Note: Descriptions are shown in the official language in which they were submitted.
1 320464
X-6594M -1-
ANTIBIOTIC A80190
This invention relates to a new polyether
antibiotic A80190, to certain derivatives thereof, and
to its preparation by cultivation of novel strains of
microorganisms.
Westley (John W. Westley, "Polyether Anti-
biotics: Naturally Occurring Acid Ionophores, Vol. 2,
Chemistry," Marcel Dekker: New York, 1983) has sepa-
rated existing polyethers by class and type. Using
Westley's system, A80190 is a new member of the Class
lb, type (1), group of polyethers because it has one
spiroketal system. Other members of this group include
A-28695 A and B (U.S. Patent No. 3,839,558); A204I and
II (U.S. Patent No. 3,705,238) and A-32887 (U.S. Patent
No. 4,133,876).
A80190 has the following structure:
OMe
! o
OMe ~/
Me ~\Me () Me Me
y~ ; ~ /Me H\o/~l 0~/ ~ / \
~,
1 3204h4
X-6594M -la-
Brief Description of the Drawinqs
Figure 1 shows the infrared absorption spectrum
of antibiotic A80190 in chloroform.
Figure 2 shows the infrared absorption spectrum
in choroform of the sodium salt of antibiotic A80190.
Figure 3 shows the infrared absorption spectrum
in chloroform of acetyl A80190.
Figure 4 shows the infrared absorption spectrum
in chloroform of propionyl A80190.
1 320464
X-6594M -2-
Characteristics of A80190
Antibiotic A80190 has been assigned structure
1 based on X-ray crystallographic studies. A80190 (in
its free acid form) has the following characteristics:
State: white crystals (from acetone-water)
Mp: 98-100C or 120-122C (more freguently);
probably varies with the degree of
solvation
pKa: = 6.2 (66% aqueous dimethylformamide)
[~]25D: -26 (c 1, CHC13)
Molecular weight: 828 (field desorption mass
spectrometry)
Empirical formula: C44H76O14
W : no absorbance
IR: (CHC13) Figure l; shows absorption
at the following frequencies (cm ): 3019,
2970, 2936, 2827, 1721, 1457, 1402, 1376,
1314, 1163, 1105, 109~, 1083, 1056, 1022,
1006, 989, 980, 945, 934, 917, 892 and 859
Elemental Analysis:
Found Calcd
Carbon 63.35 63.77
Hydrogen 9.17 9.18
oxygen 27.10 27.05
Solubility: Insoluble in water; soluble in lower
alcohols such as methanol, ketones
such as acetone, esters such as
ethyl acetate, halogenated hydro-
carbons such as chloroform and
1 320464
X-6594M -3-
hydrocarbons such as diethyl ether,
benzene, toluene and warm hexane.
A80190 has an acid function capable of forming
~alts and ester derivatives and has at least one hydroxyl
group which can be esterified or which can form ether
derivatives or urethane derivatives. The acyl and alkyl
ester and alkyl ether derivatives of A80190, urethane
derivatives, and the pharmaceutically-acceptable salts
of A80190 and of these derivatives are also useful as
antibiotics, coccidiostats and as agents which increase
feed-utilization efficiency.
According to one aspect of the invention there
is provided a compound of formula (I):
OMe
f ;\~Me
5~Me
Me/ \~e ~ ~e ~e
Me i~lé /~ ~ \Me
wherein R is hydrogen or -CONHRl and Rl is
alkyl, aryl, alkyl-aryl, arylalkyl, haloaryl, nitroaryl,
1 32(~464
X-6594M -4-
haloaryl-alkyl, alkoxyaryl, aryloxyaryl, arylcycloalkyl,
acylaryl and cycloalkyl; and acyl, alkyl ester or alkyl
ether derivatives, or salts thereof.
A80190, and its derivatives, are useful as
antibacterial and anticoccidial agents. They improve
feed-utilization efficiency in ruminants and act as a
growth promotant in monogastric animals. In addition,
they have insecticidal, herbicidal and antiviral
activity. Further, they are useful as ionophores.
Methods and compositions pertaining to these uses are
also provided.
This invention further relates to synergistic
compositions of A80190, its acyl and alkyl ester, alkyl
ether and urethane derivatives or a pharmaceutically
acceptable salt thereof (an "A80190 compound"), together
with a compound selected from nicarbazin, 4,4'-dinitro-
carbanilide, metichlorpindol and certain naphthalenamine
or benzenamine compounds. These compositions are useful
in controlling coccidiosis in animals.
The term "acyl" means a Cl to C7, preferably
a C1 to C4, alkanoic acid moiety, i.e., radicals of the
formula
Rla ICI
O
wherein R a is C1 to C6-alkyl or hydrogen, e.g., formyl,
acetyl, propionyl, butyryl and the like.
The term "cycloalkyl" means cyclic hydrocarbon
groups containing from 3 to 7 carbon atoms, such as,
cyclopropyl, cyclobutyl, cyclohexyl and the like, cyclo-
hexyl being preferred. The cycloalkyl group may be sub-
1 3204~4
X 6594M -5-
stituted by an aryl residue, as defined herein, to form
an arylcycloalkyl residue, e.g., 2-(phenyl)cyclopropyl.
The term "alkoxy" means a C1 to C7 lower alkyl
group having an oxygen function substituted therein,
such as, methoxy, ethoxy, propoxy and the like.
The term "aryl" denotes an aromatic residue
derived by the removal of a hydrogen atom from an
aromatic hydrocarbon, such as, for example, phenyl,
pyridyl or furyl, especially phenyl. The "aryl" residue
may be substituted by various groups. A substitutent
on a phenyl nucleus is preferably on the 4-position.
Examples are 4-alkylaryl, e.g., 4-methylphenyl (4-tolyl);
4-halophenyl, e.g., 4-chlorophenyl; 4-nitrophenyl;
4-aryloxy-aryl, e.g., 4-phenoxyphenyl; 4-alkoxyphenyl,
e.g., 4-methoxyphenyl; and 4-(alkyl-carbonyl)phenyl,
e.g., 4-(methylcarbonyl)phenyl or 4-(phenylcarbonyl)-
phenyl.
The term "alkyl" means a C1 to C7 straight or
branched chain hydrocarbon, preferably a C1 to C4 hydro-
carbon, e.g., methyl, ethyl, propyl, isopropyl, n-butyl,
etc. The alkyl group may be substituted by an aryl
residue, as defined suPra, to form an arylalkyl residue,
e.g., phenylethyl or 2-phenylethyl or by a haloaryl
residue to form a haloarylalkyl residue, e.g.,
4-bromophenethyl.
The salts of A80190 and of its derivatives are
useful for separating and purifying the antibiotics.
The pharmaceutically-acceptable salts are particularly
useful. Such salts should have little or no toxicity
towards warm-blooded animals. Examples of salts are the
1 320~64
X-6594M -6-
alkali-metal, alkaline-earth-metal and amine salts of
A80190 and of its derivatives.
Representative and suitable alkali-metal and
alkaline-earth metal salts of A80190 include the sodium,
potassium, lithium, cesium, rubidium, barium, calcium
and magnesium salts. Suitable amine salts of A80190
include the ammonium and the primary, secondary, and
tertiary Cl-C4-alkylammonium and hydroxy-C2-C4-alkyl-
ammonium salts. Illustrative amine salts include those
formed by reaction of A80190 with ammonium hydroxide,
methylamine, sec-butylamine, isopropylamine, diethyl-
amine, di-isopropylamine, ethanolamine, triethylamine,
3-amino-l-propanol and the like.
It is well known in the veterinary phar-
maceutical art that the form of an antibiotic is notordinarily 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 signifi-
cance. The salt form may, however, be chosen for
reasons of economy, convenience, and lack of toxicity.
Antibiotic A80190 can be produced by culturing
an A80190-producing strain of Actinomadura oligospora
under submerged aerobic conditions in a suitable culture
medium until substantial antibiotic activity is pro-
duced. The antibiotic is recovered using various iso-
lation and purification procedures understood in the
art.
1 320~6~
X-6594M -7-
The new Actinomadura oliqosPora microorganism
of this invention, which produces antibiotic A80190, was
isolated from a soil sample from India. Subsequently, a
substantially improved variant which produces increased
amounts of A80190 was isolated by LaVerne D. Boeck.
Cultures of these two A80190-producing organ-
isms have been deposited and made part of the stock
culture collection of the Northern Regional Research
Center, Agricultural Research, North Central Region,
1815 North University Street, Peoria, Illinois, 61604,
from which they are available to the public under the
accession numbers NRRL 15877 (parent strain) and NRRL
15878 (the variant strain).
Taxonomic studies of the variant were carried
out by Frederick P. Mertz of the Lilly Research Labora-
tories. Based on these studies, the two new organisms
are classified as members of a new species of the genus
Actinomadura for which the name Actinomadura oligospora
sp. nov. is proposed. This classification is based on
direct laboratory comparisons with similar species and
examination of published descriptions [M. Goodfellow and
G. Alderson, "Numerical Taxonomy of Actinomadura and
Related Actinomycetes," J. Gen. Microbiol. 112:95-111
(1970); M. Goodfellow and K. P. Schaal, "Identification
Methods for Nocardia, Actinomadura and Rhodococcus,"
p. 261-276 In F. A. Skinner and D. W. Lovelock (ed.),
"Identification Methods for Microbiologists," 2nd ed.,
The Society for Applied Microbiology Technical Series
No. 14, Academic Press, New York, 1979; L. H. Huang,
"Actinomadura macra sp. nov., the Producer of Anti-
1 3~04h4
X-6594M -8-
biotics CP-47,433 and CP-47,434," Int. J. Svst. Bacteriol.
