WO1987000832A2 - Tetracycline antibiotics, their preparation, antibiotic compositions containing them, and microorganisms useful in their preparation - Google Patents

Tetracycline antibiotics, their preparation, antibiotic compositions containing them, and microorganisms useful in their preparation Download PDF

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WO1987000832A2
WO1987000832A2 PCT/US1986/001587 US8601587W WO8700832A2 WO 1987000832 A2 WO1987000832 A2 WO 1987000832A2 US 8601587 W US8601587 W US 8601587W WO 8700832 A2 WO8700832 A2 WO 8700832A2
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antibiotic
chloro
atcc
var
brunnea
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PCT/US1986/001587
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French (fr)
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WO1987000832A3 (en
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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
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Schering Corporation
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Priority claimed from US06/764,275 external-priority patent/US4752605A/en
Application filed by Schering Corporation filed Critical Schering Corporation
Publication of WO1987000832A2 publication Critical patent/WO1987000832A2/en
Priority to DK176787A priority Critical patent/DK176787A/en
Publication of WO1987000832A3 publication Critical patent/WO1987000832A3/en

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    • 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
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    • 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

  • 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 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.
  • 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).
  • 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 concentration of the novel antibiotic in topical formulations is from about 1% to about 5%, preferably about 1% to about 3%.
  • 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. 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.
  • 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 microorganism used for the production of antibiotic ES-119 is a biologically pure culture of Actinomadura brunnea var. antibiotica var. nov.
  • the microorganism used for the production of antibiotic Tet. 7 is a biologically pure culture ofActinomadura brunnea var. antibiotica var. nov.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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) 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.
  • 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.

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Abstract

7-Chlorotetracycline derivatives as novel antibiotics preparable by cultivation of strains of Actinomadura brunnea and Dactylosporangium vescum. They are active against gram-positive and gram-negative aerobes.

