) 新規抗生物質
US4597968A (en) Antibiotic 13-384 complex from Micromonospora carbonacea var africana
CA1225053A (fr) Methodes et composes pour le traitement des infections a protozoaires a l'aide d'un nouvel antibiotique
US4735903A (en) Micromonospora carbonacea var africana
US4293546A (en) Anthracycline antibiotics produced by Streptosporangium fragilis Shearer sp. nov. ATCC 31519
US4752605A (en) 7-chloro-4a-hydroxy-8-methoxytetracycline, antibiotic compositions containing them and a method of using
US4358584A (en) Antibiotics A6888C and A6888X
US5427941A (en) Actinomadura brunnea var. antibiotica strains
EP0232373A1 (fr) Antibiotiques a base de tetracyclines, leur preparation, compositions antibiotiques les contenant et microorganismes utiles pour leur preparatioin
CA1194439A (fr) Production d'erythromycine d et de ses esters par fermentation
EP0121328B1 (fr) Dérivés de macrolides
EP0376609B1 (fr) Antibiotique A80915 et procédé de préparation
JP3830964B2 (ja) 海洋放線菌から単離した新規なチオデプシペプチド
US4859598A (en) Antibiotic LL-D42067β
EP0109750B1 (fr) Nargénicine C1
US4774184A (en) Culture and process for producing antibiotic LL-D42067alpha
US4628046A (en) Antibiotic LL-C23201δ
US5183758A (en) Production of 7-chloro-3-methoxy-2'-N-methyltetracycline with Actinomadura brunnea
US4656258A (en) Macrocin derivatives
US4876273A (en) Antibotic A80577 and process for its production

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

17P Request for examination filed

Effective date: 19870306

A5 Separate publication of the ep or int. search report
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19890301

RIN1 Information on inventor provided before grant (corrected)

Inventor name: VAUGHAN, RICHARD, WILLIS

Inventor name: KALYANPOR, MANOHAR, GOPAL

Inventor name: WAITZ, JAY, ALLAN

Inventor name: RYAN, MICHAEL, JOSEPH

Inventor name: MARQUEZ, JOSEPH, ANTHONY

Inventor name: MUNAYYER, HANAN, KARAMAN

Inventor name: MILLER, GEORGE, HENRY

Inventor name: SMITH, ELIZABETH, BARGMANN

Inventor name: HORAN, ANN, CAMILLE

Inventor name: PATEL, MAHESH, GORDHANDAS