30:565-568 (1980); and H. A. Lechevalier and M. P.
Lechevalier, "A Critical Evaluation of the Genera of
Aerobic Actinomycetes," p. 39~-405, In H. Prauser (ed.),
"The Actinomycetales," Gustav Fischer Verlog, Jena].
Methods Used
The methods recommended by the International
Streptomyces Project (ISP) for the characterization of
Streptomyces species [E. B. Shirling and D. Gottlieb,
"Methods for Characterization of Streptomvces Species,"
Int. _ SYst. Bacteriol. 16:313-340 (1966)] have been
followed along with certain supplementary tests
(D. J. Blazevic and G. M. Ederer, "Principles of
Biochemical Tests in Diagnostic Microbiology," John
Wiley and Sons, Inc., New York, 1975).
Carbon utilization was determined with ISP
No. 9 basal medium to which filter-sterilized carbon
sources were added to equal a final concentration of 1.0
percent. Plates were incubated at 30C and read after
14 days.
Melanoid pigment production (chromogenicity)
was determined with ISP No. 1 (tryptone-yeast extract
broth), ISP No. 6 (peptone-yeast extract iron agar), ISP
No. 7 (tyrosine agar) and modified ISP No. 7 which has
tyrosine removed.
Starch hydrolysis was determined by testing
for the presence of starch with iodine on ISP No. 4
(inorganic salts-starch agar) plates (See Blazevic and
Ederer, supra).
1 3~0464
X-6594M -9-
Morphology was studied using an optical light
microscope. A scanning electron microscope (SEM) ~as
used to study the spore surface ornamentation.
NaCl tolerance was measured by adding NaC1
to ISP No. 2 agar to equal the concentration desired.
ICSS-NBS Centroid Color Charts, standard
sample No. 2106 (National Bureau of Standards, 1958,
U.S. Department of Commerce, Washington, D.C.) and the
Color Harmony Manual (4th ed., Container Corporation of
America, Chicago, Illinois, 1958) were used to assign
color names.
The isomers of diaminopimelic acid (DAP) and
the carbohydrates in hydrolysates of whole cells were
established by the chromatographic methods of Becker et
al. [B. Becker, M. P. Lechevalier, R. E. Gordon, and
H. A. Lechevalier, "Rapid Differentiation between Nocardia
and strePtomyces by Paper Chromatography of Whole-cell
Hydrolysates," Appl. Microbiol. 12:421-423 (1964)]
and of Lechevalier [M. P. Lechevalier, "Identifica-
tion of Aerobic Actinomycetes of Clinical Importance,"J. Lab. Clin. Med. 71:934-944 (1968)].
Resistance to lysozyme was measured by methods
recommended by Gordon [R. E. Gordon and D. A. Barnett,
"Resistance to Rifampin and Lysozyme of Strains of Some
Species of Mvcobacterium and Nocardia as a Taxonomic
Tool," Int. ~. Syst. Bacteriol. 27, 176-178 (1977)].
Resistance to antibiotics was measured by
padding antibiotic sensitivity discs onto the surface of
seeded ISP No. 2 agar plates.
Phosphatase and urease were determined by
methods described by Blazevic, su~ra.
~- ~ 32~464
X-6594M -10-
Mycolic acids were determined by a method
based on techniques described by Minnikin [D. E.
Minnikin, L. Alshamaony and M. Goodfellow, "Differenti-
ation of MYcobacterium, Nocardia, and Related Taxa by
Thin-Layer Chromatographic Analysis of Whole-organism
Methanolysates," J. Gen. Microbiol. 88:200-204 (1975)].
Phospholipid analysis was performed as de-
scribed by Lechevalier [M. P. Lechevalier, C. De Bievre
and H. Lechevalier, "Chemotaxonomy of Aerobic Actino-
mycetes: Phospholipid Composition," Biochemical
Systematics and Ecoloqy 5, 249-260 (1977)].
Cultural Characteristics
Growth of the organism was generally poor on chemically
defined media but better on complex organic media.
Aerial mycelia were absent except for trace amounts on
ISP No. 4 and sodium butyrate agar. When spores were
present, their color was oyster white in the Tresner and
Backus System [~. D. Tresner and E. J. Backus, "System
of Color Wheels for Streptomycete Taxonomy," APpl.
Microbiol. 11:335-338 (1956)]. The color of the reverse
side was yellowish gray to brown. No soluble pigments
were produced, except for the production of a very light
brown soluble pigment in ISP No. 2 and a dark brown
soluble pigment in yeast-dextrose agar. Table I presents
these cultural characteristics.
1 32 0 ~ h 4
X-6594 -11-
oo
o
10 1~_ ~ oy
C U ~ .,.~ ~ o
E ~C e ~ ~ ~ ~ ~
00 . ~ Co Ll ~ ~ o C ~ E C C o oo o o
o~o ~:1 <~Z~ ~u~Z Z ~U')Z Z C~ZZ
Z ~q U~
Wo o~
~ ~ U ~ U
U W C s~ s~
3 O ~ 1~ _
J~ ~ O ~ ~ ~
P rl ~ ~ .~
e~ U ~ ~ U~rl
~J CO .~'q c ~ ;~ ~ 3 u~ :> :>-
u oo o s~ ~o ~3 ~ a~ ^1 3 ~:1 L~l
u~ ~ a~ ,, ~ ~ c s~ ~ C s~
.1 ~ ~ C!~ w,~ t~ C:~
C ~ ~ ~ ~ ~ U U h ~ U ~J 1~
C ~ ~ O C O ~ O ~ C O C C
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........ ...... .. ...... .. ........
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E~ 'C ~ Z ~ z ~ Z ~ Z
1 3204~)~
X-6594 -12-
,C
o ~1
u o C~ m ~ ~ ~ ~
e ~ ~ C c ~ ~ c c ~o ,~ ~ c ~o ~ ~ c ~o ~ ~ c
. ~ x o o ~ ~ o o o ~o ~ o o ~o ~ o o o~ ~ o
~Zz ~o~ZZ ~E~Z C~Z ~E~Z
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a~ ~ ~ o ~ ~ ~g _
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C ~ ~ 6 ~ ~ ~ oO ~ ~ C ~ ~ ~
~:: o ~ C o~ ~,~ C c o ~ a C o ~ C C ~ ~ C C
QS O cr~ o ~rl ~ ~ O O O r~ o o o o o o ~ ~ o o
Z ~ I~ Z ~ t4 c~ Z Z C~ 1~ Z Z ~ c~ z Z ~ z z
C
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1 320~4
X- 6 5 9 4 - 1 3 -
e ~ .~ ~ c ~ ~ ~ c c _
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u~ ~ ~ C~ ~1
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........ c~z ~zz
. ~ ~ ~ ~ a ~ ~ ~
10 ~ ~ O
E-~ h ~ ~ al
1 320464
X-6594 - -14-
Morphological Characteristics
Spore chains were sparsely produced on ISP No. 4 and
sodium butyrate agar. Sporophores contained
approximately 10 spores per chain, and were generally
flexuous as in the Rectus-flexibilis (RF) configuration.
However, hooked sporophores were also observed. The
aerial hyphae had a tendency to clump together. Spore
shape was oblong and ranged in size from 0.5-0.7 ~m by
0.9-1.3 ~m. The average spore size measured 1.1 by 0.6
~m. The spore surface ornamentation was smooth.
Phvsiological Characteristics
Table II lists the carbohydrate utilization pattern of
the strain as compared to that of _. macra. ISP medium
No. 9 was used as a basal medium. The addition of
vitamin Bl or the u~e of Luedemann's medium [C. M.
Luedemann and B. Brodsky, "Micromonospora carbonacea sp.
n., an Everinomicin-producing Organism," Antimicrob.
Agents Chemother. 1964, 47-52] produced slightly better
growth, but no change in carbon utilization. Adonitol,
cellobiose, glucose, and ribose were utilized. Ques-
tionable utilization was noted with fructose and xylose.
Arabinose, cellulose, dextran, galactose, i-inositol,
inulin, lactose, mannitol, mannose, melizitose, melibiose,
raffinose, rhamnose, salicin, sucrose, trehalose, and
xylitol were not utilized for growth.
1 320464
X-6594 -lS-
Table II: Carbon Utilization Pattern of
NRRL 15878 and A. macra
Carbon Source NRRL 15878 A. macra
control - -
adonitol +
L-arabinose
cellobiose +
cellulose - ND
dextran - ND
D-fructose
D-galactose - -
D-glucose + +
i-inositol
inulin - ND
D-lactose
mannitol
D-mannose
D-melezitose - -
D-melibiose
raffinose
L-rhamnose - -
ribose + +
salicin
sucrose - +
trehalose - +
xylitol - ND
D-xylose
+ = utilized, - = not utilized,
_ doubtful if utilized, ND = not done
1 3204h4
X-6594 - -16-
Table III lists the resistance of the strain to various
antibiotics at the concentrations indicated and compares
it to that of A. macra.
Table III: Resistance to Antibiotics by NRRL 15878
and A. macra
Antibiotic Concentration NRRL 15878 A. macra
Bacitracin10 units +
Cephalothin30 ~g +
Gentamicin10 ~g - -
Lincomycin2 ~g ~ +
Neomycin30 ~g
Oleandomycin 15 ~g
Penicillin G lO units + t
Rifampin5 ~g + +
Streptomycin 10 ~g
Tetracycline 30 ~g - -
Tobramycin10 ~g
Vancomycin30 ~g
+ = resistant (no zones of inhibition)
- = sensitive (zones of inhibition)
1 320464
X-6594 -17-
NRRL 15878 grew at temperatures from 15-42C,
and tolerated up to 2 percent NaCl. It produced catalase,
phosphatase and urease.