Description

"TETRACYCLINE ANTIBIOTICS, THEIR PREPARATION,
ANTIBIOTIC COMPOSITIONS CONTAINING THEM, AND
MICROORGANISMS USEFUL IN THEIR PREPARATION"
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.
The above-identified 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.
All these tetracycline derivatives can together be represented by the general formula
Figure imgf000004_0001
wherein R is a hydrogen atom or a hydroxy group and R1 is a hydrogen atom or a methyl group, with the proviso that at least one of R and R1 is a hydrogen atom. The invention also comprises pharmaceutically acceptable salts of tetracyclines of formula I.
For completeness, the systematic names of these tetracyclines will now be given, although the simple names given above will be used throughout the specification:
7-chloro-8-methoxytetracycline (R=R1=H):
7-chloro-4-dimethylamino-8-methoxy1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12, 12apentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide;
7-chloro-4a-hydroxy-8-methoxytetracycline (R=OH, R1=H):
7-chloro-4-dimethylamino-8-methoxy1,4,4a,5,5a,6,11,12a-octahydro-3,4a,6,10,12,12ahexahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide;
7-chloro-8-methoxy-2'-N-methyltetracycline (R=H, R1=CH3):
7-chloro-4-dimethylamino-8-methoxy1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12apentahydroxy-6-methyl-1,11-dioxo-2-(2'-Nmethylnaphthacene)-carboxamide.
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, in addition, 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.
BRIEF DESCRIPTION OF THE FIGURES
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 1H NMR spectrum of 7-chloro-8methoxy-2'-N-methyltetracycline in a mixture of acetonedg and methanol-d4;
Figure 5 is the infrared spectirum of 7-chloro4a-hydroxy-8-methoxytetracycline in KBr;
Figure 6 is the 1H NMR spectrum of 7-chloro-4ahydroxy-8-methoxytetracycline in dimethyl sulfoxide-d6;
Figure 7 is the fully-decoupled proton 13C-NMR spectrum of 7-chloro-4a-hydroxy-8-methoxytetracycline in dimethyl sulfoxide-d6; and
Figure 8 is the chemical ionization mass spectrum of 7-chloro-4a-hydroxy-8-methoxytetracyclipe.
The antibiotics and microorganisms useful in their preparation will now be described in turn. ISOLATION AND PURIFICATION OF THE ANTIBIOTIC COMPLEXES AND INDIVIDUAL ANTIBIOTICS
A. ISOLATION AND PURIFICATION OF THE ANTIBIOTIC
COMPLEX 81-47 CONTAINING 7-CHLORO-8-METHOXY-2'N-METHYLTETRACYCLINE AND OF THIS ANTIBIOTIC ITSELF
A1. 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:
(a) Adjust the pH of the whole broth to 2 and filter;
(b) Adjust the pH of the filtrate to 8.5;
(c) Extract the filtrate using two volumes of organic solvent ( e . g . ethyl ace tate ) each time for each volume of broth;
(d) Combine the organic solvent extracts and remove the organic solvent by stripping to yield a solid residue;
(e) Dissolve the residue in acetone and filter off the insolubles;
(f) Add a mixture of 1:4 (v/v) ethyl ether: hexane to the filtrate until a precipitate forms;
(g) Collect the precipitate.
Using the above procedure, 100 mg of antibiotic complex were obtained from 3.5 L of fermentation broth. Since this antibiotic complex is made up of at least two dissimilar components, no relevant physico-chemical data can be determined for the complex. A2. SEPARATION OF THE ANTIBIOTIC COMPLEX
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, including 7-chloro-8methoxy-2'-N-methyltetracycline, 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.
A3. The physical and spectroscopic data for 7chloro-8-methoxy-2'-N-methyltetracycline are presented in Table I below.
Figure imgf000009_0001
Figure imgf000010_0001
a - indicates peaks buried under DMSO peak, but observed when spectrum was run in D2O/Dioxan.
b - The resonance of C-8 carbon in the 13C-NMR of 7chlorotetracycline appears at 140 ppm. The shift of C-8 carbon in 7-chloro-8-methoxy-2'-Nmethyltetracycline to 163.2 is indicative of the presence of the novel methoxy substituent.
Based on the above data, the structure of this compound (without specifying stereochemistry) is the following:
Figure imgf000011_0001
A4. Biological Properties of Antibiotic Complex and 7-Chloro-8-Methoxy-2'-N-Methyltetracycline This antibiotic complex containing at least two components, including 7-chloro-8-methoxy-2'-Nmethyltetracycline, is active against a variety of grampositive and gram-negative bacteria when tested in vitro. In comparative in vitro antibacterial activity tests using 7-chloro-8-methoxy-2'-N-methyltetracycline and tetracycline performed via conventional microtiter dilution methods in Mueller-Hinton broth, 7-chloro-8methoxy-2'-N-methyltetracycline showed activity against 91 gram-positive tetracycline-susceptible organisms with a Geometric Mean Minimum Inhibitory Concentration (GMM, mcg/mL) of 0.36, which is better than the GMM for tetracycline (0.46). 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 5% sheep blood).
7-Chloro-8-Methoxy-2'-N-methyltetracycline can be isolated from Antibiotic ES-119 and (in trace amounts) from Antibiotic Tet. 7 as descxibed in Section B below:
Bl. ISOLATION AND PURIFICATION OF THE
ANTIBIOTIC ES-119
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:
(a) Adjust the pH of the whole broth to 2 and separate the mycelia from a solution containing antibiotic ES-119 and biologically inactive organic compounds;
(b) Separate said solution into a solution containing antibiotic ES-119 and liquor containing biologically inactive organics; and te) Remove the solvents from the solution containing antibiotic ES-119 to produce antibiotic ES-119 as a solid residue.
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. 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.
B2. ISOLATION AND PURIFICATION OF THE
ANTIBIOTIC TET. 7
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.
B3. PURIFICATION OF THE ANTIBIOTICS ES-119 AND TET. 7 AND ISOLATION OF 7-CHLORO-8METHOXYTETRACYCLINE 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 C18 columns e.g. u-Bondapak C18, 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-methoxytetιracycline 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.
The two strains of Actinomadura brunnea (ATCC 53108 and 53180), mentioned here and described in more detail in and with reference to Tables IX through XII, are representative of separate subspecies of the species Actinomadura brunnea.
B4. The physical and spectroscopic data for 7chloro-8-methoxytetracycline are presented in Table II hereinbelow.
Figure imgf000015_0001
Figure imgf000016_0001
a - indicates peaks buried under DMSO peak, but observed when spectrum was run in D2O/Dioxan.
b - the shift of the C-8 carbon from 139.7 ppm in 7chlorotetracycline. Compound C, to 163 ppm in 7chloro-8-methoxytetracycline. Compound A, indicates the presence of an O-CH3 group. Based on the above physico-chemical data, the structure of 7-chloro-8-methoxytetracycline (without specif ic stereochemistry) is the following:
Figure imgf000017_0001
B5. BIOLOGICAL PROPERTIES OF ANTIBIOTIC ES-119,
