U.S. patent number RE32,333 [Application Number 06/780,516] was granted by the patent office on 1987-01-20 for a-21978 antibiotics and process for their production.
This patent grant is currently assigned to Eli Lilly and Company. Invention is credited to Robert L. Hamill, Marvin M. Hoehn.
United States Patent |
RE32,333 |
Hamill , et al. |
January 20, 1987 |
A-21978 Antibiotics and process for their production
Abstract
Antibiotic A-21978 complexes, in particular the A-21978C
complex, comprising microbiologically active, related factors
C.sub.0, C.sub.1, C.sub.2, C.sub.3, C.sub.4, and C.sub.5. A-21978
complex and A-21978C complex are produced by submerged aerobic
fermentation of Streptomyces roseosporus NRRL 11379. The individual
A-29178C factors are separated and isolated by chromatography. The
A-21978 and A-21978C complexes; the A-21978C factors; and
pharmaceutically acceptable salts thereof are antibacterial agents
and improve growth promotion in poultry.
Inventors: |
Hamill; Robert L. (Greenwood,
IN), Hoehn; Marvin M. (Indianapolis, IN) |
Assignee: |
Eli Lilly and Company
(Indianapolis, IN)
|
Family
ID: |
27119720 |
Appl.
No.: |
06/780,516 |
Filed: |
September 26, 1985 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
399655 |
Jul 19, 1982 |
RE031396 |
Sep 27, 1983 |
|
Reissue of: |
951695 |
Oct 16, 1978 |
04208403 |
Jun 17, 1980 |
|
|
Current U.S.
Class: |
530/321; 930/190;
435/71.3; 435/886; 930/20; 930/21; 930/200; 930/270 |
Current CPC
Class: |
C12N
1/205 (20210501); C07K 7/08 (20130101); A61K
38/00 (20130101); C12R 2001/465 (20210501) |
Current International
Class: |
C07K
7/08 (20060101); C07K 7/00 (20060101); A61K
38/00 (20060101); C12D 009/14 (); C07C 103/52 ();
A61K 037/00 () |
Field of
Search: |
;435/71 ;260/112.5 |
Other References
J Shoji, et al., "The Total Structure of Cerexin A", Journal of
Antibiotics, 29 (12) 1268-1274 (1976). .
J. Shoji, et al., "The Structure of Cerexin B", ibid., 29 (12)
1275-1280 (1976). .
J. Shoji, et al., "The Structure of Brevistin", ibid., 29 (4)
380-389 (1976)..
|
Primary Examiner: Shapiro; Lionel M.
Attorney, Agent or Firm: Harrison; Nancy J.
Claims
We claim: 1. The A-21978 antibiotic complex which is produced by
submerged aerobic cultivation of Streptomyces roseosporus NRRL
11379 or an A-12978-producing mutant thereof. 2. The method of
producing A-21978 complex which comprises cultvating Streptomyces
roseosporus NRRL 11379 or an A-21978-producing mutant thereof in a
culture medium containing assimilable sources of carbohydrate,
nitrogen, and inorganic salts under submerged aerobic fermentation
conditions until a substantial amount of antibiotic activity is
produced. 3. The method of claim 2 which includes the additional
step of separating A-21978 complex from the culture medium. 4. The
method of claim 3 which includes the additional step of isolating
the A-21978C complex from the separated A-21978 complex. 5. The
method of claim 4 which includes the additional step of isolating
A-21978C factor C.sub.0 from the separated A-21978C complex. 6. The
method of claim 4 which includes the additional step of isolating
A-21978C factor C.sub.1
from the separated A-21978C complex. 7. The method of claim 4 which
includes the additional step of isolating A-21978C factor C.sub.2
from the separated A-21978C complex. 8. The method of claim 4 which
includes the additional step of isolating A-21978C factor C.sub.3
from the separated A-21978C complex. 9. The method of claim 4 which
includes the additional step of isolating A-21978C factor C.sub.4
from the separated A-21978C complex. 10. The method of claim 4
which includes the additional step of isolating A-21978C factor
C.sub.5 from the separated A-21978C complex. 11. The A-21978C
antibiotic complex, which is produced by submerged aerobic
cultivation of Streptomyces roseosporus NRRL 11379 or an
A-21978C-producing mutant thereof; and which comprises factors
C.sub.0, C.sub.1, C.sub.2, C.sub.3, C.sub.4 and C.sub.5, which have
the following structural formula: ##STR4## wherein 3MG represents
L-threo-3-methylglutamic acid, and R represents a specific fatty
acid moiety as follows: 8-methyldecanoyl (C.sub.1), C.sub.10
-methylundecanoyl (C.sub.2), 10-methyldodecanoyl (C.sub.3),
C.sub.10 -alkanoyl (C.sub.0), C.sub.12 -alkanoyl (C.sub.4), and
.[.C.sub.13 -alkanoyl.]. .Iadd.C.sub.12 -alkanoyl
C.sub.5).Iaddend..
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
Although there are many known antibacterial agents, the need for
improved antibiotics continues. Antibiotics differ in their
effectiveness against pathogenic organisms, and organism strains
which are resistant to currently used antibiotics continually
develop. In addition, individual patients often suffer serious
reactions to specific antibiotics, due to hypersensitivity and/or
to toxic effects. There is, therefore, a continuing need for new
and improved antibiotics.
2. Prior Art
The A-21978C antibiotics are closely related, acidic peptide
antibiotics. Members of this class of antibiotics which were
previously known include crystallomycin, amphomycin, zaomycin,
aspartocin, and glumamycin [see T. Korzybski, Z. Kowszyk-Gindifer
and W. Kurylowicz, "Antibiotics-Origin, Nature and Properties,"
Vol. I. Pergamon Press, New York, N.Y., 1967, pp. 397-401 and
404-408]; tsushimycin [J. Shoji, et al., J. Antibiotics 21, 439-443
(1968)]; laspartomycin [H. Naganawa, et al., J. Antibiotics 21,
55-(1968)]; brevistin [J. Shoji and T. Kato, J. Antibiotics 29,
380-389 (1976)]; cerexin A [J. Shoji, et al., J. Antibiotics 29,
1268-1274 (1976)] and cerexin B [J. Shoji and T. Kato, J.
Antibiotics 29, 1275-1280 (1976)]. Of these antiobiotics,
brevistin, cerexin A and cerexin B are believed to be the prior art
antibiotics which are most closely related to the new A-21978C
antibiotics.
SUMMARY OF THE INVENTION
This invention relates to antibiotic substances. In particular, it
relates to antibiotic examples comprising several factors. The
A-21978 complex contains major factor C and as yet uncharacterized
factors A, B, D and E. A-21978 factor C is a complex of closely
related antibiotic factors, including individual A-21978C factors
C.sub.0, C.sub.1, C.sub.2, C.sub.3, C.sub.4, and C.sub.5. A-21978
factor C is, therefore, designated herein as A-21978C complex. The
salts of the A-21978 and A-21978C complexes and of individual
A-21978C factors C.sub.0, C.sub.1, C.sub.2, C.sub.3, C.sub.4 and
C.sub.5 are also part of this invention.
The term "complex" as used in the fermentation art and in this
specification refers to a mixture of coproduced individual
antibiotic factors. As will be recognized by those familar with
antibiotic production by fermentation, the number and ratio of
individual factors produced in an antibiotic complex will vary,
depending upon the fermentation conditions used. In the A-21978C
complex, factors C.sub.1, C.sub.2, and C.sub.3 are major factors,
and factors C.sub.0, C.sub.4, and C.sub.5 are minor factors.
The antibiotic substances of this invention are arbitrarily
designated herein as A-21978 antibiotics. In discussions of
utility, the term "A-21978 antibiotics" will be used, for the sake
of brevity, to denote a number selected from the group consisting
of A-21978 complex, A-21978C complex and A-21978C factors C.sub.0,
C.sub.1, C.sub.2, C.sub.3, C.sub.4, and C.sub.5, and the
pharmaceutically acceptable salts thereof.
The A-21978 complex is produced by culturing Streptomyces
roseosporus NRRL 11379 under submerged aerobic fermentation
conditions until a substantial level of antibiotic activity is
produced. The A-21978 complex is separated by filtering the
fermentation broth, lowering the pH of the filtrate to about pH 3,
allowing the complex to precipitate, and separating the complex by
filtration. The separated complex may be further purified by
extraction techniques. For isolation of the individual A-21978C
complex and factors, chromatographic separations are required. The
A-21978 antibiotics of this invention inhibit the growth of
pathogenic organisms, especially gram-positive bacteria.
DESCRIPTION OF THE DRAWINGS
Infrared absorption spectra (KBr pellet) of the following A-21978C
antibiotics (as sodium salts) are presented in the accompanying
drawings as follows:
FIG. 1--A-21978C Complex
FIG. 2--A-21978C Factor C.sub.1
FIG. 3--A-21978C Factor C.sub.2
FIG. 4--A-21978C Factor C.sub.3
FIG. 5--A-21978C Factor C.sub.0
FIG. 6--A-21978C Factor C.sub.4
FIG. 7--A-21978C Factor C.sub.5
DETAILED DESCRIPTION OF THE INVENTION
The A-21978C factors of this invention are closely related peptide
antibiotics. As many as six antibiotic factors are recovered from
the fermentation and are obtained as a mixture, the A-21978C
complex. Individual factors C.sub.0, C.sub.1, C.sub.2, C.sub.3,
C.sub.4 and C.sub.5 are isolated as individual compounds as
hereinafter described.
