Antibiotics GE 23077, pharmaceutically acceptable salts and...

Details Antibiotics GE 23077, pharmaceutically acceptable salts and... Antibiotics GE 23077, pharmaceutically acceptable salts and...

2001-11-09

2003-07-01

Lilling, Herbert J. (Department: 1651)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C424S115000, C514S010100, C514S451000, C435S071300, C435S169000, C435S252100, C530S317000

Reexamination Certificate

active

06586393

ABSTRACT:

The present invention concerns an antibiotic substance of microbial origin, arbitrarily denominated GE23077 complex and the individual factors that constitute it, namely GE23077 factor A1, GE23077 factor A2, GE23077 factor B1 and GE23077 factor B2, a mixture of said factors in any proportion, the pharmaceutically acceptable salts and compositions thereof, and their use as an antibacterial agent with a selective inhibitory activity against
E. coli
RNA polymerase.
Another object of the present invention is a process for preparing GE23077 complex, namely GE23077 factor A1, GE23077 factor A2, GE23077 factor B1 and GE23077 factor B2, a mixture of said factors in any proportion, hereinafter reported as GE23077 compounds.
STRAIN AND FERMENTATION
Actinomadura sp. DSMZ 13491 was isolated from a soil sample and deposited on May 22, 2000, with the DSMZ, (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany), under the provision of the Budapest Treaty. The strain was accorded accession number DSMZ 13491.
The production of compounds GE23077 factors A1, A2, B1 and B2, is achieved by cultivating an Actinomadura strain capable of producing them, i.e. Actinomadura sp. DSMZ 13491 or a variant or mutant thereof; isolating the resulting antibiotic from the mycelium and or the culture broth; purifying the isolated antibiotic; and separating the antibiotic four factors A1, A2, B1 and B2 by chromatographic means. Wishing to produce the GE23077 complex, the separating step is evidently unrequired. In any case, it is preferred to produce compounds GE23077 under aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic salts, purifying the resulting compounds by means of a chromatographic technique. Many of the nutrient media usually employed in the fermentation field can be used, however certain media are preferred.
Preferred carbon sources are glucose, xilose, cellobiose, cellulose, starch, corn meal, and the like. Preferred nitrogen sources are ammonia, nitrates, soybean meal, peptone, meat extract, yeast extract, tryptone, aminoacids, hydrolized casein and the like. Among the inorganic salts which can be incorporated in the culture media, there are the customary soluble salts capable of yielding sodium, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride, carbonate, sulphate, phosphate, nitrate, and the like ions.
Preferably, the strain producing compounds GE23077 is pre-cultured in a fermentation tube or in a shake flask, then the culture is used to inoculate jar fermentors for the production of substantial quantities of substances. The medium used for the pre-culture can be the same as that employed for larger fermentations, but other media can also be employed. The strain producing compounds GE23077 can be grown at temperature between 20° C. and 40° C., preferably between 28° C. and 37° C.
During the fermentation, the GE23077 factors can be monitored by bioassay on susceptible microorganisms and/or by testing treated broth samples against or
E. coli
RNA polymerase and/or by HPLC analyses. Maximum production of GE23077 compounds generally occurs between the fourth and the seventh day of fermentation.
Compounds GE 23077 are produced by cultivating Actinomadura sp. DSMZ 13491 or a variant or mutant thereof producing compounds GE23077, and are found in the culture broths and/or in the mycelium.
MORPHOLOGICAL CHARACTERISTICS OF Actinomadura sp. DSMZ 13491