ANTIBIOTIC Tet. 7 AND
7-CHLORO-8-METHOXYTETRACYCLINE
The antibiotic ES-119 and antibiotic Tet. 7 and the common component of each, 7-σhloro-8-methoxytetracycline, were tested in vitro and found to be active against a variety of gram-positive and gram-negative bacteria.
In comparative in vitro antibacterial activity tests performed via conventional microtiter dilution methods in Mueller-Hinton broth, 7-chloro-8methoxytetracycline showed activity against 91 grampositive (tetracycline-susceptible) organisms with a Geometric Mean Minimum Inhibitory Concentration (GMM, mcg/mL) of 0.12, which is better than the GMM for tetracycline (0.46) and the GMM for 7-chloro-8-methoxy2'-N-methyltetracycline (0.36). 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).
C. ISOLATION AND PURIFICATION OF THE
ANTIBIOTIC 7273 COMPLEX
Cl. 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:
(a) Adjust the pH of the whole broth to 4;
(b) Extract the whole broth using two volumes of organic solvent (e.g. ethyl acetate) each time for each volume of broth;
(c) Combine the organic solvent extracts and remove the organic solvent by stripping to yield a solid residue;
(d) Dissolve the residue in acetone and filter off the insolubles;
(e) Add a mixture of 1:4 (v/v) ethyl ether: hexane to the filtrate until a precipitate forms; and
(f) Collect the precipitate.
Using the above procedure, 12.5g of antibiotic 7273 complex were obtained from 80 L of fermentation broth. Since the antibiotic 7273 complex is made up of at least two dissimilar components, no relevant physicochemical data can be determined for the complex. C2. SEPARATION OF THE ANTIBIOTIC 7273 COMPLEX
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.
C3. The physical and spectroscopic data for 7chloro-4a-hydroxy-8-methoxytetracycline are presented in Table III below.
Figure imgf000020_0001
Figure imgf000021_0001
a - The resonance of the C-4a carbon in the 13C-NMR of 7-chloro-8-methoxytetracycline and 7chlorotetracycline appears at about 34-35 ppm. The shift of the C-4a resonance in 7-chloro-4a-hydroxy8-methoxytetracycline (Compound B) to 76.9 ppm is indicative of the novel 4a-hydroxy substituent.
b - Indicates peaks buried under DMSO peak, but observed when spectrum was run in D2O/Dioxan.
c - The resonance of the C-8 carbon in the 13C-NMR of 7-chlorotetracycline appears at 140 ppm. The shift of C-8 carbon in 7-chloro-4a-hydroxy-8methoxytetracycline to 163.1 is indicative of the novel methoxy substituent.
Based on the above data, the structure of 7chloro-4a-hydroxy-8-methoxytetracycline (without specifying stereochemistry) is the following:
Figure imgf000022_0001
C4. Biological Properties of Antibiotic 7273 Complex and 7-Chloro-4a-Hydroxy-8-Methoxytetracycline
The antibiotic 7273 complex containing at least two biologically active components, including 7-chloro4a-hydroxy-8-methoxytetracycline, is active against a variety of gram-positive and gram-negative bacteria when tested in vitro. In comparative in vitro antibacterial activity tests using 7-chloro-4a-hydroxy-8-methoxytetracycline and tetracycline performed via conventional microtiter dilution methods in Mueller-Hinton broth, 7-chloro-4ahydroxy-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). 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).
In another sequence of comparative in vitro antibacterial activity tests using 7-chloro-4a-hydroxy-8methoxytetracycline and chlortetracycline, performed via conventional dilution methods in Mueller-Hinton agar, 7chloro-4a-hydroxy-8-methoxytetracycline showed activity against 20 gram-positive chlortetracycline-susceptible (MIC<4) strains, including 2 of B. subtil is, 15 of Staphylococcus and 3 of Streptococcus. In 24-hour tests, 7-chloro-4a-hydroxy-8-methoxytetracycline had a GMM of 1.3, compared to a GMM for chlortetracycline of 0.175. 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.
In addition to the above-mentioned activities of the novel 7-chloro-8-methoxy tetracyclines, particularly favorable activities are illustrated in the follow Tables:
MICs of 7-Chloro-8-Methoxy tetracyclines Against Streptococci
Geometric Mean MICs (mcg/ml, 24 h, MHB + 5% Horse Serum, Microtitre)
Strains No. B TC_ POX MIN
S. pneumoniae 5 0.04 0.07 0.12 0.14 0.12 2.0
S. viridans 2 0.12 0.25 0.71 0.71 0.50 4.0
Group A 5 0.05 0.14 0.29 0.29 0.25 1.3
Group B 1 0.12 0.25 1.0 1.0 0.50 2.0
Group C 3 0.04 0.12 0.25 0.32 0.25 2.0
Group G 3 0.08 0.12 0.40 0.50 0.20 2.5
S. faecalis 2 0.12 0.35 5.7 0.71 0.50 1.4
S. faecium 4 0.06 0.25 1.2 0.50 0.30 2.0
All Streptococci 25 0.06 0.15 0.45 0.36 0.25 1.9 MICs of 7-Chloro-8-Methoxytetracyclines Against Neisseria Gonorrhoea e
Geometric Mean MlCs (mcg/ml, 24 h. Chocolate Agar + 1% IsoVitaleX)
Strains No. A D TC
N. gonorrhoeae 13 0.43 0.29 0.73
Abbreviations:
MHB: Mueller-Hinton Broth; A, B, D: as identified in Tables IIA and IIIA; TC: tetracycline; DOX: doxycycline; MIN: minocycline.
The 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:
LD50s in mice, i.v., mg/kg:
Compound A B D C
LD50 85 90 110 110
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. By the term "eliciting" is meant treating or preventing susceptible bacterial infection.
The methods of this invention are implemented using pharmaceutical 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. For oral administration, 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.
Oral administration of the antibiotics of this invention is preferred.
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.
Generally, 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.
Generally, the concentration of the novel antibiotic in topical formulations is from about 1% to about 5%, preferably about 1% to about 3%.
Generally, the 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.
In treating certain patients with the compounds of this invention, other pharmaceutically active ingredients can be included in the same dosage unit. THE MICROORGANISMS Each microorganism will be described in turn.
Actinomadura brunnea ATCC 39216
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.
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
Luedemann and Brodsky, in "Micromonospora carbonacea sp. nov, an everninoinicin-producing organism", Antimicrob. Agents Chemotherapy, pp 47-52, 1964; by Horan and Brodsky, "A Novel Antibiotic-Producing Actinomadura, Actinomadura kijaniata sp. nov.", International Journal Syst. Bacterial., Vol. 32, pp 195-200, 1982; by Becker et al., "Chemical Composition of Cell Wall Preparations from Strains of Various Genera of Aerobic Actinomycetes", Applied Microbiology, Vol. 13, pp 236-243, 1966; by Lechevalier and Lechevalier, "Chemical Composition as a Criterion in the Classification of Aerobic Actinomycetes", International Journal Syst. Bacterial. Vol. 20, pp 487-493, 1970; by. Shirling and Gottlieb, "Methods for Characterization of Streptomyces Species", International Journal Syst. Bacterial, Vol. 16, pp 313340, 1966; and by Waksman, The Actinomycetes, Vol. 2, (The Williams & Wilkins Co., Baltimore, Md., 1961).