The A-21978C factors are closely related, acidic, cyclic
polypeptide antibiotics bearing a fatty acid acyl group at the
terminal amino group. Upon hydrolysis, each of the factors yielded
the following amino acids:
______________________________________ Amino Acid No. of moles
______________________________________ Aspartic acid* 4 Glycine 2
Alanine 1 Serine 1 Threonine 1 Tryptophan 1 Ornithine 1 Kynurenine
1 3-Methylglutamic acid** 1 ______________________________________
*one of which could be asparagine **could be from
3methylglutamine
Each of the A-21978C factors contains a fatty acid. Table I
summarizes carbon content, and the identity where known, of the
fatty acid contained by each of the A-21978C factors.
TABLE I ______________________________________ Fatty Acid A-21978C
Carbon Factor Content Identity
______________________________________ C.sub.1 C.sub.11
8-methyldecanoic acid C.sub.2 C.sub.12 10-methylundecanoic acid
C.sub.3 C.sub.13 10-methyldodecanoic acid C.sub.0 C.sub.10 --
C.sub.4 C.sub.12 -- C.sub.5 .[.C.sub.13 .]..Iadd.C.sub.12 --
______________________________________
Subtractive Edman degradation reactions indicate that tryptophan is
the N-terminal amino acid and that an aspartic acid moiety is the
next adjacent amino acid.
Gas-chromatographic mass-spectral studies on A-21978C factor
C.sub.2 indicate that one of the two following sequences could be
the structure of this factor (Asx indicates aspartic acid or
aparagine and MeGlx indicates 3-methylglutamic acid or
3-methylglutamine): ##STR1##
Enzymatic hydrolysis of A-21978C factor C.sub.2, using
carboxypeptidase Y confirmed that kynurenine is the C-terminal
amino acid and that the C-terminal COOH group may esterify the
hydroxyl group of the threonine moiety.
Based on the foregoing studies, the structure of the A-21978C
antibiotics is tentatively believed to be as follows: ##STR2##
wherein 3 MG represents L-threo-3-methylglutamic acid, and R
represents a specific fatty acid moiety, the specific R groups of
the factors being as follows:
______________________________________ A-21978C Factor R Moiety
______________________________________ C.sub.1 8-methyldecanoyl
C.sub.2 10-methylundecanoyl C.sub.3 10-methyldodecanoyl C.sub.0
C.sub.10 --alkanoyl* C.sub.4 C.sub.12 --alkanoyl* C.sub.5
.[.C.sub.13 .]..Iadd.C.sub.12 --alkanoyl*
______________________________________ *Identity not yet
determined
The A-21978C complex and factors (as Na salts) are soluble in water
and in acidic and alkaline solutions, except at pH levels of below
about pH 3.5; in lower alcohols such as methanol, ethanol,
propanol, and butanol; and in dimethylformamide, dimethyl
sulfoxide, dioxane, and tetrahydrofuran but are only slightly
soluble or are insoluble in acetone, chloroform, diethyl ether,
benzene, ethyl acetate, and hydrocarbon solvents. The salt forms of
the A-21978C complex and factors are soluble in water, methanol,
dimethylformamide, and dimethyl sulfoxide; but are insoluble in
solvents such as ethanol, butanol, and dioxane.
Table II summarizes the approximate percentage elemental
composition of the sodium salt of each of the A-21978C factors.
TABLE II
__________________________________________________________________________
A-21978C Factor C.sub.0 C.sub.1 C.sub.2 C.sub.4 C.sub.3 C.sub.5
Element Calcd Found Calcd Found Calcd Found Found Calcd Found Found
__________________________________________________________________________
Carbon 52.61 52.07 52.89 52.47 53.17 51.87 52.73 53.44 54.18 52.76
Hydrogen 6.07 5.95 6.14 5.93 6.21 6.05 5.99 6.28 6.35 6.71 Nitrogen
13.63 12.73 13.52 13.38 13.41 13.66 14.07 13.29 13.34 13.97 Oxygen
26.28 25.84 26.06 26.19 25.84 25.86 25.81 25.63 25.06 25.60 Sodium*
1.40 3.41 1.39 2.03 1.38 2.56 1.40 1.36 1.07 0.96
__________________________________________________________________________
*by difference
The infrared absorption spectra of the A-21978C complex and factors
(as the Na salts) in KBr pellet are shown in FIGS. 1-7 of the
accompanying drawings. Table III summarizes the most significant
absorption maxima for each of these.
TABLE III ______________________________________ IR Maxima
(cm.sup.-1) of the A-21978C Complex and Factors Complex C.sub.0
C.sub.1 C.sub.2 C.sub.3 C.sub.4 C.sub.5
______________________________________ 3310 3300 3300 3310 3310
3320 3300 3050 3050 3040 3050 3040 3050 3045 2910 2910 2910 2910
2910 2920 2910 2840 2840 2840 2840 2835 2850 2840 1655 1650 1650
1665 1650 1655 1650 1540 1540 1535 1535 1535 1525 1525 1450 1445
1450 1450 1450 1455 1445 1395 1395 1395 1400 1395 1395 1390 1310
1240 1215 1220 1225 1225 1220 1215 1160 1155 1160 1160 1160 1160
1155 1065 1060 1065 1065 1060 1065 1055 745 745 745 745 745 740 735
645 555 518 ______________________________________
The approximate molecular weights and molecular formulas of the
three major A-21978C factors are summarized in Table IV.
TABLE IV
__________________________________________________________________________
A-21978C Factor Molecular Weight Formula
__________________________________________________________________________
C.sub.0 .[..[. 1622 .]..]. .Badd.1621.Baddend. .[..[. C.sub.72
H.sub.100 N.sub.16 O.sub.27 .]..]. C.sub.72 H.sub.101 N.sub.17
O.sub.26 C.sub.1 .[..[. 1636 .]..]. .Badd.1635.Baddend. .[..[.
C.sub.73 H.sub.102 N.sub.16 O.sub.27 .]..]. C.sub.73 H.sub.103
N.sub.17 O.sub.26 C.sub.2 .[..[. 1650 .]..]. .Badd.1649.Baddend.
.[..[. C.sub.74 H.sub.104 N.sub.16 O.sub.27 .]..]. C.sub.74
H.sub.105 N.sub.17 O.sub.26 C.sub.3 .[..[. 1664 .]..].
.Badd.1663.Baddend. .[..[. C.sub.75 H.sub.106 N.sub.16 O.sub.27
.]..]. C.sub.75 H.sub.107 N.sub.17 O.sub.26 C.sub.4 .[..[. 1650
.]..]. .Badd.1649.Baddend. .[..[. C.sub.74 H.sub.104 N.sub.16
O.sub.27 .]..]. C.sub.74 H.sub.105 N.sub.17 O.sub.26 C.sub.5 .[..[.
.[.1664.]..Iadd.1650.Iaddend. .]..]. 1649 .[..[. C.sub.74 H.sub.104
N.sub.16 O.sub.27 .]..]. C.sub.74 H.sub.105 N.sub.17 O.sub.26
__________________________________________________________________________
Table V summarizes the absorption maxima of the ultraviolet
absorption spectra of the three major A-.Badd.21978C factors (Na
salt forms) in neutral ethanol.
TABLE V ______________________________________ UV Maxima
(ethanol-neutral) E.sub.1cm.sup.1% nm C.sub.1 C.sub.2 C.sub.3
______________________________________ 223 307 303 300 260 62 62 63
280 39 41 42 290 35 36 38 360 33 33 32
______________________________________
Table VI summarizes the the electrometric titration data, as
determined in 66% aqueous dimethylformamide, for the three major
A21978C factors and the A-21978C complex (Na salt forms).
TABLE VI ______________________________________ Titration (66% DMF)
A-21978C pK.sub.a Values* ______________________________________
Factor C.sub.1 ** 5.7, 5.9; 7.2, 7.6 Factor C.sub.2 ** 5.8, 5.93;
7.6, 7.63 Factor C.sub.3 ** 5.73, 5.75; 7.54, 7.58 Complex 5.62;
7.16 ______________________________________ *All have lesser groups
at 11.5-12 **Two determinations
The optical rotations of the A-21978C factors (Na salts),
[a].sub.D.sup.25, when determined in water are summarized in Table
VII.
TABLE VII ______________________________________ Optical Rotations
A-21978C Factor Rotation ______________________________________
C.sub.0 +11.9.degree. (c 0.7, H.sub.2 O) C.sub.1 +16.9.degree. (c
0.7, H.sub.2 O) C.sub.2 +18.6.degree. (c 0.9, H.sub.2 O) C.sub.3
+20.9.degree. (c 0.4, H.sub.2 O) C.sub.4 +14.8.degree. (c 0.7,
H.sub.2 O) C.sub.5 +17.9.degree. (c 0.7, H.sub.2 O)
______________________________________
The A-21978C factors may be separated by high-performance liquid
chromatography (HPLC), using the following conditions:
Column: glass 1.times.21 cm
Packing: silica gel/C.sub.18 (Quantum LP-1)
Solvent: water:methanol:acetonitrile (95:30:75) containing 0.2%
acetic acid and 0.2% pyridine
Detector: UV at 285 nm
Pressure: 100 psi
The retention times for the A-21978C factors (Na salts) are
summarized in Table VIII.