Actinomadura sp. DSMZ 13491 grows well on many standard solid media. Microscopic examination and cell dimensions were measured using the culture grown on one-tenth strength humic acid medium (H. Nonomura, 1984—Design of a new medium for isolation of soil actinomycetes. The Actinomycetes 18, 206-209).
After seven days incubation at 28° C., the strain revealed extensively branched vegetative hyphae (0.5 &mgr;m in diameter). No fragmentation was observed. The aerial mycelium contained slightly twisted chains of large spores (1.2-1.4 &mgr;m in diameter). No pseudosporangia were observed. Spore dimensions exceeded those of the mycelium, giving rise to a “segmented” appearance of the spore chain.
CULTURAL CHARACTERISTICS OF Actinomadura sp. DSMZ 13491
Actinomadura sp. DSMZ 13491 was grown for seven days in AF-MS liquid medium (see Example 1 for medium composition). The mycelium was harvested by centrifugation and washed twice in sterile one quarter strength Ringer's solution (OXOID). Subsequently, sufficient Ringer's solution was added to the mycelium to provide a suitable inoculum. Aliquots of the suspension were streaked in a cross-hatched manner onto various media recommended by Shirling and Gottlieb (E. B. Shirling and D. Gottlieb, 1966—Method for Characterization of Streptomyces species—Int. J. Syst. Bacteriol., 16, 313-340) and several media recommended by Waksman (Waksman S. A., 1961—The Actinomycetes—The Williams and Wilkins Co., Baltimore. Vol 2, pp 328-334).
The ability to use a variety of carbohydrates as a carbon and energy source was determined in ISP8 medium (Shirling and Gottlieb, ibid) containing the carbon source at a final concentration of 2% (w/v). All media were incubated at 28° C. for 21 days. Colour was assessed in natural daylight, using the Colour Atlas of Maerz and Paul (A. Maerz and M. R. Paul, 1950—A Dictionary of Colour, 2nd edition. McGraw-Hill Book Co. Inc., New York).
Colonial appearance, substrate and aerial mycelium colour and pigment production for strain GE23077 are recorded in Table I. Physiological characteristics of the strain are presented in Table II. The ability to utilise various carbohydrates for growth is shown in Table III.
TABLE I
Cultural characteristics of Actinomadura sp. DSMZ 13491
VEGETATIVE
AERIAL
DIFFUSIBLE
MEDIUM
GROWTH
a)
MYCELIUM
b)
MYCELIUM
PIGMENT
Bennet's
+ + +
Pink patches, glutinous texture,
absent
None
translucent, amorphous with a
diffuse edge
Calcium malate
+ + +
very pale pink, matt texture with a
white, abundant
None
diffuse edge
Czapek Glucose
+ + +
dull pink, glutinous texture,
absent
None
convoluted with an entire edge
Czapek Sucrose
+ + +
pink, glutinous texture with an
light pink,
None
entire edge
abundant
Egg albumin
+ + +
very pale pink 6 opaque, glutinous
white
None
texture, with edge entire;
discolouration at growing edge
Glucose
+ +
light pink 6 white speckles,
absent
None
asparagine
glutinous texture with edge entire
Hickey and
+ + +
pale yellow 6 opaque glutinous
white, sparse
None
Tresner
texture (matt at edges), convoluted
with an entire edge
ISP2
+ + +
pale pink speckles, glutinous
absent
None
texture with edge entire
ISP3
+ + +
pink (2-E-9), flat, smooth
Pale pink 6
None
white tufts
ISP4
+ + +
Deep pink (2-1-10), dimpled with
absent
None
edge entire
ISP5
+ +
very pale pink (2-C-1) speckles 6
white tufts, more
None
opaque, glutinous texture with
concentrated at
edges diffuse
edges
ISP6
+ + +
pale yellow (deeper yellow
absent
None
patches) 6 opaque glutinous
texture, convoluted with an entire
edge
ISP7
+ + +
pink 6 opaque, glutinous texture
white tufts
None
with edges diffuse
(mainly at
growing edge)
Nutrient
+ + +
Yellow 6 opaque glutinous
white tufts
None
texture, convoluted with an entire
(mainly at
edge
growing edge)
Oatmeal
+ +
very pale pink (4-C-1), flat, smooth,
absent
None
dimpled, amorphous
Potato
+ + +
pink (4-D-8), glutinous texture,
Absent
None
convoluted with an entire edge
Sabouraud
+ +
opaque glutinous texture, with an
Absent
None
entire edge
Skimmed milk
+ + +
pink (4-F-8), glutinous texture, with
Absent
None
an entire edge; clearing zones
aroun

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