NRRL 15878 degraded casein, DNA, esculin, and
gelatin, but not adenine, calcium malate, chitin,
elastin, guanine, hippurate, hypoxanthine, keratin,
starch, testosterone, tyrosine or xanthine.
Cell-wall Analysis
Hydrolyzed whole cells contained the meso
isomer of diaminopimelic acid. Sugars present in whole
cell hydrolysates were as follows: glucose, mannose,
madurose, and ribose. The cell-wall type according to
Becker, supra, is type III, and the sugar pattern is
type B (Lechevalier, 1968). A qualitative analysis of
whole-cell methanolysates for mycolic acids yielded
questionable results. It is doubtful that the culture
contains mycolic acids. A type PI phospholipid pattern
was found. Type PI contains no nitrogenous phospho-
lipids and is characteristic of the genus Actinomadura
(Lechevalier, 1977).
IdentitY of the Strain
NRRL 15878 has a Type III cell wall, Type B
whole-cell sugar pattern, and a Type PI phospholipid
pattern. This chemotaxonomic information plus its
general cultural characteristics are consistent with the
assignment of the strain to the genus Actinomadura
Lechevalier and Lechevalier (Lechevalier, 1970).
1 32()4~
X-6594 -18-
Comparison of its characteristics to those in
published descriptions of known species of Actinomadura
show that the culture is similar to _. pelletieri
(Laveran 1906) Lechevalier and Lechevalier, 1970 and
to A. macra (Huang, 1980).
The culture resembles Actinomadura eelletieri
mainly by the absence, or at least the rare occurrence,
of aerial mycelia. Morphology of sporophores, when
produced, is similar to that described in the litera-
ture (Goodfellow, 1979). The two cultures also possessa number of physiological characteristics in common.
However, the cultural and physiological differences are
sufficient to separate them as distinct species.
According to Lechevalier (1970), _. pelletieri
is represented exclusively by a group of clinical
isolates. The original description of A. pelletieri by
Gordon tR. E. Gordon, "Some Criteria for the Recognition
of Nocardia madurae (Vincent) Blanchard," J. Gen. Microbiol
45:355-364 (1966)] describes it as being a bright red
culture. NRRL 15878 does not produce this pigment. A.
Pelletieri degrades elastin, hypoxanthin, keratin,
tyrosine, utilizes trehalose, reduces nitrate, and grows
at 45C; but NRRL 15878 does not have these characteristics.
NRRL 15878 utilizes adonitol and cellobiose, degrades
esculin and DNA, and is resistant to lysozyme; but A.
pelletieri does not have these characteristics. The
new culture is, therefore, considered to be a different
species than A. pelletieri.
Because of the culture's similarity to A.
macra, simultaneous laboratory comparisons were made.
1 3~0~64
X-6594 -19-
NRRL 15878 and A. macra share many properties. Both
are unable to degrade adenine, calcium-malate, chitin,
elastin, guanine, keratin, starch, testosterone,
tyrosine, and xanthine. Neither produces H2S or
melanoid pigments. Both cultures degrade casein, DNA
and gelatin, produce catalase and phosphatase, and
synthesize a polyether antibiotic. They have the same
tolerance to NaCl; both grow on sodium butyrate; and
they have the same cell-wall type. A. macra and NRRL
15878 differ in carbon-utilization pattern, esculin and
hypoxanthine degradation, resistance to antibiotics,
temperature range, urease production, and the reduction
of nitrate.
A. macra and NRRL 15878 share many cultural
characteristics, notably the absence of aerial mycelia.
However, there are significant differences. The reverse
side of NRRL 15878 is gray to a yellowish brown; _. macra
on many media produces a red color. This distinction is
most clearly seen on glucose asparagine agar. On this
medium A. macra produces a pink aerial growth, whereas
NRRL 15878 produces none. These cultural comparisons are
shown in Table I.
The morphology of NRRL 15878 is similar to that
of A. macra. Both have poorly developed aerial mycelia
that belongs in the Rectus-flexibilis (RF) section of
Pridham [T. G. Pridham, C. W. Hesseltine, and R. C.
Benedict, "A Guide for the Classification of Streptomycetes
According to Selected Groups," ApPl~ Microbiol. 6:52-79
(1957)]. Spore surface ornamentation is smooth.
The differences and similarities between NRRL
15878 and _. macra are summarized in Table IV.
1 320464
X-6594 - -20-
Table IV: Summary Comparison of NRRL 15878 and A. macra
Similarities Differences
Morphology Antibiotic resistance
Physiological
properties Carbon-utilization pattern
Polyether synthesis Cultural characteristics
Scarcity of aerial
hyphae Degradation of esculin
Degradation of hypoxanthine
Nitrate reduction
Spore shape and size
Temperature range
Urease production
Table V shows these similarities and differences in
greater detail.
1 320464
X-6594 -21-
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1 320~64
X-6594 -22-
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1 3204h4
X-6594 -23-
These comparisons indicate that the NRRL 15877 and NRRL
15878 cultures are significantly different from other
species of Actinomadura and represent strains of a new
species for which the name Actinomadura oliqosPora sp.
nov. is proposed. The specific epithet (O.ligo.spor.us:
L. Adj. oliqo few, L.n. sporus spored, oliqospora few
spored) refers to the relative absence of sporophores in
the organism. Strain NRRL 15878 is the type strain of
A. oliqospora.
As is the case with other organisms, the
characteristics of the A80190-producing culture of this
invention, Actinomadura oliqospora NRRL 15877, are
subject to variation. Recombinants, mutants or variants
of the strain may be obtained by methods in the art.
For example, mutants can be obtained by treatment with
various known physical and chemical mutagens such as
ultraviolet light, X rays, gamma rays and chemicals
such as N-methyl-N -nitro-N-nitrosoguanidine. Natural
and induced variants, mutants and recombinants of
Actinomadura oligospora NRRL 15877 which retain the
characteristic of A80190 production are considered part
of this invention.
The culture medium used to grow the Actino-
madura oliqospora cultures 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
carbohydrate source in large-scale fermentation is
glucose, although ribose, xylose, fructose, galactose,
mannose, mannitol, potato dextrin and the like can also
1 320464
X-6594 -24-
be used. Glycerol and lipids support little or no
growth or antibiotic production when used as the primary
carbon source. In combination with glucose, they
enhance biomass, but depress antibiotic production.
A preferred nitrogen source is collagen
hydrolysate, although enzyme-hydrolyzed casein, meat
peptones, fish meal, liver meal, and the like are also
useful. Among the nutrient inorganic salts which can
be incorporated in the culture media are the customary
soluble salts capable of yielding zinc, sodium,
magnesium, calcium, ammonium, chloride, carbonate,
sulfate, nitrate and like ions.
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 substituents of
the medium in amounts sufficient to meet the growth
requirements of the organism. Foaming is not usually a
problem, but small amounts (i.e. 0.2 ml/L) of an anti-
foam agent such as polypropylene glycol may be added tolarge scale fermentation media if needed.
For production of substantial quantities of
antibiotic A80190, submerged aerobic fermentation in
tanks is preferred. Small quantities of A80190 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
1 320464
X-6594 -25-
mycelial fragments of the organism to obtain a fresh,
actively growing culture of the organism. The vegeta-
tive inoculum is then transferred to a larger tank.
The vegetative inoculum medium can be the same as that
used for larger fermentations, but other media are also
suitable.
A80190 is produced by the A80190-producing
organism when grown at temperatures between about 25
and about 37C. An optimum temperature for A80190
production appears to be about 30-32C.
As is customary in submerged aerobic culture
processes, sterile air is blown into the vessel from the
bottom while the medium is stirred with conventional
turbine impellers. The maximum oxygen uptake of the
fermentation under the conditions used thus far has not
exceeded about 0.2 mM/L/minute. In a fully baffled
165-liter fermentor containing approximately 115 liters
of broth, an aeration rate of 0.125 v/v/m with an agita-
tion rate of 200 rpm is sufficient to maintain the
level of dissolved oxygen at or above 30% of saturation.
Production of antibiotic A80190 can be fol-
lowed during the fermentation by testing samples of the
broth for antibiotic activity against organis~s known to
be sensitive to the antibiotic. One assay organism
useful in testing A80190 is Bacillus subtilis ATCC 6633.
The bioassay is conveniently performed by the agar-well
plate test.
Following its production under submerged
aerobic fermentation conditions, A80190 can be recovered
from the fermentation medium by methods used in the
.,,
1 320464
X-6594 - -26-
fermentation art. The antibiotic activity produced
during fermentation of the A80190-producing organism
occurs both in the filtered broth and in the mycelial
mass. Maximum recovery of A80190 is accomplished,
therefore, by initially filtering the medium to separate
the broth from the mycelial mass. The filtered broth
and the mycelial mass can then be purified separately to
give their respective portion of A80190. A variety of
techniques may be used in this purification. A pre-
ferred technique for purification of the filtered brothinvolves adjusting it to a pH of about 9 and extracting
with a suitable solvent such as, for example, ethyl
acetate. The extracting solvent can then be evaporated
under vacuum to give the broth portion of A80190. A
preferred method of purifying the mycelial mass is to
extract the separated mycelial filter cake with a
suitable solvent such as, for example, methanol or
acetone. The extracting solvent is then evaporated
under vacuum to give a concentrated aqueous solution.