TABLE VIII ______________________________________ HPLC Retention
Times A-21978C Time Bioassay (Micrococcus luteus) Factor (minutes)
(units/mg) ______________________________________ C.sub.0 6 966
C.sub.1 8 1663 C.sub.4 9 1410 C.sub.2 13 1390 C.sub.5 14 1246
C.sub.3 19 803 ______________________________________
The A-21978C complex can be separated and dintinguished from
A-21978 factors A, B, D and E by using silica-gel thin-layer
chromatography (TLC). Acetonitrile:water (3:1) is a preferred
solvent system, and bioautography with Micrococcus luteus is a
preferred detection method. The approximate R.sub.f values of these
A-21978 factors (Na salt forms) are given by Table IX.
TABLE IX ______________________________________ A-21978 Factor
R.sub.f Value ______________________________________ A 0.66 B 0.57
C complex 0.31 D 0.51 E 0.48
______________________________________
The factors of the A-21978C complex can be separated and
distinguished from each other most conveniently using
reversed-phase silica-gel TLC (Quantum, C.sub.18). A preferred
solvent system is water:methanol:acetonitrile (45:15:40) which
contains (0.2 percent pyridine and 0.2 percent acetic acid.
Long-wave UV light (365 nm) may be used for detection. The
approximate R.sub.f values of the A-21978C factors (Na salt forms)
in this system are given in Table X.
TABLE X ______________________________________ A-21978C Factor
R.sub.f Value ______________________________________ C.sub.0 0.71
C.sub.1 0.64 C.sub.2 0.56 C.sub.3 0.47 C.sub.4 0.63 C.sub.5 0.53
______________________________________
The A-21978C factors and the A-21978C complex are stable in
solutions having a pH of 2-9 at 5.degree. C. and 25.degree. C. for
at least seven days. They are unstable at pH 11 after four hours
(total inactivation) at both 5.degree. C. and 25.degree. C.
The A-21978 and A-21978C complexes and individual A-21978C factors
C.sub.0, C.sub.1, C.sub.2, C.sub.3, C.sub.4 and C.sub.5 are capable
of forming salts. These salts are also part of this invention. Such
salts are useful, for example, for separating and purifying the
complexes and the individual factors. In addition, pharmaceutically
acceptable salts are especially useful.
"Pharmaceutically-acceptable" salts are those in which the toxicity
of the compound as a whole toward warm-blooded animals is not
increased relative to the non-salt form.
It will be appreciated that the A-21978 antibiotics have as many as
five free carboxyl groups which can form salts. Partial, mixed and
complete salts are, therefore, contemplated as part of this
invention. In preparing these salts, pH levels greater than 10
should be avoided due to the instability of the antibiotics at such
levels.
The A-21978 antibiotics also have two free amino groups and can,
therefore, form mono- or di-acid-addition salts.
Pharmaceutically-acceptable alkali-metal, alkaline-earth-metal and
amine salts and acid-addition salts are particularly useful.
Representative and suitable alkali-metal and alkaline-earth metal
salts of the A-21978 antibiotics include the sodium, potassium,
lithium, cesium, rubidium, barium, calcium and magnesium salts.
Suitable amine salts of the A-21978 antibiotics include the
ammonium and the primary, secondary, and tertiary C.sub.1 -C.sub.4
-alkylammonium and hydroxy-C.sub.2 -C.sub.4 -alkylammonium salts.
Illustrative amine salts include those formed by reaction of an
A-21978 antibiotic with ammonium hydroxide, methylamine,
sec-butylamine, isopropylamine, diethylamine, di-isopropylamine,
ethanolamine, triethylamine, 3-amino-1-propanol and the like.
The alkali-metal and alkaline-earth-metal cationic salts of the
A-21978 antibiotics are prepared according to procedures commonly
used for the preparation of cationic salts. For example, the free
acid form of A-21978C factor C.sub.1, is dissolved in a suitable
solvent such as warm methanol or ethanol; a solution containing the
stoichiometric quantity of the desired inorganic base in aqueous
methanol is added to this solution. The salt thus formed can be
isolated by routine methods, such as filtration or evaporation of
the solvent.
The salts formed with organic amines can be prepared in a similar
manner. For example, the gaseous or liquid amine can be added to a
solution of A-21978C factor C.sub.1 in a suitable solvent such as
acetone; the solvent and excess amine can be removed by
evaporation.
Representative and suitable acid-addition salts of the A-21978
antibiotics include those salts formed by standard reaction with
both organic and inorganic acids such as, for example,
hydrochloric, sulfuric, phosphoric, acetic, succinic, citric,
lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic,
d-glutamic, d-camphoric, glutaric, glycolic, phthalic, tartaric,
lauric, stearic, salicylic, methanesulfonic, bezenesulfonic,
sorbic, picric, benzoic, cinnamic and like acids.
It is well known in the veterinary pharmaceutical art that the form
of an antibiotic is not ordinarily of great significance when
treating an animal with the antibiotic. In most cases, conditions
within the animal change the drug to a form other than that in
which it was administered. The salt form in which it may be
administered is, therefore, not of great significance. The salt
form may, however, be chosen for reasons of economy, convenience,
and toxicity.
The novel antibiotics of this invention are produced by culturing
an A-21978-producing strain of Streptomyces roseosporus under
submerged aerobic conditions in a suitable culture medium until
substantial antibiotic activity is produced. The antibiotics are
recovered by the use of various isolation and purification
procedures recognized in the fermentation art.
THE MICROORGANISM
The microorganism of this invention was studied and characterized
by Frederick P. Mertz and Ralph E. Kastner of the Lilly Research
Laboratories.
The new organism useful for the preparation of the A-21978C
antibiotics was isolated from a soil sample collected on Mount
Ararat, Turkey. This organism is classified as a novel strain of
Streptomyces roseosporus. Falcao de Morias and Dailia Maia 1961.
This classification is based on a comparison with published
descriptions [R. E. Buchanan and N. E. Gibbons, "Bergey's Manual of
Determinative Bacteriology," The Williams and Wilkins Company, 8th
Ed., 1974; and E. B. Shirling and D. Gottlieb, "Cooperative
Description of Type Strains of Streptomyces," Intern. Journal of
Systematic Bacteriol., 808-809 (1972)].
This classification is based on methods recommended for the
International Streptomyces Project [E. B. Shirling and D. Gottlieb,
"Methods of Characterization of Streptomyces Species," Intern.
Journal of Systematic Bacteriol. 16, 313-340 (1966)] along with
certain supplementary tests. Carbon utilization was determined on
ISP #9 basal medium to which carbon sources were added to equal a
final concentration of 1.0%. The carbon sources were sterilized by
filtration; the basal medium was sterilized by autoclaving. Plates
were read after 14 days incubation at 30.degree. C. The cell-wall
sugars were determined using a modification of the procedure of
Lechevalier, (M. P. Lechevalier, "Chemical Methods as Criteria for
the Separation of Actinomycetes into Genera," Workshop sponsored by
the Subcommittee on Actinomycetes of the American Society of
Microbiology, Dr. Thomas G. Pridham, Convenor; held at the
Institute of Microbiology, Rutgers University, The State University
of New Jersey, New Brunswick, New Jersey, 1971.) The isomer of
diaminopimelic acid was determined using the method of Becker et
al. [B. Becker, et al., "Rapid Differentiation Between Norcardia
and Streptomyces by Paper Chromatography of Whole Cell
Hydrolysates," Appl. Microbiol. 11, 421-423 (1964)]. Amino acid
analysis was determined with washed cell-wall fragments. Melanoid
pigments were determined using ISP #1 (tryptone-yeast extract
broth), ISP #6 (peptone-yeast extract iron agar), ISP #7 (tyrosine
agar), ISP #7 modified (ISB #7 without tyrosine), and a tyrosine
assay [Yuzuru Mikami, et al., "Modified Arai and Mikani Melanin
Formation Test of Streptomyces," Intern. Journal of Systematic
Bacteriol. 27(3), 290 (1977)]. Starch hydrolysis was determined by
testing for the purpose of starch with iodine.
Temperature range, NaCl tolerance, pH range, and antibiotic
sensitivity were done using ISP #2 agar medium. The range of
temperatures were: 25.degree., 28.degree., 30.degree., 34.degree.,
37.degree., 40.degree., 45.degree., 50.degree. and 55.degree. C.
NaCl tolerance was measured by adding NaCl to the agar to equal: 0,
1, 2, 3, 4, 5, 6, 8, 10 and 12%. These were incubated at 30.degree.
C. The pH range was measured by adjusting the agar from pH 3.0 to
11.0 at increments of 1.0 pH units, just prior to pouring.
Antibiotic sensitivity was determined using sensitivity discs
padded onto seeded agar plates.
Color names were assigned according to ISCCNBS method (K. L. Kelly
and D. B. Judd, "The ISCCNBS Methods of Deisgnating Colors and a
Dictionary of Color Names," U.S. Department of Commerce Circ. 553,
Washington, D.C., 1955).
Figures in parentheses refer to the Tresner and Backus color series
[H. D. Tresner, and E. J. Backus, "System of Color Wheels for
Streptomycete Taxonomy," Appl. Microbiol. 11, 335-338 (1956)].