Figure imgf000030_0001
microorganism in Table V, two color designators are employed. The first is a color name taken from the "Descriptive Color Name Dictionary" by Taylor, Knoche and Granville published by the Container Corporation of America (1950) U.S.A., with a color chip number corresponding to the color name, the chip number being taken from "The Color Harmony Manual", 4th Edition, 1958, also published by the Container Corporation of America. The second designator consists of a color name and number which refers to the synonym and near synonym found in the National Bureau of Standards, Circular 553, 'November 1, 1965 (U.S.A.). This comment applies to Table X and XIV also.
Growth of the microorganism, Actinomadura brunnea NRRL 15216, ATCC 39216, on various carbon compounds is reported in Table VI.
Physiologic characteristics of the microorganism Actinomadura brunnea NRRL 15216, ATCC 39216 are reported in Table VII.
A comparison of the characteristics of Actinomadura brunnea NRRL 15216, ATCC 39216 with those of other species of Actinomadura is listed in Table VIII.
Whole cell analysis of the microorganism Actinomadura brunnea NRRL 15216, ATCC 39216 found mesodiaminopimelic acid as the characteristic cell wall amino acid, trace amounts of the L,L isomer and madurose (3-0methyl-D-galactose) as the characteristic whole cell sugar.
Growth of the microorganism occurs from 27° to 40°C on yeast-dextrose agar. Poor growth occurs at 45°C, with optimum growth at from 27° to 35°C.
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Based on the cell wall analysis and morphologic characteristics reported in Tables TV and VIII, 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, Actinomadura brunnea ATCC 39216 (hereinafter A. brunnea), 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-β-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.
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.
It is understood that in accordance wi-th the rules of Nomenclature of Bacteria (S.P. Lapage et al. Ed. 1975, International Code of Nomenclature of Bacteria, 1976 revision) A. brunnea is the type strain and-, should another strain be found, the type strain would also be the type subspecies.
FERMENTATION OF THIS MICROORGANISM 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. To determine when peak antibiotic production has been reached, 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. Under these conditions the microorganism produces an antibiotic complex of this invention containing at least two components as determined by bioautography against both S. aureus and E. coli of the complex after development of a thin layer chromatography plate in 2:2:1 (v/v/v) chloroform: methanol: pH 3.5 acetate buffer.
The foregoing media are exemplary of the nutrients utilized by Actinomadura brunnea to produce an antibiotic complex of this invention. However, it is obvious to those skilled in the fermentation art that, 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 7.5.
The fermentation is initiated by addition of the inoculum to the broth. Generally, 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..
Actinomadura brunnea var. antibiotica var. nov.
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.
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. coli OLA 290R5. 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. Representatives of the mutated population of Actinomadura brunnea were allowed to grow on 600 agar plates such as starch yeast agar until single colonies had grown to 3 to 4 mm in diameter. Thereafter, all 600 plates were directly overlaid with agar containing an appropriate gram-negative indicating organism, e.g., E. coli OLA 290R5. The desired mutant colonies of Actinomadura brunnea var. antibiotica var. nov. were recognized by the clear zone where the gram-negative indicator strain was unable to grow. A spontaneous tetracycline-resistant derivative was selected and its antibiotic product was designated antibiotic Tet-7. Antibiotic Tet. 7 comprises 7-chloro-8-methoxytetracycline containing less than about 1% of 7-chloro-8-methoxy-2'-N-methyltetracycline.
Description of the Producing Strains: Actinomadura brunnea var. antibiotica var. nov. ATCC 53108 and ATCC 53180.
Figure imgf000050_0001
Microscopic Characteristics of ATCC 53108 and ATCC 53180 Long, branching aerial mycelia, 0.5 to 0.8 microns in diameter, are formed on water agar after 10 to 14 days at 30°C. The aerial mycelia fragment into long, straight to flexous chains of more than 20 elliptical spores, 0.8 to 1.2 microns in diameter by 1.0 to 2.0 microns in length. The culture characteristics of the microorganisms Actinomadura brunnea var antibiotica ATCC 53108 and ATCC 53180 on various standard descriptive media are reported in Table X. In the description of the growth characteristics of the microorganisms in Table X, two color designators are employed, as mentioned above for Table V.
Growth of the microorganisms, Actinomadura brunnea var. antibiotica ATCC 53108 and ATCC 53180 and Actinomadura brunnea ATCC 39216, on various carbon compounds is reported in Table XI.
Physiologic characteristics of the microorganisms Actinomadura brunnea var. antibiotica ATCC 53108 and ATCC 53180 and Act inoma dura brunnea ATCC 39216 are reported in Table XII;
Whole. cell analysis of the microorganisms Actinomadura brunnea var. antibiotica ATCC 53108 and ATCC 53180 found meso-diaminopimelic acid as the characteristic cell wall amino acid together with trace amounts of the L,L isomer, and madurose ( 3-0-methyl-Dgalactose) as the characteristic whole cell sugar.
Growth of the microorganisms 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.
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000057_0001
a) Observations made after 14-21 days at 30°C.
b) G = Growth; S = Surface Character istics; AM = Aerial Mycelia, DFP = Diffusible Pigments; and C = Color
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Actinomadura brunnea var. antibiotica ATCC 53108, but not ATCC 53180, differs from the parent strain, A. brunnea ATCC 39216, in sensitivity to rifamycin (ATCC 53108 is very sensitive to this antibiotic whereas the parent strain, ATCC 39216, and A. brunnea var. antibiotica ATCC 53180 are not), in vegetative mycelial pigmentation and degree of growth on some descriptive media and in the production of the antibiotic 7-chloro-8-methoxytetracycline. The mutant strains (ATCC 53108 and ATCC 53180) and the parent microorganism (ATCC 39216) produce 7-chloro-8-methoxy-2'N-methyltetracycline.
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.
It is understood that in accordance with the rules of Nomenclature of Bacteria (S.P. Lapage et al. Ed. 1975, International Code of Nomenclature of Bacteria, 1976 revision) A. brunnea var. antibiotica is the type strain and, should another strain be found, the type strain would also be the type subspecies.
FERMENTATION OF THESE MICROORGANISMS