This aqueous solution is then adjusted to a pH of about
9 and is extracted with a suitable solvent such as, for
example, ethyl acetate. The extracting solvent is then
concentrated under vacuum to give the mycelial portion
of A80190. The broth and mycelial portions of the
A-80190 complex are further purified by similar procedures.
A preferred procedure involves silica gel chromatography.
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 A80190. For example, after
1 320464
X-6594 -27-
production of A80190, the whole fermentation broth can
be dried by lyophilization, by drum-drying, or by
azeotropic distillation and drying. The dried broth is
then mixed directly into a feed premix.
The alkali-metal and alkaline-earth-metal
cationic salts of A80190 are prepared according to pro-
cedures commonly used for the preparation of cationic
salts. For example, the free acid of A80190 iB dis-
solved in a suitable solvent such as acetone; a 1/3
volume of water is added; and this solution is adjusted
to a pH of about 9 to 10 with the base of the desired
cationic salt (e.g. NaOH, KOH). The salt thus formed
can be isolated by routine methods, such as filtration
or evaporation of the solvent.
A preferred method of forming salts is to
dissolve A80190 (acid form) in a water-immiscible
solvent such as ethyl acetate, add an equal volume of
water, and adjust the mixture to pH 10 with the cor-
responding cationic base (e.g. NaOH, XOH, etc.) The
separated organic phase is washed with water and con-
centrated to dryness. The residue is lyophilized from
dioxane. The salt can be crystallized from an appro-
priate solvent, such as pentane.
The salts formed with organic amines can be
prepared similarly. For example, the gaseous or liquid
amine can be added to a solution of A80190 in a suit-
able solvent such as acetone; the solvent and excess
amine can be removed by evaporation.
A80190 acyl-ester derivatives are prepared
by treating A80190 with a corresponding acid anhydride
1 3~0464
X-6594 -28-
or acid chloride. Esterification occurs at one of the
A80190 hydroxyl groups. Such esters are typically
prepared by reacting A80190 with, for example, the
corresponding acid anhydride at room temperature.
A80190 alkyl ester derivatives are prepared by
esterification of the carboxyl group, using standard
procedures. The A80190 alkyl ester derivatives are
typically less active when tested ln vitro. When
administered to an animal, however, such esters can act
as pro drugs which are converted to A80190 in vivo.
The alkyl ether derivatives of A80190 are
those compounds wherein one or more of the hydroxyl
groups has been replaced by a YR group wherein:
Y represents o or S; and
R represents C1-C6-alkyl,
Cl-C4-alkoxy-C2-C5-alkyl,
Cl-C4-alkoxycarbonyl-C2-C5-alkyl,
amino-C2-C5-alkyl,
mercapto-C2-C5-alkyl,
hydroxyalkyl,
haloalkyl, or
- (R')m-phenyl(CH2)n~,
wherein R' represents C1-C4-alkyl, C1-C4-alkoxy, or
hydroxy;
m represents 0-2; and
n represents 0-3.
The terms alkyl and alkoxy have the meaning discussed
supra, but are limited to the number of carbon atoms
specified.
r~
~ j
1 320464
X-6594 -29-
The term "hydroxyalkyl" refers either to a
monohydroxy-C2-C5-alkyl moiety or, when Y is O, to the
2,3-dihydroxyprop-1-yl moiety.
The term "haloalkyl" refers to a C2-C5-alkyl
moiety having from one to three halogen substituents,
selected from the group consisting of bromine, chlorine,
and fluorine. When the alkyl moiety is dihalo- or
trihalo-substituted, the halo-substituents must be the
same halogen moiety.
Preferred A80190 ether derivatives are those
compounds wherein Y represents O and R represents Cl to
C6 alkyl. The ether derivatives are prepared by react-
ing A80190, or a salt thereof, with a corresponding
primary alcohol or thiol.
With some of the starting alcohols or thiols
it may be necessary to add an acid catalyst to the reac-
tion. Suitable catalysts include hydrochloric acid,
sulfuric acid, methanesulfonic acid, benzenesulfonic
acid, toluenesulfonic acid, selenium dioxide, and boron
trifluoride.
A solvent such as, for example, water, acetone,
benzene, ether, tetrahydrofuran, or dioxane may be added
to facilitate the reaction. Reactions generally occur
at room temperature, although higher temperatures, for
example up to 100C, may be used.
Although ordinary reaction work-up procedures
are sometimes sufficient, additional purification may be
required to obtain the compounds of this invention.
Such purification may be accomplished by well-known
methods, such as, for example, column chromatography,
1 320464
X-6594 ~30
thin-layer chromatography, fractional crystallization
and the like.
The A80190 urethane derivatives of formula I
can be prepared by treating A80190 or an A80190 salt
with an isocyanate of formula 3
R-NC0
wherein R is as defined supra.
Preferably, a salt of A80190, in particular
the sodium salt, is used. The isocyanate of formula 3
should be added in slight excess, e.g., about 10%
excess, in order to form the mono derivative in optimum
quantity. The reaction is preferably carried out in an
inert solvent such as a chlorinated hydrocarbon, e.g.,
carbon tetrachloride, methylene chloride or chloroform,
ether, ethyl acetate or in an aromatic hydrocarbon
solvent such as benzene or toluene. The reaction
temperature is not critical but can be between above
0C. and the boiling point of the reaction mixture, but
is preferably about room temperature.
The A80190 compounds inhibit the growth of
bacteria and fungi which are pathogenic to animal and
plant life. The inhibitory activity of A80190 is
illustrated in Table VI. Activity was measured by the
conventional disc-diffusion method (6-mm pads were
dipped in solutions containing 1 mg of compound per ml
of solution; pads were placed on agar plates seeded with
test organism).
1 320464 ~
X-6594 -31-
Table VI: Antibacterial Activity of A80190
Zone of Inhibition
Test Organism (mm diameter)
Staphylococcus aureus 17
Bacillus subtilis 23
Micrococcus luteus 20
Mycobacterium avium 21
Saccharomvces astorianus 10
Neurospora crassa 14
Candida albicans 14
One important aspect of the antimicrobial
activity of A80190 compounds relates to their activity
against anaerobic bacteria. The minimal inhibitory
concentrations (MIC's) at which A80190 inhibits various
anaerobic bacteria, as determined by standard agar-dilu-
tion assay, are summarized in Table VII. End points
were read after 24-hour incubation.
~ 1 320464
X-6594 -32-
Table VII: Susceptibility of Anaerobic Bacterial
Isolates to A80190
Anaerobic Bacteria MIC (~g/ml)
Clostridium difficile 2994 <0.5
Clostridium perfring ns 81 <0.5
Clostridium septicum 1128 <0.5
Eubacterium aerofaciens 1235 <0.5
Peptococcus asaccharolyticus 1302
Peptococcus pre~70ti 1281 <0 5
Peptostreptococcus anaerobius 1428
Peptostreptococcus intermedius 1624
Propionibacterium acnes 79
Bacteroides fragilis 111 >128
Bacteroides fragilis 1877 >128
Bacteroides fragilis 1936B >128
Bacteroides thetaiotaomicron 1438 >128
Bacteroides melaninogenicus 1856/28 >128
Bacteroides melaninogenicus 2736 >128
Bacteroides vulgatis 1211 16
Bacteroides corrodens 1874 >128
Fusobacterium symbiosum 1470 8
Fusobacterium ~ e~ 6054A 16
IBI
1 320464
X-6594 -33-
Anticoccidial activity is an important
property of the A80190 compounds. Table VIII summarizes
the results of an in vitro tissue culture screen of
A-80190 compounds vs. Eimeria tenella.
Table VIII: Activity of A80190 Compounds vs.
Eimeria tenella In Vitro
Concentration (~g/ml)
Compound 5 1 0.2 0.04 0.008
A80190 Ca C A A A
Acetyl A80190 A A A A N
Propionyl A80190 A A A A S
aC = cytotoxic
A = active
S = slightly active
N = not active
For treating coccidiosis in poultry, a non-
toxic anticoccidial amount of an A80190 compound is
administered to infected or susceptible birds, pre-
ferably orally on a daily basis. The A80190 compound
can be supplied in many ways, but it is most con-
veniently supplied with a pharmaceutically acceptable
carrier, preferably ~he feed ingested by the birds.
Although a variety of factors must be considered in
determining an appropriate concentration of A80190
1 32046~
X-6594 -34-
compound, the rates of administration are generally in
the range of about 2 to about 100 ppm in the feed and
are preferably in the range of about 15 to about 50 ppm
of feed ration. In another aspect, this invention
relates to compositions for treating coccidiosis com-
prising an effective amount of an A80190 compound for
treating coccidiosis, together with a suitable vehicle.
Compositions for controlling coccidiosis in
poultry of this invention comprise:
1) a compound of formula (I)
in combination with
2) a compound selected from the group consisting of
a) nicarbazin,
b) 4,4'-dinitrocarbanilide,
c) anaphthalenamine compound of formula 4:
R2
R4 ~ \t~
H
C F ~ ~,~N 2
~ / \Rs
NO2
-
....