Color tab designations are underlined. The Maerz and Paul color
blocks are enclosed in brackets (A. Maerz and M. R. Paul,
"Dictionary of Color," McGraw-Hill Book Company, Inc., New York,
N.Y. 1950).
CHARACTERIZATION OF A-21978-PRODUCING STRAIN
Morphology
The morphology of culture A-21978.6, the culture which produces the
A21978 antibiotics, consists of sporophores which are of the
Rectus-Flexibilis (RF) classification. Spore chains have >10
spores per chain. Spore surface is smooth.
Culture A-21978.6 is characterized by the production of a
predominantly red aerial spore mass color, with a reddish-brown
reverse color. A light-brown water-soluble pigment is also present.
These characteristics are exhibited on three of 14 agar plating
media (ISP #2, ISP #7, TPO). These three media are the only ones
which supported abundant serial and vegetative growth.
Two agar plating media, ISP #4 and glucoseasparagine agar,
producing a white-to-gray aerial spore mass color, with a yellow
reverse color. No water-soluble pigment was observed. These two
media supported good, but not abundant, serial and vegetative
growth.
Nine other agar plating media were used, but these gave poor-to-no
growth and sporulation. Aerial color when present, although poor,
was in the white-to-gray color series.
Melanoid pigments are absent. Major constituents of the cell wall
are: LL-DAP, glycine, glucose, and ribose. This indicates a Type I
cell wall, and type C sugar pattern (R. E. Buchanan, and N. E.
Gibbons, Eds., "Bergey's Manual of Determinative Bacteriology," The
Williams & Wilkins Company, 8th Edition, 1974, p. 658).
The following five cultures were compared in laboratory tests to
A-21978.6:
Streptomyces albovinaceous ISP 5136; ATCC 15833
Streptomyces candidus ISP 5141; ATCC 19891
Streptomyces moderatus ISP 5529; ATCC 23443
Streptomyces roseosporus ISP 5122; ATCC 23958
Streptomyces setonii ISP 5395; ATCC 25497
These cultures belong to the white and red color series, have RF
type sporophore morphology, smooth spore surface ornamentation,
and, according to the ISP descriptions, are melanin negative and do
not have a distinctive reverse color or water-soluble pigments.
These characteristics, together with carbon-utilization pattern and
other secondary features, match those of culture A-21978.6.
When these cultures were compared with A-21978.6 under laboratory
conditions, four were rejected. S. candidus and S. setonii
exhibited a yellow serial spore mass on many media, thereby
differing from culture A-21978.6 S. albovinaceous and S. moderatus
exhibited dark distinctive reverse color, water-soluble pigments,
and produced melanoid pigments, all of which were different from
culture A-21978.6. The ISP description of S. moderatus refers to
reddish brown or strong-brown reverse color, but does not refer to
such a characteristic for S. albovinaceous. Neither culture is
listed as melanin positive.
Culture A21978.6 was classified, therefore, as a strain of
Streptomyces roseosporus, Falcao de Morias and Dalia Maia 1961.
This classification was based on comparison with published
description and direct laboratory comparisons. The following
cultural characteristics summarize the direct comparison
studies.
__________________________________________________________________________
CULTURAL CHARATERISTICS Morphology A21978.6 S. roseosporus
Sporophores straight to flexuous (RF), with no hooks, loops or
spirals observed. Chains of spores > 10. The spore surface
smooth as determined by scanning electron microscopy. Spores:
Oblong to oval Oblong to cylindrical Average: 0.85 .times. 1.78
.mu.M 1.01 .times. 2.47 .mu.M Range: 0.65-0.97 .times. 0.97-2.6
.mu.M 0.97-1.3 .times. 1.63-3.25 .mu.M Growth Color Growth Color
__________________________________________________________________________
Carrot Plugs Aerial: good gray - c pink none none Vegetative:
Abundant brown good yellow-brown no soluble pigment no soluble
pigment Potato Plugs Aerial: good gray - c pink none none
Vegetative: abundant brown fair orange-brown dark brown soluble
pigment no soluble pigment ISP #1 (Tryptone-yeast ext. agar)
Aerial: fair (W) a white poor (W) a white Vegetative: good [10A1]
pale yellow green poor [10B2] pale yellow green no soluble pigment
no soluble pigment ISP #2 (Yeast-malt extract agar) Aerial:
abundant (R) 5cb gy. yellow pink abundant (R) 3ca pale orange
yellow Vegetative: abundant [5D10] lt. red brown abundant [12L7]
lt. olive brown light brown sol. pigment light brown sol. pigment
ISP #3 (Oatmeal agar) Aerial: fair (W) a white poor (W) a white
Vegetative: fair [10A2] pale yellow pink fair pale greenish gray
light brown sol. pigment no soluble pigment ISP #4 (Inorganic salts
starch agar) Aerial: good (W) b white good 3c2 pale orange yellow
Vegetative: good [10B1] pale yellow-green abundant [1115] grayish
yellow light brown sol. pigment no soluble pigment ISP #5
(Glycerol-asparagine agar) Aerial: fair (W) 13ba purplish white
fair (W) b white Vegetative: good [3B7] gy. yellow pink good [10C2]
grayish yellow gy. pink sol. pigment light brown sol. pigment ISP
#7 (Tyrosine agar) Aerial: abundant (R) 5cb gy. yell. pink abundant
(R) 5cb gy. yell. pink Vegetative: abundant [7L12] mod. red brown
abundant [11E5] yellow-brown dark brown sol. pigment light brown
sol. pigment Bennett's modified agar Aerial: none -- abundant (R)
5cb gy. yell. pink Vegetative: poor pale yellow br. abundant [11D4]
grayish yellow no soluble pigment light brown sol. pigment Calcium
malate agar Aerial: none -- poor (W) a white Vegetative: fair
[7L12] mod. red brown poor pale yellow-green light brown sol.
pigment pale yell.-green sol. pigment Czapek's solution agar
Aerial: poor (W) a white none -- Vegetative: poor off-white none --
no soluble pigment Emerson's agar Aerial: poor -- abundant (R) 5cb
gy. yell. pink Vegetative: abundant [13L6] abundant [1115] gy.
yellow no soluble pigment light brown sol. pigment
Glucose-asparagine agar Aerial: poor (W) b white fair (W) b white
Vegetative: good [12B2] gy. yellow good [12B2] pale yell. green no
soluble pigment no soluble pigment Glycerol-glycine agar Aerial:
poor -- abundant (W) b white Vegetative: abundant [8L12] dk. gy.
brown abundant [10G3] light yellow brown soluble pigment light
brown sol. pigment Nutrient agar Aerial: none -- fair (W) b white
Vegetative: poor pale yellow-gray good pale yellow gray no soluble
pigment no soluble pigment (Tomato-paste Oatmeal agar) Aerial:
abundant (R) 5cb gy. yell. pink abundant (R) 5cb gy. yell. pink
Vegetative: abundant [8L12] dk. gy. brown abundant [12L7] yell.
brown brown soluble pigment brown soluble pigment
__________________________________________________________________________
______________________________________ Carbon Utilization Substrate
A21978.6 S. roseosporus ______________________________________
L-Arabinose + + D-Fructose + - D-Galactose + + D-Glucose + +
i-Inositol - - D-Mannitol + - D-Raffinose - - L-Rhamnose + +
Salicin + + Sucrose - - D-Xylose + +
______________________________________ Key: + = Positive
utilization - = Negative utilization
______________________________________ S. roseo- Characteristic
A21978.6 sporus ______________________________________ Melanoid
Pigments - - ISP #1 (tryptone-yeast ext.) - - ISP #6 (peptone-yeast
ext. iron) - - ISP #7 (tyrosine agar) - - ISP #7 mod. (ISP #7 minus
tyrosine) - - Tyrosine assay - - Gelatin liquefaction + + Skim milk
action slight slight hydrolysis hydrolysis Starch hydrolysis + + pH
range 5-11 5-11 Temperature range 25-40.degree. C. 25-45.degree. C.
Nitrate reduction - + NaCl tolerance: growth up to 10% 6%
______________________________________
______________________________________ Antibiotic Sensitivity
Conc./ S. roseo- Antibiotic Disc Class A21978.6 sporus
______________________________________ Erythromycin 15 .mu.g
Macrolide + + Cephalothin 30 .mu.g .beta.-Lactam + + Lincomycin 2
.mu.g Glycoside - - Nystatin 100 units Polyene - - Polymyxin B 300
units Peptide + - Streptomycin 10 .mu.g Aminoglycoside + +
Tetracycline 30 .mu.g Tetracycline + + Vancomycin 30 .mu.g
Glycopeptide + + ______________________________________ + =
sensitive (zones of inhibition) - = resistant (no zones of
inhibition)
Certain characteristics of the A-21978-producing S. roseosporus,
NRRL 11379 strain differ from the characteristics published for S.
roseosporus. Culture A21978.6 differs from the published strain in
spore size, carrot-and potato-plug growth, NaCl tolerance, and in
nitrate reduction.
The Streptomyces roseosporus culture useful for the production of
the A-21978 antibiotics has been deposited and made a part of the
stock culture collection of the Northern Regional Research Center,
U.S. Department of Agriculture. Agricultural Research Service,
Peoria, Illinois, 61604, from which it is available to the public
under the number NRRL 11379.