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°. Accordingly, 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. However, 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. To determine when peak antibiotic production has been reached, 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. Under these conditions 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. 7 containing 7σhloro-8-methoxytetracycline substantially free of 7chloro-8-methoxy-2'-N-methyltetracycline; this can be determined by bioautography against both S. aureus, ATCC 209P and E. coli, OLA 290R5 of the antibiotics after development of a thin layer chromatography plate in, for example, 2:2:1 (v/v/v) chloroform: methanol: 0.2M sodium acetate buffer, pH 3.5 lower phase.
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. However, it is obvious to those skilled in the fermentation art that 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. Generally, 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.
Dactylosporangium vescum ATCC 39499
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.
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.
Description of the Producing Strain: Dactylosporangium vescum sp. nov. ATCC 39499
Figure imgf000066_0001
The culture characteristics of the microorganism Dactylosporangium vescum ATCC 39499 on various standard media are reported in Table XIV. In the description of the growth characteristics of the microorganism in Table XIV, two color designators are employed, as mentioned above for Table V.
Growth of the microorganism, Dactylosporangium vescum ATCC 39499, on various carbon compounds is reported in Table XV.
Physiologic characteristics of the microorganism Dactylosporangium vescum ATCC 39499 are reported in Table XVI.
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.
Growth of the microorganism occurs from 27° to 40°C on yeast-dextrose agar. Poor growth occurs above about 40°C, with optimum growth at from 27° to 35°C.
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Based on the formation of finger-like sporangia each containing a single row of two to four motile spores, abundant formation of sporangioles both on agar and in broth, the presence of hydroxydiaminopimelic acid in the cell wall and xylose and arabinose in hydrolyzed whole cells, the organism is identified as a member of the genus Dactylosporangium.
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.
It is understood that in accordance with the rules of Nomenclature of Bacteria (S.P. Lapage et al. Ed. 1975, International Code of Nomenclature of Bacteria, 1976 revision) D. vescum is the type strain and, should another strain be found, the type strain would also be the type subspecies.
FERMENTATION OF THIS MICROORGANISM 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. To determine when peak antibiotic production has been reached, 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. Under these conditions, the microorganism produces antibiotic 7273 complex containing at least two biologically active components as determined by bioautography against both S. aureus and E. coli of the complex after development of a thin layer chromatography plate in 2:2:1 (v/v/v) chloroform: methanol: pH 3.5 acetate buffer.
The foregoing media are exemplary of the nutrients utilized by Dactylosporangium vescum to produce antibiotic 7273 complex. However, it is obvious to those skilled in the fermentation art that 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. Generally, 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 1 - Preparation of Antibiotic Complex 81-47
A. Inoculum Preparation
1) Initial Stage
Prepare ten 250 mL Erlenmeyer flasks with 50 mL of the following germination medium:
Beef Extract 3 g
Tryptone 5 g
Yeast Extract 5 g
Dextrose 1 g
Potato Starch 24 g
Tap Water 1000 mL
Adjust the pH of the germination broth .to 7.5. Sterilize the. broth and. after cooling add 2.5 mL of a frozen whole broth sample from a previously prepared inoculum of A. brunnea ATCC 39216 to the broth. Incubate at 30°C with continual agitation at 300 rpm for 48 hours.
2) Second Stage
Transfer 25 ml of the first stage germination broth to each of twenty 2-liter Erlenmeyer flasks, each containing 500 mL of the same germination medium which had been previously adjusted to pH 7.5 and sterilized. Incubate at 30°C with continual agitation at 300 rpm for 48 hours.
B. Fermentation
In 14 L fermentors, add 10 L of the following medium:
Yeast Extract 5 g
Casein Hydrolysate 5 g
Cerelose 10 g Soluble Starch 20 g
Calcium Carbonate 4 g
Cobalt (II) Chloride 2.4 x 10-4g
Tap water 1000 mL
Adjust the pH of the medium to 6.7 and then sterilize the medium. After sterilization, adjust the pH of the medium to 7.0 with a sterile alkaline solution. Inoculate the fermentation medium with 5% volume of the second stage inoculum preparation of Step A. Incubate the fermentation mixture at 30°C with 0.35 WM of air and 350 rpm agitation for about 96 hours.
C. Isolation
Adjust the pH of the whole fermentation broth of step B to 2 and filter off the insolubles. Adjust the pH of the filtrate to 8.5. Extract 3.5 L of the filtrate twice with 3.5 L of ethyl acetate. Combine the ethyl acetate solutions, dry them over anhydrous sodium sulfate and remove the solvent by stripping to give a residue. Dissolve the dry residue in 35 mL of acetone and then add 350 mL of a 1:4 (v/v) mixture of ethyl ether:hexane until a precipitate results. Filter off the precipitate and dry in a vacuum to give 279 mg. of the (crude) antibiotic complex.
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. 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.
Obtain the novel 7-chloro-8-methoxy-2'-Nmethyltetracycline by combining desired fractions (e.g. 26 to 32). Lyophilize the pooled fractions to provide a light yellow powder. Crystallize a portion of the light yeliow powder from a 30:70 (v/v) methylene chloride: hexane mixture to provide the title compound as a yellow powder having physico-chemical data summarized in Table I.
Example 3 - Preparation of Antibiotic ES-119
A. Inoculum Preparation
1) First Stage
Prepare a 25 mm tube with 10 mL of the following germination medium:
Beef Extract 3 g
Tryptone 5 g
Yeast Extract 5 g
Cerelose 1 g
Soluble Starch 24 g
Calcium Carbonate 2 g
AF-1 Antifoam 1 mL
Tap Water 1000 mL
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
A. brunnea var antibiotica ATCC 53180 to 10 mL of the medium. Incubate at 30°C with continual agitation at 300 rpm for 48 hours.
2) Second Stage (optional)
Transfer 25 mL of the first stage germination broth to each of two liter Erlenmeyer flasks, each containing 500 mL of the initial stage germination medium which had been previously pH adjusted and sterilized. Incubate at 30°C with continual agitation at 300 rpm for 48 hours.
B. Fermentation
To each of four two liter Erlenmeyer flasks, add 500 mL of the following medium:
Yeast Extract 5 g
Casein Hydrolysate (NZ-Amine A) 5 g
Cerelose 10 g
Soluble Starch 20 g
Calcium Carbonate 4 g
Cobalt (II) Chloride 2.4 x 10-4 g
(1 mL of 0.001 M solution)
AF-1 Antifoam as necessary 1 mL
Tap water 1000 mL