- 1 320464
X-6594 -35-
wherein:
R2 is C1-C4 alkyl;
R3 is halogen, Cl-C4 fluoroalkyl, C1-C4
fluoroalkoxy or C1-C4 fluoroalkylthio;
R is halogen;
R5 is hydrogen or halogen;
m is 0, 1 or 2; and
n is 0 or 1;
with the proviso that, when an R4 substituent0 exists, it is at other than the 2-position;
d) a benzenamine selected from 2,4-dinitro-
N-[4-(trifluoromethoxy)phenyl]-6-(trifluoromethyl)benz-
enamine; 2,4-dinitro-N-[4-(1,1,2,2-tetrafluoroethoxy)-
phenyl]-6-(trifluoromethyl)benzenamine or 2,4-dinitro-
5 N-[4-(pentafluoroethoxy)phenyl]-6-(trifluoromethyl)-
benzenamine;
e) metichlorpindol; or
f) a pharmaceutically acceptable salt of an
(a)-(e) compound.
In formula _, C1-C4 alkyl includes methyl,
ethyl, n-propyl, isopropyl, _-butyl, sec.-butyl,
isobutyl, t-butyl, and the like.
The term "halogen" represents fluorine,
chlorine, bromine and iodine.
C1-C4 Fluoroalkyl is a C1-C4 alkyl group
bearing one or more fluorine atoms. Such fluoroalkyl
groups include trifluoromethyl, 1,1,2,2-tetrafluoro-
ethyl, pentafluoroethyl, 1,2,3,3-tetrafluoropropyl,
nonafluorobutyl, and the like.
1 320464
X-6594 -36-
Cl-C4 Fluoroalkoxy is a C1-C4 alkoxy group
bearing one or more fluorine atoms. Such fluoroalkoxy
groups include difluoromethoxy, trifluoromethoxy,
l-fluoroethoxy, 1,1,2,2-tetrafluoroethoxy,
pentafluoroethoxy, 1,2,2,3,3-pentafluoropropoxy,
heptafluoropropoxy, 4,4,4-trifluorobutoxy, and the
like.
C1-C4 Fluoroalkylthio is a C1-C4 alkylthio
group bearin~ one or more fluorine atoms. Such
fluoroalkylthio groups include trifluoromethylthio,
1,1,2,2-tetrafluoroethylthio, pentafluoroethylthio,
4,4,4-trifluorobutylthio, and the like.
Preferred formula _ compounds are those
wherein m and n are 0 and R5 is hydrogen.
Typical formula _ compounds are:
4-Fluoro-N-[2,4-dinitro-6-(trifluoromethyl)-
phenyl~-1-naphthalenamine
4-Iodo-N-[2,4-dinitro-6-(trifluoromethyl)-
phenyl]-1-naphthalenamine
4-Trifluoromethyl-N-[2,4-dinitro-6-(trifluoro-
methyl)phenyl]-1-naphthalenamine
4-Pentafluoroethyl-N-[2,4-dinitro-6-(trifluoro-
methyl)phenyl-1-naphthalenamine
6,7-Dimethyl-4-(1,1,2,2-tetrafluoroethoxy)-N-
t2,4-dinitro-6-(trifluoromethyl)phenyl]-1-naphthalenamine
2-Isopropyl-4-chloro-N-[3-chloro-2,4-dinitro-
6-(trifluoromethyl)phenyl]-1-naphthalenamine
8-_-Butyl-4-(4,4,4-trifluorobutox,)-N-[3-bromo-
2,4-dinitro-6-(trifluoromethyl)phenyl]-1-naphthalenamine
3-Methyl-6-propyl-4-heptafluoropropyl-N-[2,4-
dinitro-6-(trifluoromethyl)phenyl]-1-naphthalenamine
1 320464
X-6594 -37-
3,4-Dichloro-N-[2,4-dinitro-6-(trifluoro-
methyl)phenyl]-l-naphthalenamine
4-(1,1-Difluoroethoxy)-N-[2,4-dinitro-6-(tri-
fluoromethyl)phenyl]-l-naphthalenamine
4-(1,1,2,2-Tetrafluoroethoxy)-N-[2,4-dinitro-
6-(trifluoromethyl)phenyl]-1-naphthalenamine and
4-(1,1,2,2-Tetrafluoroethylthio)-N-[2,4-
dinitro-6-(trifluoromethyl)phenyl]-1-naphthalenamine.
Nicarbazin and 4,4'-dinitrocarbanilide are
taught in U.S. Patent 2,731,382. Nicarbazin is a
complex of 4,4'-dinitrocarbanilide and 2-hydroxy-4,6-
dimethylpyrimidine, but the 4,4'-dinitrocarbanilide
alone exhibits anticoccidial activity. See Science
122, 244 (1955).
The components of the combinations of an
A80190 compound with compounds 2(a)-(e) are used in
amounts which, in combination, are synergistic as to at
least one coccidiosis-causing organism. In general, the
maximum amounts to be used in the combinations are the
same as the maximum amounts for anticoccidial treatment
by the individual components. The lower limits are
generally less than that required for therapy by the
individual components. Accordingly, the present inven-
tion is generally practiced with compositions containing
1) from about 2 to about 100 ppm of an A80190 compound
and 2) a) from about 5 to 125 ppm of nicarbazin, b) from
about 25 to about 150 ppm of 4,4'-dinitrocarbanilide, c)
from about 1 to about 1000 ppm of the specified naphthal-
enamine, d) from about 5 to about 125 ppm of a specified
benzenamine, or e) from about 20 to about 70 ppm of
1 32046~
X-6594 -38-
metichlorpindol. The A-80190 compounds are particularly
effective when administered with nicarbazin. Preferred
combinations contain from about 2 to about 20 ppm of an
A80190 compound with from about 5 to about 50 ppm of
nicarbazin.
Another important property of the A80190
compounds is the ability to improve feed-utilization
efficiency in animals. For example, the A80190 com-
pounds improve feed-utilization efficiency in ruminants
which have a developed rumen function.
The efficiency of feed use can be monitored by
observing the production and concentration of propionate
compounds in the rumen using the method described by
Arthur P. Raun in U.S. Patent 3,794,732 (see especially
Example 5). Table IX shows the ratio of volatile-fatty-
acid (VFA) concentrations in A80190-treated flasks to
concentrations in control flasks in this test.
1 320464
X-6594 -39
1 ~ 0 0 ~ r~
O ~ ~1 a) o o ~ u~ o ~ ~ ~1
~1 ~i ~ ~ ~ CD a~ o ~ ~ ~ ~ ~ o
.. .. .. . .. . . .. . a~
00 0~1 00 0 00~1 0 00 0
E~ u
~o o~
~ V~
N a
~ ~ ~ 10 r1 ~ O O a~ ~ ~ ~ ~ N ~ ~ .
_~ ~ ?~ 0 c~ co a ~ ~ r ~ u~
.. .. .. . .. . . .. . o
m oo oo oo o oO o o oO O
V o
~ P~ ~
a~~e~ * ~ 00
O u~ 1 ~ ~ ~ ~ ~ ~ CD
~ ~ aD a~ ~ o ~ ~ ~ ~ ~ t~
~~ ~ a~ CD cr ~ o o o o ~ o o a~ ~ o
~1 a) . . . . . . . . .. . , ~ aD
X ~ O o o o ,~ o~ o o o ~ O
'~ ~0 ~
~3 ~ .,~
~: ~ ~ ~
~ ~ ~ * * * * * * * * * * * *
O ~ ~ ~ O ~ ~ ~ ~ U~
h O aD o
tq n~ ~ ~ O o ~ ~ I I
~a ,, ~ . . . . . . . . . . . . . . ~ ~ ,,
o o~ ,~ ~ ,~~1 ~1 ~
g~ ~
~ ~ 0
v t~ ~ u m
~ ~ ~ ~ ~ o
o~q . . . ,~ ~ ~ U s~
~ O ~ O O O
,1C:
o ~ ~ ~ 11 11
o o o o o o o o :~ ~ a~ a~
a~ ~ ~ ~ ,~ ,~
O O O ~1 ~1 0 ~1 ~ O ~1 ~1 O r~l _I h O O O
a~ ~ooa~oo~oo a~oo
o ~ o ~ ~: o ~ ~: o ~: ~ O ~ ~ a ~
aJ 0 al c) h ~o U h ~ r~ h ~o t~ h ~ :~ E~ ~ t4
~ ~ ~ ~ ~ * ~ ~
1 320464
X-6594M -40-
The A80190 compounds are typically effective
in increasing propionates and, thereby, the efficiency
of feed utilization when administered to ruminants
orally at rates of from about 0.02 mg/kg/day to about
1.5 mg/kg/day. Preferable rates of administration are
from about 0.05 mg/kg/day to about 0.5 mg/kq/day.
A preferred method of administration is to mix
the compound with the animals' feed; however, it can be
administered in other ways, for example, tablets,
drenches, boluses, or capsules. Formulation of these
various dosage forms can be accomplished by methods well
known in the veterinary pharmaceutical art. Each
individual dosage unit should contain a quantity of a
compound of this invention directly related to the
proper daily dose for the animal to be treated.
This invention further relates to feed compos-
itions adapted to increase feed utilization comprising
feed ration and from 2.5 to 25 grams per ton of an A80190
compound.
As described sUPra, A80190 compounds are
active against anaerobic bacteria, particularly
Clostridium perfringens. A80190 compounds are,
therefore, beneficial in the treatment or prevention of
enteritis in chickens, swine, cattle and sheep. A80190
compounds are also useful in the treatment of entero-
toxemia in ruminants.
The A80190 compounds are also anthelmintic
agents. For example, A80190 is active against
Caenorhabditis eleqans at levels as low as 20 ppm.
1 320464
X-6594M -41-
In another aspect the A80190 compounds are
useful in the treatment of swine dysentery. A80190
inhibits the growth of Treponema hYodysenteriae~ a
causative agent of swine dysentery, at levels as low as
0.39 mcg/ml. A preferred method of administration to
swine is by incorporation of an appropriate amount of an
A80190 compound into the feed ration or drinking water.