As is the case with other organisms, the characteristics of the
A-21978-producing culture, Streptomyces roseosporus NRRL 11379, are
subject to variation. For example, artificial variants and mutants
of the NRRL 11379 strain may be obtained by treatment with various
known mutagens such as ultraviolet rays, X-rays, high-frequency
waves, radioactive rays and chemicals. All natural and artificial
variants and mutants of Streptomyces roseosporus NRRL 11379 which
produce the A-21978 antibiotics may be used in this invention.
The culture medium used to grow Streptomyces roseosporus NRRL 11379
can be any one of a number of media. For economy in production,
optimal yield, and ease of product isolation, however, certain
culture media are preferred. Thus, for example, a preferred carbon
source in large-scale fermentation is tapioca dextrin, although
glucose, fructose, galactose, maltose, mannose, cottonseed oil,
methyl oleate, glycerol, refined soybean oil, and the like can also
be used. A preferred nitrogen source is enzyme-hydrolyzed casein,
although soluble-meat peptone, soybean flour, soybean hydrolysate,
soybean grits, yeast, amino acids such as L-asparagine and
DL-leucine, and the like are also useful. Nutrient inorganic salts
which can be incorporated in the culture media are the soluble
salts capable of yielding potassium, ammonium, chloride, sulfate,
nitrate and like ions. Among these, K.sub.2 SO.sub.4 is especially
useful for antibiotic production. Molasses ash, ash dialysate and
synthetic mineral mix are also useful.
For production of the A-21978 antibiotics, it is preferable to use
distilled or deionized water in the fermentation medium. Some of
the minerals in tap water, such as, for example, calcium and
carbonate, appear to discourage antibiotic production.
Essential trace elements necessary for the growth and development
of the organism should also be included in the culture medium. Such
trace elements commonly occur as impurities in other constituents
of the medium in amounts sufficient to meet the growth requirements
of the organism.
It may be necessary to add small amounts (e.g., 0.2 ml/L.) of an
antifoam agent such as polypropylene glycol to large-scale
fermentation media if foaming becomes a problem.
For production of substantial quantities of the A-21978
antibiotics, submerged aerobic fermentation in tanks is preferred.
Small quantities of the A-21978 antibiotics may be obtained by
shake-flask culture. Because of the time lag in antibiotic
production commonly associated with inoculation of large tanks with
the spore form of the organism, it is preferable to use a
vegetative inoculum. The vegetative inoculum is prepared by
inoculating a small volume of culture medium with the spore form or
mycelial fragments of the organism to obtain a fresh, actively
growing culture of the organism. The vegetative inoculum is then
transferred to a larger tank.
The A-21978-producing organism can be grown at temperatures between
about 20.degree. and about 37.degree. C. Optimum A-21978C
production appears to occur at temperatures of about
30.degree.-32.degree. C.
As is customary in aerobic submerged culture processes, sterile air
is dispensed through the culture medium. For efficient production
of the A-21978 antibiotics the percent of air saturation for tank
production should be above 20%, preferably above 30% (at 30.degree.
C. and one atmosphere of pressure).
For tank fermentation, it is preferable to maintain the pH level of
the fermentation medium in a range of from about 6.5-7.0. This can
be done by the addition of appropriate amounts of, for example,
sodium hydroxide (in the early stages) and hydrochloric acid (in
the later stages).
Production of the A-21978 antibiotics can be followed during the
fermentation by testing samples of the broth or of extracts of the
mycelial solids for antibiotic activity against organisms known to
be sensitive to the antibiotics. One assay organism useful in
testing these antibiotics is Micrococcus luteus. The bioassay is
preferably performed by paper-disc assay on agar plates.
Following their production under submerged aerobic fermentation
conditions, the A-21978 antibiotics can be recovered from the
fermentation medium by methods recognized in the fermentation art.
The antibiotic activity produced during fermentation of an
A-21978-producing organism generally occurs in the broth. Maximum
recovery of the A-21978 antibiotics is accomplished, therefore, by
an initial filtration to remove the mycelial mass. The filtered
broth can be purified by a variety of techniques to give the
A-21978 complex. A preferred method involves extraction and
precipitation to give the A-21978 complex.
Further purification and separation of the A-21978C complex and the
individual A-21978C factors includes additional adsorption and
extraction procedures. Useful adsorptive materials for the
purification of the A-21978C complex and factors include: (1)
Anion-exchange resins--(a) strongly basic; polystyrene, BioRad AG 1
& 2, Bio-Rex, Dowex 1 and 2, Amberlite IRA 400, 401, 410; (b)
moderately basic; epoxypolyamine Bio-Rex 5, and Duolite A30b, (c)
weakly basic; polystyrene or phenolic polyamine Bio-Rad AG3,
Duolite A-6, A-7, Amberlite IRA 68, , IR-45, IR-4B; (2) silica gel;
(3) florisil; (4) polymeric adsorbents (XAD-2 and 4); (5) high
porous polymer (Diaion HP-20); (6) Sephadex G-10, G-25, and G-50;
Bio-Gel P-2 and P-10; (7) reversed-phase resins, silica
gel/C.sub.18 and silica Gel/C.sub.8 ; (8) carbon; (9) DEAE
cellulose, DEAE Sephadex; (10) polyamide; (11) alumina; and (12)
microcellulose. Sources: Bio-Rad and Bio-Gel resins-Bio Rad
Laboratories, Richmond, Cal.; Amberlite and XAD resins-Rohm and
Haas Co., Philadelphia, Pa.; Duolite resins-Diamond Shamrock
Chemical Co., Redwood City, Cal.; Sephadex resins-Pharmacia Fine
Chemicals AB, Uppsala, Sweden; Dowex resins-Dow Chemical Co.,
Midland, Mich.; Diaion-Mitsubishi Chemical Industries Ltd., Tokyo,
Japan; XAD resins; silica gel/C.sub.18 and silica gel/C.sub.8 -E.
Merck, Darmstadt, Germany.
Alternatively, the culture solids, including medium constituents
and mycelium can be used without extraction or separation, but
preferably after removal of water, as a source of A-21978
antibiotics. For example, after production of A-21978 antibiotic
activity, the culture medium can be dried by lyophilization and
mixed directly into feed premix.
Activity of the A-21978 Antibiotics
The A-21978C complex and the individual A-21978C factors used in
the tests herein discussed were always in the sodium salt form.
The A-21978 and A-21978C complexes and individual A-21978C
antibiotic factors C.sub.0, C.sub.1, C.sub.2, C.sub.3, C.sub.4 and
C.sub.5 inhibit the growth of certain pathogenic organisms,
particularly gram-positive bacteria. The minimal inhibitory
concentrations (MIC's) at which the A-21978C factors and the
A-21978C complex inhibit selected bacteria, as determined by
standard agar-dilution tests, are summarized in Table XI.
TABLE XI
__________________________________________________________________________
MIC (.mu.g/ml) Organism (aerobic) Complex C.sub.0 C.sub.1 C.sub.2
C.sub.3 C.sub.4 C.sub.5
__________________________________________________________________________
Staphylococcus aureus 3055 0.13 1.0 0.5 0.13 0.06 0.25 0.13 Group D
Streptococcus 282 0.25 2.0 1.0 0.25 0.13 1.0 0.13 Streptococcus
pyogenes C203 0.13 0.25 0.13 0.13 0.25 0.13 0.06 Streptococcus
pneumoniae Park I 0.13 0.5 0.13 0.25 0.13 0.5 0.06 Viridans
Streptococcus 9943 0.5 1.0 0.5 1.0 0.5 1.0 0.13 Neisseria
gonorrhoeae 111076-4 8.0 NT* 16.0 4.0 4.0 NT NT
__________________________________________________________________________
NT = not tested
The minimal inhibitor concentrations at which A-21978C complex and
the major A-21978C factors inhibit selected bacteria, as determined
by standard broth-dilution tests are summarized in Table XII.
TABLE XII ______________________________________ MIC (.mu.g/ml)
Organism (aerobic) Complex C.sub.1 C.sub.2 C.sub.3
______________________________________ Staphylococcus aureus 3055
0.25 1.0 0.5 0.13 Group D Streptococcus 282 0.25 2.0 1.0 0.13
Streptococcus pyogenes C203 0.13 0.5 0.25 0.13 Streptococcus
pneumoniae Park I 0.5 2.0 1.0 .5 Viridans Streptococcus 9943 8.0
32.0 16.0 32.0
In one important aspect, the A-21978C antibiotics inhibit the
growth of organisms which are resistant to other antibiotics. Table
XIII summarizes agar-dilution MIC values of A-21978C factors
C.sub.0, C.sub.1, C.sub.2, C.sub.3, C.sub.4, and C.sub.5 against
representative organisms, using the ICS agar-dilution
techniques.