Adjust the pH of the medium to 6.7 and then sterilize the medium. After sterilization, adjust the pH of the medium to 7.0 with a sterile alkaline solution. Inoculate the fermentation broth with 25 ml of the first (or optionally of the second) stage inoculum preparation of Step A. Incubate the fermentation mixture at 30°C with 0.35 VVM of air and 300 rpm agitation for about 96 hours. C. Isolation (Small Scale)
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.
D. Isolation (Large Scale)
Adjust the pH of the whole broth of Step B to 2 and filter off the insolubles. Adjust the pH of the filtrate to 7 and pass through a 7.62 cm (internal diameter) x 45.72 cm (height) chromatography column containing 450 mL of XAD-16 resin (a neutral polystyrene resin available from Rohm & Haas, Philadelphia, PA). Elute, successively, with 3 bed volumes (1350 mL) of each of the following: 1:20, 1:3, 1:1, 3:1 (v/v) methanol: water, then pure methanol and then 500:1 (v/v), methanol: 0.02N HCl.
Monitor the antibiotic activity of the eluate by disc testing each fraction (0.02 mL) against B. subtilis and E. coli. Combine the appropriate fractions and evaporate the solvent under vacuum to obtain crude antibiotic ES-119. Example 4 - Separation of Antibiotic ES-119 - Purification of 7 -Chloro-8-Methoxy tetracycline
A. Small Scale Purification
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). Use 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. for each of the 13 runs required to process the filtrate from 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.
B. Large Scale Purification
Dissolve 350 mg of the 500:1 (v/v) methanol: 0.02N HCl eluate obtained from the XAD-16 resin column in 100 mL of 0.02N HCl and filter. Place the filtrate on a 7.62 cm (internal diameter) x 76.2 cm (height) glass chromatography column containing 500 g of Sephadex G-25 slurried in 0.02N HCl. Elute the column with 0.02N HCl. Monitor the antibiotic activity of the eluate by disc testing against gram-negative organisms. Combine the fractions having activity against gram-negative organisms and lyophilize them to obtain 7-chloro-8- methoxytetracycline as a solid having the physicochemical data presented in Table II.
Example 5 - Large Scale Preparation of Antibiotic ES-119 and Antibiotic Tet. 7
The following procedures are used for large scale preparation of antibiotic ES-119 and antibiotic Tet. 7.
A. Inoculum Preparation
1) First Stage
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.
2) Second Stage
Add 25 mL of the f i rs t s tage inoculum to 2 L Erlenmeyer shake flasks each containing 500 mL of the sterilized germination medium used in the first stage of this Example. Incubate under conditions described for the first stage.
B. Fermentation
Add 5 volume percent of the second stage inoculum to 11 liters of the sterilized fermentation medium (pH 7.0) of Example 3(B) in a 14 liter fermentor. Incubate the fermentation mixture for 90 hours at 30°C with 350 rpm agitation and an air flow rate of 3.5 L/min (with dissolved oxygen monitoring). Sample before and after inoculation and during fermentation and harvesting. Check samples for pH, growth (packed volume of cells), purity and carbohydrate utilization. Monitor activity of the fermentation by assaying the whole broth with paper discs on agar plates seeded with S. aureus, ATCC 209P (pH 8.0) and E. coli, OLA 290R5 (pH 8.0). 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.
D. Isolation
Adjust the pH of the whole broth (60 L) from step C of this Example to 4.5 and filter. Pass the filtrate through a 121.92 cm (height) x 12.7 cm (internal diameter) chromatography column containing 6 L of XAD-4 resin. Elute successively with 1:3, 1:1 (v/v) methanol: water and 100% methanol. Monitor the antibiotic activity by disc testing the fractions against S. aureus and E. coli. Combine the fractions having biological activity and evaporate to give a solid residue. Redissolve the residue in distilled water and lyophilize to give a solid.
Treat the solid with EDTA in water and extract the water phase at pH 6.5 with methylene chloride. Evaporate the organic solvent to give a solid. Adsorb the solid on a Sephadex G-25 column, eluting with 0.02N HCl. Combine the active fractions. Example 6 - Preparation of Antibiotic 7273 Complex
A. Inoculum Preparation
1) Initial Stage
Prepare a 300 mL Erlenmeyer flask with 70 mL of the following germination medium:
Beef Extract 3 g
Tryptone 5 g
Yeast Extract 5 g
Cerelose 1 g
Potato Starch 24 g
Calcium Carbonate 2 g
Tap Water 1000 mL
AF-1 Antifoam 1 mL
Adjust the pH of the germination broth to 7.5. Sterilize the broth and after cooling add 3.5 mL of a frozen whole broth sample of the microorganism D. vescum ATCC 39499 from a previously prepared inoculum to each flask broth. Incubate at 30°C with continual agitation at 300 rpm for 48 hours.
2) Second Stage
Transfer 25 mL of the first stage germination broth to each of twenty 2-liter Erlenmeyer flasks, each containing 500 mL of the same germination medium which had been previously pH-adjusted and sterilized. Incubate at 30°C with continual agitation at 300 rpm for 48 hours. B. Fermentation
In a 14 L fermentor, add 11 L of the following medium:
Yeast Extract 5 g
Casein Hydrolysate 5 g
Cerelose 10 g
Soluble Starch 20 g
Calcium Carbonate 4 g
Cobalt (II) Chloride (10-3M) 1 mL
AF-1 Antifoam 1 mL
MgCl2.6H2O 0.5 g
Tap water 1000 mL
Adjust the pH of the medium to 6.7 and thensterilize the medium. After sterilization, adjust the pH of the medium to 7.0 with a sterile alkaline solution. Inoculate the fermentation medium with 5% volume of the second stage inoculum preparation of Step A. Incubate the fermentation mixture at 30°C with 0.35 VVM (3.5 L/min) of air flow and 350 rpm agitation for about 66 hours.
C. Isolation
Adjust the pH of the whole fermentation broth of step B to 4 with 6 N H2SO4. Extract 80 L of the whole broth four times with equal volumes of ethyl acetate. Combine the ethyl acetate solutions, dry them over anhydrous sodium sulfate and remove the solvent by stripping to give a residue. Dissolve the residue in 500 mL of acetone and then add 56 L of a 1:4 (v/v) mixture of ethyl ether:hexane until a precipitate results. Filter off the precipitate and dry in a vacuum to give the antibiotic 7273 complex. Example 7 - Separation of Antibiotic 7273 Complex Isolation of 7-chloro-4a-hydroxy-8-methoxytetracycline
Suspend 12.5 g of the crude antibiotic 7273 complex from Example 6C in 100 mL of H2O and adjust the pH to 1.5 with 5% HCl and filter. Add 10 g of ethylenediaminetetracetic acid to the filtrate and adjust the pH of the filtrate to 6.0 with concentrated ammonium hydroxide. Extract the aqueous phase so formed four times with equal volumes of methylene chloride. Separate, combine and dry the organic phase over anhydrous sodium sulfate. Remove the methylene chloride to give 3.9 g of purified antibiotic 7273 complex. Adsorb a solution of 3.9 g of the purified complex in 30 mL of 0.02N HCl on a 7.62 cm (internal diameter) x 101.6 cm (height) chromatography column containing 300 mL of Sephadex G-25 filtration gel (medium;, dry particle size 50-150μm). Elute the column with 0.02N HCl at a flow rate of about 1 mL per minute. Monitor the activity of each fraction 10 mL) against S. aureus ATCC 209P (pH 8.0) 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.