An appropriate amount will relate to whether the treat-
ment is to prevent or to cure infection. Usually, a
lower concentration of active compound is needed to
prevent infection than is required for to eliminate
infection in animals already afflicted. In general,
amounts in the range of from about 20 to about 100 grams
of A80190 compound per ton of feed are effective to
prevent infection. Amounts in the range of from about
100 to about 500 g of A80190 compound per ton of feed
are recommended for treating swine suffering from
dysentery. These amounts provide from about 1 to about
5 mg/kg of body weight per day (prophylactic treatment)
or from about 5 to about 25 mg/kg of body weight per day
(treatment of infected animal). When added to the
drinking water, amounts of from about 0.04 to about
0.2 g (prophylactic) or from about 0.2 to about 1 g
(therapeutic) of A80190 compound per gallon of water
are recommended.
This invention further relates to feed com-
positions for treating swine dysentery comprising swine
ration and an effective amount of an A80190 compound
for this purpose. As discussed, an effective amount is
typically one in the range of from about 20 to about
500 grams of A80190 compound per ton of feed.
1 320464
X-6594M -42-
The A80190 compounds can be adminis~ered to
animals orally or parenterally. They can also be
administered by insufflation, i.e. by blowing the
antibiotic, in the form of a medicated dust, into an
enclosed space or room wherein the animals or poultry
are held. The animals or poultry breathe the medicated
dust present in the air; the medicated dust also is
taken into the body through the eyes (a process called
intraocular injection).
The most practical way to administer the
A80190 compounds is by formulation into the feed supply.
A variety of feeds, including the common dry feeds,
liquid feeds, and pelleted feeds, may be used. Although
the preferred method of administration is by mixing it
with the animals' feed, it can also be administered in
other ways, for example, tablets, drenches, boluses, or
capsules. Each individual dosage unit should contain a
quantity of A80190 compound directly related to the
proper daily dose for the animal to be treated.
The methods of formulating drugs into animal
feeds are well known. A preferred method is to make a
concentrated drug premix which in turn is used to
prepare medicated feeds. Typical premixes may contain
from about 1 to about 200 grams of drug per pound of
premix. Premixes may be either liquid of solid prepara-
tions.
The final formulation of feeds for animals or
poultry will depend upon the amount of drug to be
administered. The common methods of formulating,
mixing, an pelleting feeds may be used to prepare feeds
containing an A80190 compound.
1 320464
X-6594M -43-
The A80190 compounds may be formulated for
parenteral administration by methods recognized in the
veterinary pharmaceutical art. Effective injectable
compositions containing the A80190 compounds may be in
either suspension or solution form. In the solution
form, the A80190 compound is dissolved in a physio-
logically acceptable carrier. Such carriers comprise a
suitable solvent, preservatives such as benzyl alcohol,
if needed, and buffers. Useful solvents include, for
example, alcohols, glycols, or inert oils such as
vegetable oils or highly refined mineral oils.
Injectable suspension compositions are pre-
pared using a nonsolvent for the compound with adjuvants,
as a carrier. The nonsolvent can be, for example, water
or a glycol such as polyethylene glycol.
Suitable physiologically acceptable adjuvants
are necessary to keep the compound suspended in suspen-
sion compositions. The adjuvants may be chosen from
among thickeners such as carboxymethylcellulose, poly-
vinylpyrrolidone, gelatin, and the alginates. Manysurfactants are also useful for suspending the com-
pounds. Lecithin, alkylphenol polyethylene oxide
adducts, naphthalenesulfonates, alkylbenzenesulfonates,
and the polyoxyethylene sorbitan esters are useful
suspending agents in liquid nonsolvents.
Many substances which affect the hydro-
philicity, density, and surface tension of the liquid
nonsolvent can assist in making injectable suspensions
in individual cases. For example, silicone antifoams,
glycols, sorbitol, and sugars can be useful suspending
agents.
1 32046~
X-6594M -44-
In the preparation of dusts or powders for
administration by insufflation, the compounds are
typically mixed with talc, diatomaceous earth, or some
other inert substance as an adjuvant.
The A80190 compounds are also useful as
insecticides and acaricides. For example, A80190 is
active against the following insects as levels as low
as the level indicated in parenthesis: mosquito larvae
(<50 ppm), blowfly larvae (35 ppm), adult housefly
(35 ppm), and Mexican bean beetle and southern armyworm
(<100 ppm). A80190 is also active against mites such as
two-spotted spider mite when applied at a rate as low as
25 ppm.
In another aspect, the A80190 compounds have
herbicidal activity. For example, A80190 is active
against crabgrass, mustard and pigweed at 2 lb/acre
(200 gpa) either pre-emergence or post-emergence.
The A80190 compounds are active against polio
virus. For example, tissue-culture tests show that
A80190 is active against Polio III virus at a level of
2000 ~g/ml.
Antibiotic A80190 exhibits ion-transport
properties and is, therefore, an ionophore (ion-bearer)
(see B. C. Pressman, Alkali metal chelators the iono-
phores, in "Inorganic Biochemistry," Volume 1, G. L.
Eichhorn, Elsevier, 1973). At a 0.5 ~g/ml concentration
A80190 showed ionophorous activity; its activity with
Na , K and Rb was better than that with Cs or Li .
At a lower concentration (0.2 ~g/ml), however, A80190
exhibited an even greater ionophorous effect with Cs
than with Na+ as cation.
1 320464
X-6594M -45-
A80190 can be used, therefore, when the selec-
tive removal of particular cations is desired. Examples
of such uses include the removal and recovery of silver
ions from solutions in photography, the removal of toxic
cations from industrial waste streams before such
streams are discharged to the environment, and desaliniz-
ation of sea water. A80190 can be used as one component
of an ion-specific electrode (see O. Kedem, et al., U.S.
Patent 3,753,887, Aug. 21, 1973).
A80190 alters the cation permeability of both
natural and artificial membr-~nes. A80190 can be used,
therefore, as a component in a membrane used for the
selective transport of cations against a concentration
gradient. One potential application of this property is
in recovery of heavy and precious metals on a commercial
basis [see E. L. Cussler, D. F. Evans, and Sister M. A.
Matesick, Science 172, 377 (1971)].
In yet another aspect, the A80190 compounds
are active as inhibitors of the enzyme ATPase. ATPase,
an alkali-metal-sensitive enzyme found in cell mem-
branes, is involved in the energy necessary for active
transport. "Active transport" refers to the energy
requiring series of operations whereby intracellular and
extracellular fluids maintain their compositions.
Inhibitors of ATPase reduce the energy required for
active transport. In vitro tests have shown that A80190
inhibits mitochondrial ATPase with a half-effective
concentration (IC50) of 0.5 ~g/ml.
The A80190 compounds are also potential
cardiotonic agents.
1 320464
X-6594M -46-
In order to illustrate more fully the opera-
tion of this invention, the following examples are
provided:
Example 1
Preparation of A80190
A. Shake-flask Fermentation of A80190
The culture Actinomadura oliqosDora NRRL
15878, either as a lyophilized pellet or as a suspension
maintained in liquid nitrogen, is used to inoculate
a seed medium having the following composition:
SEED MEDIUM
In ~ t Amount (%)
Glucose 1.0
Soluble starch 2.0
Yeast extract 0.5
Enzymatic hydrolysate
of casein* 0.5
CaCO3 0.1
Deionized water q.s. 1 liter
NaOH was added to raise the pH of the
medium to about 7.2 before sterilizing.
*NZ Amine A, Sheffield Chemical Co.,
Norwich, N.Y.
1. Trade Mark
. .
1 3204 64
X-6594M -47-
Slants or plates are prepared by adding 2.5%
agar to the seed medium. The inoculated slant is
incubated at 30C. for from about 10 to about 14 days.
The mature slant culture is scraped with a sterile
tool to loosen the spores and remove and macerate the
mycelial mat. About one-fourth of the loosened spores
and culture growth thus obtained is used to inoculate
50 ml of a first-stage ceed medium.
The inoculated first-stage medium i9 incubated
in a 25Q-ml Erlenmeyer flask at 30C. for about 48 hours
on a shaker orbiting in a two-inch (5.08 cm) ~ircle at
250 rp~.
This incubated first-stage medium (0.4 ml) is
used to inoculate 50 ml of a production medium having
the following composition:
Inqredient Amount (%)
Glucose 3.0
~N-Z Amine A~ 0.4
Collagen hydrolysate* 0.5
MgSO4-7H2O 0.05
CaCO3 0.2
Cold tap water q.s. 1 liter
(Presterilization pH adjusted to 7.0)
*IPC 3, Inland Industrial Molasses Co.,
Dubuque, Iowa
The inoculated production medium is incubated
in a 250-ml wide-mouth Erlenmeyer flask at 30-32C. for 8
to 10 days on a shaker orbiting in a two-inch circle at
250 rpm.
1 320464
X-6594M -48-
B. Tank Fermentation of A80190
In order to provide a larger volume of
inoculum, 10 ml of incubated first-stage medium, pre-
pared as described in Section A, is used to inoculate400 ml of a second-stage growth medium having the same
composition as that of the first-stage medium. This
second-stage vegetative medium i6 incubated in a two-
liter wide-mouth Erlenmeyer flask for about 48 hours
at 30C. on a shaker orbiting in a two-inch circle at
250 rpm.