TABLE XIII
__________________________________________________________________________
EFFECTIVENESS OF A-21978C FACTORS AGAINST CLINICAL ISOLATES Minimum
Inhibitory Concentration (.mu.g/ml)** Test Organism* A21978C.sub.0
A21978C.sub.1 A21978C.sub.2 A21978C.sub.3 A21978C.sub.4
A21978C.sub.5
__________________________________________________________________________
Staphylococcus aureus (10) 1.0[10] 0.5[10] 0.12-0.25[10]
0.06-0.12[10] 0.25-0.5[10] 0.06-0.25[10] Staphylococcus epidermidis
(12) 1-2[12] 0.13-0.25[9] 0.13-0.25[9],0.5 0.06-0.25[11],1
0.25-1.0[12] 0.13-0.5[12] Streptococcus pyogenes (7)
0.25-5[5],32,>32 0.12[5],8,16 0.06-0.12[5],4,8 0.06-0.25[6],8
0.13[5],16.32 0.06[5],4,16 Group D Streptococcus (9) 2-4[8],>32
1-2[8],>16 0.25-0.5[8],8 0.12-0.25[8],4 0.5-1.0[8],32
0.13-0.25[8],>32 Streptococcus pneumoniae (8) 0.13-1.0[7],4
0.12-1[7],8 0.12[5],0.5,4[2] 0.06-0.25[7],4 0.5[7],2 0.06[7],2
Viridans Streptococcus (2) 1-4[2] 0.5,8 0.5,4 0.5[2] 1-2[2]
0.13-0.25[2] Neisseria gonorrhoeae (11) NT*** 16->128[11]
4->128[11] 4->128[9] NT*** Nt
__________________________________________________________________________
*The number in parenthesis = the number of isolates tested **The
number in brackets = the number of isolates having this MIC or MIC
range; where there is no number in brackets, only one isolate had
this MIC. ***NT = not tested
A-21978C antibiotics also inhibit the growth of certain anaerobic
bacteria. Table XIV summarizes the activity of the A-21978C complex
and A-21978C factors C.sub.1, C.sub.2 and C.sub.3 against various
anaerobic bacteria, using the standard agar-dilution test.
TABLE XIV
__________________________________________________________________________
MIC (mcg/ml) Test Organism* C.sub.0 C.sub.1 C.sub.2 C.sub.3 C.sub.4
C.sub.5 Complex
__________________________________________________________________________
Actinomyces israelii 2 4 1.0 1.0 1.0 0.5 1.0 Clostridium
perfringens 2 16 8 8 1.0 0.5 8 Clostridium septicum 4 4 1.0 1.0 1.0
0.5 1.0 Eubacterium aerofaciens 4 16 8 4 2 0.5 8 Peptococcus
asaccharolyticus 4 4 2 1.0 1.0 0.5 1.0 Peptococcus prevoti 4 2 1.0
<0.5 2 0.5 <0.5 Peptostreptococcus anaerobius 0.25 2 1.0 1.0
0.25 0.25 1.0 Peptostreptococcus intermedius 2 4 1.0 <0.5 1.0
0.25 1.0 Propionibacterium acnes 1 8 2 1.0 0.5 0.25 2 Bacteroides
fragilis >128 >128 >128 >128 >128 >128 >128
Fusobacterium symbiosum 4 >128 >128 16 4 2 >128
Fusobacterium necrophorum 2 64 64 32 4 0.5 >128
__________________________________________________________________________
The A-21978C factors have shown in vivo antimicrobial activity
against experimental bacterial infections. When two doses of test
compound were administered to mice in illustrative infections, the
activity observed as measured as an ED.sub.50 value [effective dose
in mg/kg to protect fifty percent of the test animals: See Warren
Wick, et al., J. Bacteriol, 81, 233-235 (1961)]. The ED.sub.50
values observed for A-21978C complex and A-21978C factors C.sub.1,
C.sub.2 C.sub.3, C.sub.4, and C.sub.5 are given in Table XV.
TABLE XV
__________________________________________________________________________
COMPARATIVE IN VITRO AND IN VIVO ACTIVITY Staphylococcus aureus
Streptococcus pyogenes Streptococcus pneumoniae Antibiotic
MIC.sup.1 ED.sub.50.sup.2 MIC ED.sub.50.sup.2 ED.sub.50.sup.3 MIC
ED.sub.50.sup.2
__________________________________________________________________________
A21978C.sub.1 0.5 0.22 0.13 0.064 93 0.13 0.3 A21978C.sub.2 0.13
0.16 0.13 0.032 59 0.13 0.14 A21978C.sub.3 0.06 0.08 0.06 0.032 66
0.03 0.09 A21978C.sub.0 0.25 0.16 0.5 0.88 A21978C.sub.4 0.13 0.10
0.5 0.36 A21978C.sub.5 0.06 0.053 0.06 0.17 A21978C 0.13 0.18
<0.03 0.043 0.13 0.1 complex Erythromycin 0.13 0.5 0.13 0.64 0.5
7.3
__________________________________________________________________________
.sup.1 MIC = minimum inhibitory concentration (.mu.g/ml), agar
dilution .sup.2 subcutaneous administration .sup.3 oral
administration
In an important aspect of this invention, the A-21978C factors and
A-21978C complex are effective in the treatment of pyelonephritis.
For example, in an experimental descending pyelonephritis infection
in rats, the A-21978C factors afforded protection which was
superior to that provided by vancomycin. In this test, the
bacterial culture used was Streptococcus faecalis (Guze). The
culture was grown on Trypticae soy agar (BBL), suspended in brain
heart infusion broth (BBL), divided into 0.2-ml portions, and
frozen in liquid nitrogen. Bacterial suspensions for rat
inoculations were prepared daily by seeding a 50-ml flask of
trypticase soy broth (BBL) from a frozen ampoule and growing the
culture overnight at 37C on a shaker. The S. faecalis culture was
diluted to 5.times.10.sup.8 colony-forming units per ml. Test
compounds were injected subcutaneously once daily for seven days.
All compounds were suspended in 0.125% carboxymethylcellulose.
The experimental rat infections were accomplished by the following
procedure. Female, random-bred albino rats (Cox-Wistar) weighing
190 to 210 g were anesthetized by intraperitoneal injection of 12
mg of sodium methohexital supplemented as necessary. The
experimental pyelonephritis model was based on the studies of Guze
and Beeson in which the left ureter was occluded for 20 min,
followed by injection of 0.5 ml of the test organism in the femoral
vein. Antimicrobial therapy was commenced 4 to 5 hrs postinfection.
Four hours after the last treatment the rats were sacrificed, and
the left kidney was removed and homogenized in a Duall grinder
containing 9 ml of physiological saline. This represented at
10.sup.-1 dilution of the kidney tissue. Additional 10-fold
dilutions in saline were based on the anticipated bacterial cells
present in the tissue homogenate. Finally, duplicate agar pour
plates were made from several of these dilutions, and the plates
were incubated overnight at 37C. The therapeutic results were
expressed in two ways: (i) the percentage of rats with kidney
counts of less than 10.sup. 2 per g of kidney tissue, referred to
as "cures," and (ii) the percentage of rats with at least a
4-log.sub.10 reduction in bacterial titer compared with infected
control kidneys. Control rats were treated with 0.125%
carboxymethylcellulose only. Viable cell counts in kidney tissue
from control rats with S. faecalis ranged from 1.2.times.10.sup.8
to 4.6.times.10.sup.8 per g of homogenized tissue.
The results of these studies are summarized in Table XVI.
TABLE XVI ______________________________________ STREPTOCOCCUS
FAECALIS DESCENDING RAT PYELONEPHRITIS TEST Percent of Rats Percent
Antibiotic MIC.sup.1 Rat Dose.sup.2 with a 4-Log of Rats Tested
(.mu.g/ml) mg/kg .times. 7 Titer Decrease Cured
______________________________________ Vancomycin 1.0 12.0 55 33
A21978C.sub.1 1.0 1.0 50 50 A21978C.sub.2 0.25 1.0 100 89
A21978C.sub.3 0.13 1.0 78 78 A21978C 0.25 1.0 89 89 complex
______________________________________ .sup.1 In vitro
susceptibility of the S. faecalis Guze strain .sup.2 subcutaneous
administration
Toxicity data for the major A-21978C factors and the A-21978C
complex are summarized in Table XVII.
TABLE XVII ______________________________________ TOXICITY OF
A-21978C LD.sub.50 (mg/kg) Mouse Rat A-21978C IV SC IV
______________________________________ Factor C.sub.1 >250
>365 479 .+-. 32 Factor C.sub.2 150-250 175 204 .+-. 17 Factor
C.sub.3 <50 70-75 <160 Complex 150 175-190 169 .+-. 10
______________________________________
When A-21978C complex or an A-21978C factor is used as an
antibacterial agent, it may be administered either orally or
parenterally. As will be appreciated by those skilled in the art,
the A-21978C complex or factor is commonly administered together
with a pharmaceutically acceptable carrier or diluent. The dosage
of A-21978C complex or factor will depend upon a variety of
considerations, such as, for example, the nature and severity of
the particular infection to be treated. Those skilled in the art
will recognize the appropriate dosage ranges and/or dosage units
for administration may be determined by considering the MIC and
ED.sub.50 values and toxicity data herein provided together with
factors such as the patient or host and the infecting
microorganism.
The A-21978 antibiotics also useful as growth-promoting agents in
animals. In chickens, for example, the A-21978C complex improved
weight gains and feed efficiency. Table XVIII summarizes the
results of two tests demonstrating this activity. In these tests
the A-21978C complex was given to animals at a concentration of 25
grams per ton of feed. The antibiotic was fed to four replicates of
eight birds each in a time-replicated study conducted in batteries
(total of eight replicates of eight birds, or 64 birds). The test
period was the 21-day period from 7-28 days of age of the birds.
The growth-performance data (weight gain, feed consumption and feed
efficiency) were compared to that of 40 replicates of a
contemporary control treatment.