Obtain the novel 7-chloro-4a-hydroxy-8methoxytetracycline as a yellow powder having physicochemical data summarized in Table III by combining the desired fractions and lyophilizing them. Formulations Example 8 Parenteral Formulation
Per vial: 7-chloro-8-methoxytetracycline, 7chloro-4a-hydroxy-8-methoxytetracyσline 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. Add sterile water for injection U.S. P. or bacteriostatic water for injection U.S. P., for reconstitution.
Example 9
Capsule Formulation
Item No. Ingredient mg/capsule mg/capsule
1 Drug 100 200
2 Lactose 122 244
3 Corn Starch, Dried 25.5 51
4 Magnesium Stearate 2.5 5
250 mg 500 mg
Method
Mix Items No. 1, 2, and 3 in a suitable mixer for 10-15 minutes. Add Item No. 4 and mix for 1-3 minutes. Fill the above mixture into two-piece hard gelatin capsules of required size.
Example 10
Tablet Formulation
Item No. Ingredient mg/capsule mg/capsule
1 Drug 125 250
2 Lactose 93.75 187.5
3 Corn Starch (as a 5 10
10% Paste)
4 Corn Starch, Dried 25 50
5 Magnesium Stearate 1.25 2.5
250 mg 500 mg Method
Mix Items No. 1 and 2 and a portion of Item No. 4 in a suitable mixer for 10-15 minutes. Granulate the mixture with Item No. 3. Pass the wet granulation through a coarse screen (e.g., 0.6 to 0.65 cm, or 1/4") if needed, and dry the wet granules. Mill the dried granules using a suitable milling machine. Add Item No. 5 and the remaining amount of Item No. 4 to the dried granules in a suitable blender. Mix for 5-10 minutes. Compress the mixture into tablets of required shape and size on a rotary tablet machine. The tablets may be coated using standard coating procedures.
Example 11
Topical Formulation
Item No. Ingredient mg/g
1 Drug 25
2 Ethyl Alcohol 400
3 Hydroxypropyl Cellulose 15
4 Polyethylene Glycol 400 560
Mix Items No. 1, 2 and 4 in a suitable mixer. Stir vigorously and charge Item No. 3. Maintain stirring until uniformity is achieved.
Example 12 Oral Powder for Reconstitution (I)
Part A (Powder Formulation)
Item No, Ingredient mg/g
1 Drug 46.3
2 Flavor (s) q.s.
3 Colorant q.s.
4 Preservative q.s.
5 Buffer Agents q.s.
6 Sugar q.s.
To make 1.0 g
Mix Items No. 1, 2, 3, 4 and 5 thoroughly. Charge Item No. 6 and mix until uniformity is achieved.
Part B (Reconstitution)
Charge 54 g of above formulated powder: into a. proper container and add enough water to make up 100 ml. Shake well after the addition of water. Each 5 ml (1 teaspoonful) will then contain drug equivalent to 125 mg.
Example 13 Oral Powder for Reconstitution (II)
Part A (Powder Formulation)
Item No Ingredient mg/g
1 Drug 416.7
2 Flavor (s) q. s.
3 Colorant q. s.
4 Preservative q. S.
5 Buffering Agents 28.3.
6 Saccharin q. s.
7 PVP q.s.
To make 1.0 g
Mix well Items No. 1, 2, 3, 4, 5, 6, and 7 until uniform. Part B (Reconstitution)
Charge 6.0 g of above powder into a suitable container and add enough water to make up 100 ml. Shake well until uniform. Each 5 ml will then contain drug equivalent to 125 mg.
Example 14
Oral Liquid
Item No. Ingredient mg/ml
1 Drug 25. 0
2 Sweetener q. s.
3 Flavor q. s.
4 Colorant q. s.
5 Vegetable Oil q. s.
To make 1.0 ml
Charge 90% of Item No. 5 needed into a suitable container. Charge Items No. 1, 2, 3 and 4 and mix well. Bring to final volume with the remainder of Item No. 5.
Example 15 Suppository Item No. Ingredient mg/Suppository
1 Drug 125.0
2 Witepsol H-15 1868
Melt Item No. 2 and blend Item No. 1 until uniform. Pour into mold and congeal in refrigerator. Remove suppository from mold.
per WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau
Figure imgf000106_0001
INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(51) International Patent Classification 4 : (11) International Publication Number: WO 87/ 008
C 12 N 1/20; C 07 C 103/19; C 12 P 29/00; A 61 K 31/65; // (C 12 P 29/00; C 12 R 1: 01; C 12 R 1:03; C 12 N 1/20; " A3 C 12 R 1: 01; C 12 R 1. 03) (43) International Publication Date: 12 February 1987 (12.02.
(21) International Application Number: PCT/US86/01587 HORAN, Ann, Camille ; 138 Mountain Avenue, Sum NJ 07901 (US). MARQUEZ, Joseph, Anthony ; 47 Pleas
(22) International Filing Date: 6 August 1986 (06.08.86) Avenue, Montclair, NJ 07042 (US). VAUGHAN, Richa Willis ; 3467 Stallings Island Road, Augusta, GA 309
(31) Priority Application Numbers: 763,740 (US). KALYANPOR, Manohar, Gopal ; 10, rue Odil 763,742 Redon, F-78370 Plaisir (FR). WAITZ, Jav, Allan ; 29 Sa 764,275 Maria Avenue, Portola Valley, CA 94025 (US).
(32) Priority Dates: 8 August 1985 (08.08.85) (74) Agents: K.ANSTAD, Steinar, V. et al.; Schering-Plough C
8 August 1985 (08.08.85) poration, One Giralda Farms, Madison, NJ 07940-10
9 August 1985 (09.08.85) (US).
(33) Priority Country: US (81) Designated States: AT (European patent), BE (European tent), CH (European patent), DE (European patent), D
(71) Applicant: SCHERING CORPORATION [US/US]; 2000 GalFR (European patent), GB (European patent). IT (Eu loping Hill Road, Kenil orth, NJ 07033 (US). pean patent), JP, LU (European patent), NL (European tent), SE (European patent).
(72) Inventors: SMITH, Elizabeth, Bargmann ; 46 Edgemere AvePublished nue, Plainsboro, NJ 08536 (US). MUNAYYER, Hanan, Karaman ; 494 Passaic Avenue, West Caldwell, NJ 07006 With international search report. (US). RYAN, Michael, Joseph ; 34 North ood Drive, West Before the expiration of the time limit for amending t Milford, NJ 07480 (US). MILLER, George, Henry ; 25 Tara claims and to be republished in the event of the receipt Lane, Montville, NJ 07045 (US). PATEL, Mahesh, Gordhan- amendments. das ; 42 Brentwood Drive, Verona, NJ 07046 (US).
(88) Date of publication of the international search report:
18 June 1987 (18.06.8
(54) Title: TETRACYCLINE ANTIBIOTICS, THEIR PREPARATION, ANTIBIOTIC COMPOSITIONS CONTAI ING THEM, AND MICROORGANISMS USEFUL IN THEIR PREPARATION
(57) Abstract
7-Chlorotetracycline derivatives as novel antibiotics preparable by cultivation of strains of Actinomadura brunn and Dactylosporangium vescum. They are active against gram-positive and gram-negative aerobes.
FOR THE PURPOSES OF INFORMATION ONLY
Codes usedto identify Statespartyto the PCT on the frontpages ofpamphlets publishing international applications under the PCT.
AT Austria FR France ML Mali
AU Australia GA Gabon MR Mauritania
BB Barbados GB United Kingdom M Malawi
BE Belgium HU Hungary NL Netherlands
BG Bulgaria IT Italy NO Norway
BJ Benin JP Japan RO Romania
BR Brazil KP Democratic People's Republic SD Sudan
CF Central. African Republic of Korea SE Sweden
CG Congo KR Republic of Korea SN Senegal
CH Switzerland LI Liechtenstein SU Soviet Union
CM Cameroon LK Sri Lanka TO Chad
DE Germany Federal Republic of LU Luxembourg TG Togo
DK Denmark MC Monaco US United States of America
FI Finland MG Madagascar