Incubated second-stage vegetative medium (800
ml3 thus prepared is used to inoculate 100 liters of
sterile production medium, prepared as described in
Section A. The inoculated production medium is allowed
to ferment in a 1~5-liter stirred fermentation tank for
8 to 10 days at a temperature of 30-32C. Low airflow
(0.12-0.25 v/v/m) and low rpm (150-200) in the stirred
vessel maintain a dissolved oxygen level above 30% of
air saturation.
Example 2
Isolation of A80190
Whole fermentation broths from two 100-L tanks
were combined (207 L) and filtered through a filter
press with the aid of"Hyflo Supercel'.'* The mycelial
filter cake was extracted by circulating methanol (40 L)
through the filter press. Acetone can also be used as
* Trade Mark
1 320464
X-6594M -49-
an extractant. The methanol extract, concentrated in
vacuo to a volume of about 15 L, was combined with the
broth filtrate (182 L). This was adjusted to pH 9 with
lN sodium hydroxide, and the resulting solution was
extracted with an equal volume of ethyl acetate. The
ethyl acetate extract was concentrated to a volume of
about 700 ml. Water (1 L) was added to the concentrated
extract; the pH was adjusted to 9.O with sodium
hydroxide; and the mixture was extracted twice with
toluene (1 L), maintaining the pH at 9Ø The toluene
extracts were combined and concentrated in vacuo to give
an oily residue containing A80190.
The residue was dissolved in toluene (100 ml)
and applied to a column containing 2 L of silica gel
(Woelm, 70-150 mesh) in toluene. The column was eluted
first with toluene (10 L) and then with toluene:ethanol
mixtures (49:1, 10 L) and (48:2, 10 L), collecting l-L
fractions. Elution was monitored by bioassay and TLC.
Fractions containing A80190 were combined and con-
centrated. The residue was dissolved in dioxane and
freeze-dried to yield 13.6 g of crude A80190.
Example 3
Purification of A80190
Crude A80190 (20.4 g), obtained from four
100-L tanks as described in Examples 1 and 2, was
dissolved in acetonitrile (200 ml). The solution was
applied to a column containing 2 L of silica gel (Woelm,
1 320464
X-6594M -50_
70-150 mesh) in acetonitrile. The column was washed
with acetonitrile (10 L) and eluted sequentially with
acetonitrile:acetone mixtures (95:5, 2 L), (9:1, 10 L),
(4:1, 10 L) and (7:3, 10 L), collecting l-L fractions.
Elution was monitored by bioassay, using Bacillus
subtilis. Fractions containing A80190 were combined and
concentrated. The residue was dissolved in dioxane and
freeze-dried to yield 15.8 g of purified A80190.
Exam~le 4
Crystallization of A80190
Purified A80190 (28.2 g), was dissolved in
acetone (500 ml). Water (500 ml) was added, and the pH
was adjusted to 5.0 with dilute hydrochloric acid. The
resulting solution was allowed to stand at room tempera-
ture for 20 hours for crystallization to occur. The
crystals were separated by filtration, washed with water
and dried in a vacuum oven to yield 25.9 g of crystal-
line A80190 (acid form). Figure 1 shows the infrared
absorption spectrum of A80190 in chloroform.
ExamPle 5
Preparation of A80190 Sodium Salt
A80190 (acid form, 300 mg) was dissolved in
acetone (30 ml); water (10 ml) was added; and the pH was
adjusted to 10 with NaOH. The solution was concentrated
1 320464
X-6594M -51-
to an oily residue which was dissolved in dioxane and
freeze-dried to yield a white powder (m.p. 145-150).
The molecular weight of the product by field desorption
mass spectrometry (FDMS) was 850. IR in CHCl3, shown
in Figure 2, shows absorption maxima at the following
frequencies (cm 1): 2962, 2934, 2885, 2825, 1719, 1569,
1455, 1397, 1377, 1358, 1314, 1285, 1243, 1162, 1104,
1074, 1057, 1025, 987, 979, 936, 922, 895 and 847.
ExamPle 6
Alternate PreParation of A80190 Sodium Salt
A80910 (acid form, 5 g) was dissolved in ethyl
acetate (500 mg); water (500 ml) was added. The mixture
was adjusted to pH 10 with 5N NaOH, stirring for 15
minutes to maintain the pH. The ethyl acetate phase
was separated and washed with water (500 ml) and con-
centrated under vacuum to dryness. The residue was
dissolved in dioxane (100 ml) and freeze-dried to give
4.1 g of the sodium salt of A80190.
The water wash was allowed to stand at room tem-
perature overnight, and the crystals which formed were
filtered and dried. The crystals were A80190 in the
acid form (mp 115-120C).
1 320464
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Example 7
CrYstallization of A80190 Sodium Salt
A80190 sodium salt (1 g) was dissolved in
pentane l10 ml) and allowed to stand at room temperature
for three days (solvent evaporates to about 5 ml). The
crystals which formed were filtered and dried under
vacuum to give 593 mg of crystalline A80190 sodium salt,
mp 225-230C.
Exam~le 8
Preparation of Acetyl A80190
A80190 (acid form, 200 mg) was dissolved in
pyridine (4 ml); acetic anhydride (4 ml) was added; and
the mixture was allowed to stand for 72 hours at room
temperature. Water (10 ml) was added, and the aqueous
solution was extracted with chloroform (50 ml). The
chloroform extract was washed successively with 50 ml
each of O.lN HCl, water containing 1 percent NaHCO3, and
water. The chloroform extract was then concentrated ln
vacuo to a residue, and the residue was dissolved in
acetone (100 ml). The acetone solution was concentrated
in vacuo to remove residual pyridine and acetic acid.
This step was repeated three times, and the resulting
residue was dissolved in acetone (50 ml). Water (50 ml)
was added, and the resulting solution was allowed to
stand at room temperature for crystallization to occur.
1 320464
X-6594M -53-
The crystals were separated by filtration, washed with
water and dried in a vacuum oven at 50C to yield 192 mg
of crystalline acetyl A80190 (m.p. 87-89C). Molecular
weight = 870 by fast-atom bombardment mass spectrometry
(FABMS). IR in CHC13, shown in Figure 3, shows absorption
maxima at the following frequencies (cm~1): 3021, 3018,
2935, 2825, 1726, 1454, 1373, 1311, 1244, 1162, 1104,
1078, 1058, 1024, 987, 976, 934 and 925.
Exam~le 9
Pre~aration of Propionyl A80190
A80190 (acid form, 200 mg) was dissolved in
pyridine (4 ml); propionic anhydride (4 ml) was added;
and the mixture was allowed to stand at room temperature
for 72 hours. Water (10 ml) was added, and the solution
was extracted with chloroform (50 ml). The chloroform
extract was washed successively with 50 ml each of O.lN
HCl, water containing 1 percent NaHCO3 and water. The
chloroform extract was concentrated in vacuo to a
residue which was dissolved in acetone (100 ml). The
acetone solution was concentrated in vacuo to remove
residual pyridine and acetic acid. This step was
repeated three times. The residue was dissolved in
acetone (50 ml); water (50 ml) was added; and the
mixture was allowed to stand at room temperature for
16-20 hours for crystallization to occur. The crystals
were separated by filtration, washed with water and
dried in a vacuum oven at 50~C to yield 170 mg of
1 320464
X-6594M -54-
crystalline propionyl A80190 (m.p. 83-85C). Molecular
weight of 884 by FDMS and FABMS. IR in CHC13, shown in
Fig. 4, shows absorption maxima at the following fre-
~uencies (cm l): 3018, 2976, 2935, 2827, 1725, 1460,
1376, 1313, 1160, 1105, 1082, 1058, 1024, 987, 979, 945,
935 and 893.
Examples 10-12
The following A80190 ester derivatives can be
prepared using the method of Examples 8-9:
n-Heptanoyl A80190
Valeryl A80190
tert-Butyryl A80190
Example 13
Preparation of A80190 4-Bromophenvlurethane Derivative
A80190 (acid form, 7 g) was dissolved in
benzene (100 ml), and 4-bromophenyl isocyanate (3 g) in
benzene (100 ml) was added with stirring. The mixture
was stirred at room temperature for 168 hours. The
reaction was monitored daily by silica-gel TLC, using
acetonitrile:acetone (1:1) as the developing solvent and
detecting with vanillin-H2S04 spray reagent. The reac-
tion appeared to be complete by 168 hours. The pre-
cipitate which formed was separated by filtration,
and the filtrate containing the derivative and some
unreacted A80190 was concentrated and lyophilized to
give 7.5 g of product.
1 320464
X-6594M -55~
This product was dissolved in acetonitrile
(50 ml) and applied to a 3- x 140-cm column containing
800 ml of silica gel (Woelm, 70-150 mesh) in aceto-
nitrile. The column was developed with acetonitrile,
collecting a first fraction having a volume of 500 ml,
five subsequent fractions of 100 ml each and then six
fractions of 500 ml each. Elution was monitored by
silica-gel TLC. Crystals formed in the fourth fraction
after standing at room temperature overnight. The
crystals were separated by filtration and dried under
vacuum to give 689 mg of A80190 4-bromophenylurethane
derivative.
The filtrate was dried. The residue was
dissolved in dioxane (50 ml) and lyophilized to give an
additional 477 mg of the product. The fifth through the
tenth fractions were combined and concentrated to
dryness. The residue was dissolved in acetonitrile
~50 ml) and lyophilized to give an additional 2.3 g of
amorphous A80190 4-bromophenyl urethane derivative.
This compound had the following characteristics:
mp: 142-145C
[~]25 +4O (c 10, CHCl3)
molecular weight: 1025 (fast-atom-bombardment mass
spectrometry)
Examples 14-15
The following A80190 urethane derivatives were
prepared using the method of Example 13: A80190 4-
chlorophenylurethane derivative and A80190 4-nitro-
phenylurethane derivative.