TABLE XVIII
__________________________________________________________________________
Conc. Wt. Gain. % Feed Conc. % Expt. Treatment (g/ton) (g)
Impr..sup.1 (g) Feed/Gain Impr.
__________________________________________________________________________
1 Control -- 414 -- 734 1.773 -- A21978C 25 431 4.10 750 1.741 1.80
2 Control -- 423 -- 704 1.665 -- A21978C 25 432 2.12 683 1.582 4.99
__________________________________________________________________________
##STR3##
The A-21978 antibiotics are typically effective in promoting growth
in poultry when administered with the animals' feed at rates of
from about one to about 100 grams of A-21978 antibiotic per ton of
animal feed.
In order to illustrate more fully the operation of this invention,
the following examples are provided.
EXAMPLE 1
A. Shake-flask Fermentation of A-21978C
A lyophilized pellet of Streptomyces roseosporus NRRL 11379 was
dissolved in 1-2 ml of sterilized water. This solution was used to
inoculate an agar slant having the following composition:
______________________________________ Ingredient Amount (%)
______________________________________ Glucose 0.5 Yeast extract
0.2 CaCO.sub.3 0.3 Agar 2.0 Vegetable juice* 20.0 Deionized water
______________________________________ Unadjusted pH 6.1;
postautoclaving pH 5.9 *V/8 Juice. Campbell Soup Co.
The inoculated slant was incubated at 30.degree. C. for about seven
to ten days. The mature slant culture was covered with sterile
distilled water (10 ml) and scraped with a sterile pipette to
loosen the spores. A portion (1 ml) of the resulting suspension of
spores was used to inoculate 50 ml of a vegetative medium having
the following composition:
______________________________________ Ingredient Amount (%)
______________________________________ Trypticase Soy Broth* 3.0
Dextrin 2.5 Water (deionized)
______________________________________ *Baltimore Biological
Laboratories. Cockeysville, Md.
The inoculated vegetative medium was incubated in a 250-ml
Erlenmeyer flask at 30.degree. C. for about 48 hours on a shaker
rotating through an arc two inches in diameter at 250 RPM.
This incubated vegetative medium (0.5 ml) was used to inoculate 50
ml of a production medium having the following composition:
______________________________________ Ingredient Amount (g/l.)
______________________________________ Glucose 7.5 Tapioca dextrin*
30.0 Enzymatic hydrolysate of casein** 5.0 Enzyme-hydrolyzed
casein*** 5.0 K.sub.2 SO.sub.4 17.4 L-Asparagine, anhydrous 1.32
Deionized water q.s. 1 liter
______________________________________
The inoculated production medium was incubated in a 250-ml
Erlenmeyer flask at 30.degree. C. for 6-7 days on a shaker rotating
through an arc two inches in diameter at 250 RPM.
B. Tank Fermentation of A-21978C
In order to provide a larger volume of inoculum, 10 ml of incubated
vegetative medium prepared as described above was used to inoculate
400 ml of a second-stage vegetative growth medium having the same
composition as that of the vegetative medium. This second-stage
medium was incubated in a 2-liter flask for 48 hours at 30.degree.
C. on a shaker rotating through an arc 2 inches in diameter of 250
RPM.
Inoculated second-stage vegetative medium (800 ml) thus prepared
was used to inoculate 100 liters of sterile production medium
having the same composition given in Sect. A. The inoculated
production medium was allowed to ferment in a 165-liter
fermentation tank for about 6-8 days at a temperature of 30
.degree. C. The fermentation medium was aerated with sterile air at
a pressure of one atmosphere to maintain an air saturation of about
30%, stirring with conventional agitators at 200-300 RPM.
EXAMPLE 2
Separation of A-21978C Antiobiotic Complex
Whole fermentation broth (1600 gal.), obtained as described in
Example 1, was filtered on a filter press, using 3% filter aid
(Celite 545, Johns-Manville Products Corp.). The filter cake was
washed with water to yield a total filtrate of 4100 liters assaying
230 units/ml. The pH of the filtrate was adjusted to 3.5 with HCl,
and the acidified filtrate was held at room temperature for 16
hours to allow the active factors to precipitate. Filter aid (0.75%
Celite 545) was added to the suspension; the precipitate was
separated by filtration. The filter cake was extracted twice with
410 liters of methanol, stirring each time for 1 hour before
filtering. To the combined methanol extracts (720 liters) was added
0.1 volume of water (72 liters). The pH of this solution was
adjusted to 6.5-7.0 with NaOH. The solution was concentrated under
vacuum to about 1/20th volume (30 liters) to remove the methanol;
distilled water was added as needed during the concentration.
n-Butanol (3/4 volume of 22 liters) was added with stirring. The pH
of the resulting solution was adjusted to 3.0 with HCl. The phases
were separated and the n-butanol phase, which contained the
activity, was concentrated under vacuum to a residue. This residue
was dissolved in a minimal amount of methanol; the methanol
solution was added to 30 volumes of acetone to precipitate the
major portion of the A-21978C complex. The precipitate was
separated by filtration and dried to yield 247 g of crude A-21978C
complex (780 units/mg).
The methanol-acetone filtrate containing the remaining portion of
the A-21978 complex (factors A and B) was concentrated to a
residue. The residue was dissolved in t-butanol:H.sub.2 O (5:1),
and this solution was freeze-dried to yield 169 g of A-21978
complex.
EXAMPLE 3
A. Purification of the A-21978Complex
Crude A-21978C complex (734 g), prepared as described in Example 2,
was suspended in water (25 liters); the pH of this suspension was
adjusted to 6.5 with 5N NaOH to completely dissolve the material.
This solution was applied to a column containing 27 liters of
ion-exchange (acetate cycle) resin (IRA68, Rohm & Haas Co.).
The column was washed with 4 column volumes of water (108 liters),
and then with 5 column volumes of 0.1N acetic acid (135 liters).
The active material was eluted with 0.5N acetic acid, collecting
ca. 120-liter fractions and assaying each fraction for biological
activity.
The highly active fractions were combined and freeze-dried to yield
278 g of brown-colored A-21978C complex (1100 units/mg); the
fractions with low activity were combined to yield 238 g of brown
A-21978C complex (880 units/mg).
B. Further Purification of A-21978C Complex
A portion of the more active A-21978C complex preparation (150 g)
from the IRA-68 column was suspended in water (600 ml); the pH was
adjusted to 6.5 to completely dissolve the suspended preparation; a
sufficient amount of dry silica gel (Grace, Grade 62) was added to
absorb the aqueous solution. This moist silica-gel preparation was
placed on a 30-liter silica-gel (Grace 62) column (10.times.375 cm)
packed in acetonitrile (the silica gel had been previously washed
with water to remove fine particles; the column was then packed
with the silica gel suspended in water; and the silica gel column
was washed with 30 liters of acetonitrile). After loading, the
column was washed with acetonitrile (15 liters), and then was
developed with acetonitrile:water (4:1), collecting about 4-liter
fractions. Elution was monitored by bioassay and silica-gel TLC
[CH.sub.3 CN:H.sub.2 O(3:1)] bioautogram. Fractions containing only
A-21978C complex (fractions 43-60) were combined, concentrated
under vacuum, and freeze-dried to yield 86.2 g of yellow-tan
purified A-21978C complex (1160 units/mg). Fractions 21-29,
containing factors D and C were combined and freeze-dried to yield
13 g of yellow powder with low biological activity.
The purified A-21978C complex (30 g) thus obtained was further
decolorized by suspending 30 g of the complex in a minimal amount
of water and mixing with a small amount of silica gel (Type LP-1,
10-20 microns, Quantum Industries, 341 Kaplan Drive, Fairfield,
N.J. 07006) to absorb the solution. The moist silica-gel mixture
was suspended in acetonitrile:methanol (4:1) and packed in a
4.times.30-cm (O.D.) glass lead column attached to a
6.5.times.82-cm (O.D.) glass column containing 2.8 liters of silica
gel (Quantum LP-1) packed in acetonitrile:methanol (4:1) [the
silica gel was washed previously with water and then
acetonitrile:methanol (4:1); and the column was packed with the
silica gel in acetonitrile:methanol (4:1) under 50-60 psi of
pressure]. The lead column and main column were washed with 3
liters of acetonitrile:methanol (4:1) at 50 psi. The active
material was eluted with acetonitrile:methanol:water (55:20:25),
collecting 300-ml fractions. Elution was monitored by bioassay
(Micrococcus luteus). Fractions 14-25 had the highest activity and
were combined, concentrated, and freeze-dried to yield 24 g of
light-yellow, pure A-21978C complex as the sodium salt (1250
units/mg). Fractions 26-32 were less active; they were combined,
concentrated, and freeze-dried to yield 1.6 g of less-pure A-21978C
complex (780 units/mg).