Claims

CLAIMS: 1. The compounds represented by the formula
Figure imgf000095_0001
wherein R is a hydrogen atom or a hydroxy group and R1 is a hydrogen atom or a methyl group, with the proviso that at least one of R and R1 is a hydrogen atom; and the pharmaceutically acceptable salts thereof.
2. 7-Chloro-8-methoxytetracycline, 7-chloro-4ahydroxy-8-methoxytetracycline, and 7-chloro-8-methoxy-2'N-methyltetracycline.
3. A pharmaceutical composition comprising an antibiotically effective amount of a compound or salt defined in claim 1 or claim 2 and a pharmaceutically acceptable carrier or diluent.
4. A pharmaceutical composition as claimed in claim 3 suitable for parenteral or topical administration.
5. A pharmaceutical composition as claimed in claim 3 suitable for oral administration.
6. A method of eliciting an antibiotic effect in a host having a susceptible infection which comprising administering to said host an antibiotically effective amount of a compound or salt defined in claim 1 or claim 2, or a pharmaceutical composition thereof.
7. A biologically pure culture of a microorganism of the species Actinomadura brunnea, said culture being capable of producing an antibiotic complex 81-47, containing 7-chloro-8-methoxy-2'-N-methyltetracycline, in a recoverable quantity upon fermentation under aerobic conditions in an agueous medium containing assimilable sources of nitrogen and carbon.
8. Antibiotic complex 81-47, containing 7-chloro-8methoxy-2'-N-methyltetracycline, produced by cultivating a microorganism of the species Actinomadura brunnea in a pH- and temperature-controlled agueous nutrient medium containing assimilable sources of nitrogen and carbon under aerobic conditions, until a composition of matter having substantial antibiotic activity is produced.
9. A biologically pure culture of the microorganism Actinomadura brunnea var antibiotica var nov., having the identifying characteristics of ATCC 53108, said culture being capable of producing antibiotic ES-119 comprising 7-chloro-8-methoxytetracycline and also its 2'-N-methyl derivative in a recoverable guantity upon fermentation under aerobic conditions in an agueous medium containing assimilable sources of nitrogen and carbon.
10. Antibiotic ES-119 produced by cultivating a strain of Actinomadura brunnea var antibiotica var nov., having the identifying characteristics of ATCC 53108, in a pH- and temperature-controlled agueous nutrient medium containing assimilable sources of nitrogen and carbon under aerobic conditions, until a composition of matter having substantial antibiotic activity is produced.
11. A biologically pure culture of the microorganism Actinomadura brunnea var antibiotica var nov., having the identifying characteristics of ATCC 53180, said culture being capable of producing antibiotic Tet. 7 comprising 7-chloro-8-methoxytetracycline substantially free of 7-chloro-8-methoxy-2'-N-methyltetracycline, in a recoverable guantity upon fermentation under aerobic conditions in an agueous medium containing assimilable sources of nitrogen and carbon.
12. Antibiotic Tet. 7 produced by cultivating a strain of Actinomadure brunnea var antibiotica var. nov., having the identifying characteristics of ATCC 53180, in a pH- and temperature-controlled agueous nutrient medium containing assimilable sources of nitrogen and carbon under aerobic conditions, until a composition of matter having substantial antibiotic activity and containing 7chloro-8-methoxytetracycline, substantially free of 7chloro-8-methoxy-2'-N-methyltetracycline, is produced.
13. A biologically pure culture of a microorganism of the species Dactylosporangium vescum, said culture being capable of producing the antibiotic 7273 complex in a recoverable guantity upon fermentation under aerobic conditions in an agueous medium containing assimilable sources of nitrogen and carbon.
14. Antibiotic 7273 complex produced by cultivating a microorganism of the species Dactylosporangium vescum in a pH- and temperature-controlled agueous nutrient medium containing assimilable sources of nitrogen and carbon under aerobic conditions, until a composition of matter having substantial antibiotic activity is produced.
PCT/US1986/001587 1985-08-08 1986-08-06 Tetracycline antibiotics, their preparation, antibiotic compositions containing them, and microorganisms useful in their preparation WO1987000832A2 (en)

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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

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Cited By (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

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CH373375A (en) * 1957-04-05 1963-11-30 American Cyanamid Co Process for preparing tetracyclines
US3360559A (en) * 1964-11-30 1967-12-26 American Cyanamid Co Substituted 1, 3, 4, 10, 11, 12-hexahydroxynaphthacene-2-carboxamides
FR1553651A (en) * 1962-11-05 1969-01-17

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CH373375A (en) * 1957-04-05 1963-11-30 American Cyanamid Co Process for preparing tetracyclines
FR1553651A (en) * 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

Cited By (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

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