1 320464
X-6594M -56-
Examples 16-23
The following urethane derivatives can be prepared
using the method of Example 13:
A80190 phenylurethane
A80190 4-methylphenylurethane
A80190 4-iodophenylurethane
A80190 4-fluorophenylurethane
A80190 cyclohexylurethane
A80190 2-phenethylurethane
A80190 2-(phenyl)cyclopropylurethane
A80190 4-phenoxyphenylurethane
Example 24
Preparation of A80190 Methvl Ether Derivative
A80190 in the acid form is dissolved in
methanol; water (~ volume) is added. The solution is
allowed to stand until the ether derivative is formed.
The solution is evaporated under vacuum. The product is
chromatographed using, for example, silica gel to give
to A80190 methyl ether derivative.
Examples 25 to 27
Using a procedure like that of Example 24 and
the appropriate alcohol or thiol, the following A80190
ether derivatives can be prepared:
1 3204 64
X-6594M -57-
A80190 n-Propyl Ether Derivative
A80190 Methyl Thioether Derivative
A80190 n-Butyl Ether Derivative
Example 28
Chromatoqraphic Identification of A80190
I. TLC on silica qel
System: acetonitrile:acetone (1:1)
Rf = 0.59
Detection: Bacillus subtilis
Vanillin-H2SO4 spray
II. HPLC *
Adsorbent: aBondapak C18"(4 x 300-mm column)
Solvent system: acetonitrile:tetrahydrofuran:H20
(6:1:3) containing 1 percent
H3PO4; adjusted to pH 3.0 with
NH40H
Detection: refractometer
Flow rate: 3.0 ml/min
Retention time: 9.7 min
Example 29
A80190 is prepared by the method of Example 1,
but using the Actinomadura oligosporus NRRL 15877 culture.
* Trade Mark
I 320464
X-6594M -58-
Example 30
A80190 Modified Chick Ration for Coccidiosis Control
A balanced, high-energy ration adapted to feed
- chicks for rapid weight gain is prepared by the follow-
ing recipe:
Ingredient % lbs
Ground yellow corn 50 1,000
Soybean meal, solvent-
extracted dehulled, finely
ground, 50 percent protein 31.09 621.8
Animal fat (beef tallow) 6.5 130
Dried fish meal, with
solubles (60% protein) 5.0 100
Distillers' solubles
from corn 4.0 80
Dicalcium phosphate,
25 feed grade 1.8 36
Calcium carbonate 0.8 16
1 320464
X-6594M -59-
Inqredient % lbs
Vitamin premix
(representing vitamins A,
D, E, K, and B12, choline,
niacin, pantothenic acid,
riboflavin, biotin, with
glucose bulking agent) 0.5 10
Trace mineral premix
(representing MnSO4, ZnO,
KI, FeSO4, CaCO3) 0.2 4
2-Amino-4-hydroxybutyric acid
(hydroxy analog of methionine) 0.1 2
A80190 (Na Salt) 0.01 0.2
These substances are mixed in accordance with
standard feed-mixing techniques. Chicks fed such a
ration, with water ad l bitum, are protected against
exposure to coccidiosis; weight gains are comparable to
those of coccidiosis-free chicks fed a similar, unmedi-
cated diet.
1 320464
X-6594M -60-
Example 31
A80190-ImProved Beef-Cattle Ration
A balanced high-grain beef-cattle ration is
prepared as follows:
Ingredient % lbs
Finely ground corn 67.8 1356
Ground corn cob 10 200
Dehydrated alfalfa meal,
17 percent protein 5 100
Dehulled soybean meal,
solvent extracted,
50 percent protein 9.9956199.912
Cane molasses 5 100.0
Urea 0.6 12.0
A80190 (Na salt) 0.00440.088
Dicalcium phosphate,
feed grade 0.5 10.0
Calcium carbonate 0.5 10.0
t 320464
X-6594M -61-
Inqredient % lbs
Sodium chloride 0.3 6.0
5 Trace mineral premix 0.03 0.6
Vitamin A and D2 premix* 0.07 1.4
Vitamin E premix** 0.05 1.0
Calcium propionate 0.15 3.0
*Containing per pound: 2,000,000 I.U. of vitamin A;
227,200 I.U. of vitamin D2 and 385.7 g of soybean
feed with 1% oil added
**Corn distillers dried grains with solubles containing
20,000 I.U. of d-alpha-tocopheryl acetate per pound
The mixed feed is compressed into pellets. At
an average daily ingestion rate of 15 pounds of feed per
animal, this feed supplies approximately 300 mg of
A80190 (Na salt) per animal per day.
1 3204 64
X-6594M -62-
Example 32
A80190-Improved Swine Ration
A balanced swine farrowing ration is prepared
as follows:
Inqredient %lbs/ton
Ground yellow corn 65.10 1302
Soybean oil meal, solvent
extracted dehulled 18.50 370
Dried-beet pulp 10.00 200
Dicalcium phosphate 2.90 58
Calcium carbonate 1.20 24
Swine vitamin premix1 1.10 22
Salt (NaCl) 0.55 11
Choline chloride, 25% 0.35 7
Trace-mineral premix2 0.15 3
Vitamin A premix3 0.10 2
30 Hydroxy analog of Methionine 0.05
Total 100.00 2000
1 320464
X-6594M -63-
1Each kg of premix contains the following: 77,161
USP units Vitamin D2; 2,205 I.U. of Vitamin E;
441 mg riboflavin; 1,620 mg pantothenic acid;
2,205 mg niacin; 4.4 mg Vitamin B12; 441 mg
Vitamin K; 19,180 mg choline; 110 mg folic acid;
165 mg pyridoxine; 110 mg thiamine; 22 mg biotin.
2Each kg of premix contains the following: 50 g
of manganese as manganese sulfate; 100 g of zinc
as zinc carbonate; 50 g of iron as ferrous sulfate;
5 g of copper as copper oxide; 1.5 g of iodine
as potassium iodide and 150 g maximum and 130 g
minimum calcium as calcium carbonate.
3Each kg of premix contains 6,6138,00 USP units
Vitamin A.
For 200 pounds of this ration, a premix is
prepared by adding A80190 (10 g) to a small amount of
solvent-extracted soybean feed, grinding them in a
mortar and pestle, and diluting the ground mixture to
one pound with additional solvent-extracted soybean
feed. This premix is then added to 200 lb. of the
above-described swine ration, mixing by standard
techniques. This medicated feed provides a level of 100
grams of A80190 per ton of basal ration. Medicated feed
is fed to sows for at least one day, and preferably from
seven to ten days, prior to farrowing and after farrow-
ing for as long as is desirable.
1 320464
X-6594M -64-
Larger or smaller quantities of medicated
ration with varying levels of A80190 are prepared by
varying the quantity of A80190 in the premix and/or the
quantity of basal ration.
Sows are fed at rates of from 1.0 to 10 grams
of A80190 per 100 pounds of feed. The medicated feed is
available ad libitum with water. Usually, sows consume
about 6-8 lb of ration per day.
Example 33
A80190-Formulation for Piglets
A80190 is dissolved in a small amount of
ethanol. This ethanol solution is suspended in poly-
ethylene glycol 200. The suspension is concentrated so
that each unit dose has a volume of about 0.5 to 2 ml.
Such suspensions are given to young pigs at rates of 0.5
to 50 mg per lb, three times a day by gavage.
Example 34
Im~roved Ration for Control of Swine Dysenterv
A premix is prepared by standard methods
using the following ingredients:
1 320464
X-6594M -65-
Inqredient Grams/Kiloqram
Active Compound 150.0
Calcium Silicate 20.0
Calcium Carbonate
(Oyster Shell Flour) 830.0
Total Weight 1000 gms.
This premix is added to commercial swine ration, using
standard feed-mixing techniques, to givP a final level
of active compound of 100 grams/ton.
Demonstration of the synergistic effect with
nicarbazin in combination with A80190 was obtained as
follows: one-week-old broiler chicks were alloted to
five-bird cages and were fed a medicated or control
ration, typically for one day, prior to infection with
oocysts of a coccidiosis-causing organism. The chicks
were maintained on their respective rations for a period
of time, typically seven days. There were four repli-
cates per treatment.
Test 1
The chicks were inoculated with 200,000 oocysts
of Eimeria acervulina ~Lilly strain 59), 60,000 oocysts
of Eimeria maxima (strain FS-177) and 40,000 oocysts of
Eimeria tenella (strain FS-155).
1 32()4f,4
X-6594M -66-
INTESTINAL LESION SCORES
A80190 (p.p.m.)
Nicarbazin
(p.p.m.) 0 4 8 12 16 20
0 9.13 8.63 5.94 3.50 1.19 0.0
9.13 5.13 0.19 0.13 0.0
8.88 1.63 0.50 0.0
8.90 0.56 0.0
8.56 0.0
125 0.08
15CECAL LESION SCORES
A80190 (p.p.m.)
Nicarbazin
_(p.~.m.) 0 4 8 12 16 20
0 3.44 2.63 1.75 1.19 0.19 0.0
3.19 2.38 0.31 0.56 0.06
3.50 0.94 0.19 0.0
3.17 1.00 0.58
3.19 0.54
125 0.33
WEIGHT GAIN (grams/per chicken)
30A80190 (p.p.m.)
Nicarbazin
(P.p.m.) 0 4 8 12 16 20
0 147 236 265251 272 261
159 268 284266 279
174 279 258283
211 258 289
222 276
125 279