EXAMPLE 4
Separation of A-21978C Factors
Purified A-21978C complex (2 g), obtained as described in Example
3, was dissolved in water (40 ml) and applied through a pump (FMI
LAB Pump, Fluid Metering, Inc. 48 Summit St., Oyster Bay, NY 11771)
at 50 psi onto a 4.1.times.60-cm column of reverse-phase silica gel
(Quantum LP-1 silica gel/C.sub.18) set in
water:methanol:acetonitrile (100:15:85) containing 0.15% acetic
acid and 0.15% pyridine. The column was developed at 65 psi with
this solvent, collecting 25-ml fractions. Elution of factors was
monitored by UV at 280 nm and by bioassay. Individual fractions
were assayed on an analytical column for factor purity. Typical
separations were: fractions 33-37 contained factor C.sub.0 ;
fractions 45-53 contained factor C.sub.1 ; fractions 75-92
contained factor C.sub.2 ; fractions 112-134 contained factor
C.sub.3 ; fractions 54-74 contained factors C.sub.1, C.sub.2, and
C.sub.4 ; and fractions 93-111 contained factors C.sub.2, C.sub.3,
and C.sub.5. Fractions containing mixtures were rerun on the column
to obtain further yields of C.sub.1, C.sub.2, and C.sub.3, as well
as factors C.sub.4 and C.sub.5. The fractions containing a single
factor were combined, concentrated under vacuum, and freeze-dried
to give light yellow powders of each of the factors (as Na salts).
From 60 g of complex the yields were: factor C.sub.1 =5.55 g;
factor C.sub.2 =10 g; factor C.sub.3 =6.61 g. The fractions
containing mixed factors were recycled over the reversed-phase
resin column to give additional yields; factor C.sub.0 =550 mg;
factor C.sub.1 =1.29 g; factor C.sub.2 =1.99 g; factor C.sub.3 =443
mg; factor C.sub.4 =512 mg; and factor C.sub.5 =384 mg.
EXAMPLE 5
Large-Scale Separation and Purification of A-21978C Factors
On a larger scale, the factors were separated by reverse-phase
column chromatography. Pure A-21978C complex (6 g), obtained as
described in Example 3, was dissolved in water (80 ml). The pH of
this solution was adjusted to 4.4 with acetic acid, and
tetrahydrofuran (20 ml) was added. The solution was pumped under
low pressure (Lapp Pump) onto a steel column (4.8.times.100 cm)
containing 1.77 liters of silica gel/C.sub.18 [Quantum LP-1, 10-20
microns, silylated with octadecyltrichlorosilane] packed in
water:tetrahydrofuran (THF) (4:1). The column was washed under
pressure (about 100 psi) with 150 ml of H.sub.2 O:THF (4:1). The
column was developed with water:methanol:acetonitrile
(47.5:15:37.5) containing 0.2% pyridine and 0.2% acetic acid at
about 100 psi at a flow rate of 35 ml/minute, collecting 175-ml
fractions. Elution was monitored continuously on a recorder with an
ultraviolet (uv) detector at 280 nm. Fractions containing
individual factors as indicated by the peaks on the graph were
further monitored on an analytical reversed-phase resin column.
Fractions containing a single factor were combined and
freeze-dried. A typical run is illustrated here: fractions 12-16
contained factor C.sub.0 ; fractions 20-26 contained factor
C.sub.1, fractions 38-50 contained factor C.sub.2 ; fractions 63-78
contained factor C.sub.3. Fractions 27-37 (containing factors
C.sub.1 and C.sub.4) and fractions 51-62 (containing factors
C.sub.2 and C.sub.5) were recycled through the column to obtain
pure factors C.sub.4 and C.sub.5. Column loads ranged from 6-12 g.
Yields from a total of 84 g of A-21978C complex were: 1.9 g of
C.sub.0, 3.27 g of C.sub.1, 4.97 g of C.sub.2, and 1.94 g of
C.sub.3. Higher yields of individual factors were obtained by
recycling mixed-factor fractions using appropriate HPLC solvent
systems. The choice of system varied and was dependent on
individual lots, and on the reverse-phase resin and columns.
The following are useful systems for separation of the A-21978C
factors:
A. Analytical Systems
Water:methanol:acetonitrile (50:15:35) containing 0.2% acetic acid
(HOAc) adjusted to pH 5.5 with pyridine
Water:methanol:acetonitrile (50:15:35) containing 0.2% HOAc and
0.2% pyridine
Water:methanol:acetonitrile (50:15:35) containing 0.75% ammonium
formate
Water:methanol:acetonitrile (95:30:75) containing 0.2% HOAc and
0.2% pyridine
Water:methanol:acetonitrile (105:15:80) containing 0.2% HOAc and
0.2% pyridine
Water:methanol:THF (59:15:25) containing 0.5% HOAc and 0.5%
pyridine
Water:methanol:THF (60:15:25) containing 0.5% ammonium formate
B. Preparative Systems
Water:methanol:acetonitrile (95:20:85) containing 0.15% HOAc and
0.15% pyridine
Water:methanol:acetonitrile (100:15:85) containing 0.15% HOAc and
0.15% pyridine
Water:methanol:acetonitrile (50:10:40) containing 0.1% HOAc and
0.1% pyridine
Water:methanol:acetonitrile (50:15:35) containing 0.75% ammonium
formate
Water:methanol:acetonitrile (55:10:35) containing 0.2% HOAc and
0.8% pyridine
Water:methanol:THF (52.5:15:32.5) containing 0.6% ammonium
formate
Water:methanol:THF (50:15:35) containing 0.6% ammonium formate
The advantage of acetic acid-pyridine over ammonium formate is that
the former can be removed during the freeze-drying, whereas
ammonium formate must be removed by column chromatography (Sephadex
G-25).
EXAMPLE 6
Alternate Isolation of A-21978C Complex
Whole fermentation broth (97 liters), obtained as described in
Example 1, was filtered with a filter aid (4% Hyflo Super-Cel); the
resulting filtrate (80 liters) was stirred with 2 liters of a
nonionic macroporous copolymer of styrene cross-linked with
divinylbenzene (Diaion HP-20 resin. Mitsubishi Chemical Industries
Limited, Tokyo, Japan) for 2 hours. The supernate was decanted; the
resin was washed with water (8 liters); the water was decanted. The
resin was then stirred with 8 liters of acetonitrile:water (15:85)
for 15 minutes; the solvent was removed by filtration. The A-21978C
complex was then eluted from the resin by stirring it with 8 liters
of acetonitrile:water (2:3) for 1 hour and filtering. This
procedure was repeated to remove all the A-21978C complex. The two
filtrates were combined and concentrated in vacuo to an oil. The
oil was dissolved in a minimal volume of water; two volumes of
methanol were added with warming; then 30 volumes of acetone were
added to precipitate the A-21978C complex. The precipitate was
separated by filtration and dried in vacuo to yield 13.6 g of crude
A-21978C complex (570 units/mg).
The crude A-21978C complex was purified by silica-gel column
chromatography. The complex (1 g) was dissolved in a minimal volume
of water; silica gel (Grace 62) was added to absorb the water; the
absorbent was slurried in acetonitrile. This slurry was applied to
a 1.5.times.40-cm column of silica gel (Grace, Grade 62) packed in
acetonitrile. The column was then washed with acetonitrile. The
activity was eluted with acetonitrile:water (4:1), collecting 25-ml
fractions. Fractions were monitored as described in Example 3.
Fractions 21 to 46, containing most of the A-21978C complex, were
combined, concentrated to a small volume under vacuum and
freeze-dried to yield 605 mg of purified A-21978C complex (Na salt)
(900 units/mg).
EXAMPLE 7
Preparation of the A-21978Complex (Acid Form)
A-21978C complex in the Na salt from (7 g), prepared as described
in Example 6, was dissolved in water (150 ml); n-butanol (150 ml)
was added. The pH of the mixture was adjusted to pH 3.4 with 2N
HCl, while stirring for 1 hour. The n-butanol phase was separated
and concentrated to a residue in vacuo. The residue was dissolved
in water and freeze:dried to yield 6 g of A-21978C complex (acid
form). The individual A-21978C factor salts are converted to the
corresponding acid forms by the same method.
EXAMPLE 8
Preparation of A-21978C Complex Sodium Salt from A-21978C Complex
in the Acid Form
A-21978C complex in the acid form (50 mg), prepared as described in
Example 7, was dissolved in warm absolute ethanol (5 ml); 1N NaOH
was added dropwise until the pH of the solution was 9.4; the
resulting solution was held at room temperature overnight. The
precipitate which formed was filtered off and dried in vacuo to
give 32 mg of A-21978C complex (sodium salt). The salt contained 8%
sodium by atomic-absorption assay.
EXAMPLE 9
Using the procedure described in Example 8, A-21978C complex
calcium salt was formed by adding CaCl.sub.2 in ethanol to an
ethanolic solution of A-21978C complex in the acid form.
EXAMPLE 10
Microbiological Assay for A-21978 Fermentation and Isolation
Samples
The method used to quantitate the activity of A-21978 in
fermentation broths and isolation samples was a paper-disc
agar-diffusion system, using Micrococcus luteus.
Seeded agar-diffusion plates were prepared by inoculating a
nutrient agar medium with an appropriate concentration of the test
culture, pouring 8 ml of agar into each 20.times.100-mm plastic
petri dish.
The assay reference standard was a preparation of A-21978C complex.
This preparation was used on a unit basis. Highly purified A-21978C
complex contains about 1250 units per milligram. The standard dose
response curve was prepared to contain 150-75-40-20-10 units per
ml. Diluent for the standard and samples was 0.1M pH 6.0 phosphate
buffer.
Sample and standard solutions were delivered to 12.7-mm paper discs
with an automatic pipette. Incubation was at 30.degree. C. for
16-18 hrs. Zones were read on a modified Fishcer-Lilly Antibiotic
Zone Reader.
* * * * *