DK159684B - Polyether ANTIBIOTIC REFERRED A80190 AND SALTS, METHOD FOR PRODUCTION OF THESE RELATIONS AND MICRO ORGANISM STRAIN FOR USE IN PURSUIT OF PROCEDURE, COMPOUNDS FOR USE AS VETERINAERPRODUKTER AND THE COMBAT coccidiosis in POULTRY, FREMGANGMAADE TO INCREASE FEED utilization efficiency in ruminant animal AND COMPOUND CONTAINING compounds - Google Patents

Description

in

DK 159684 B

This invention relates to a novel polyether anti-biotic named A80190 and pharmaceutically acceptable salts thereof with bases, a process for the preparation of these compounds and a novel microorganism strain for use in the practice of the process. The invention further relates to the compounds for use as veterinary products and in the control of coccidiosis in poultry, methods for increasing the feed utilization efficiency of ruminant animals and for promoting the growth of monogastric animals, and animal feed mixtures containing the compounds.

Westley (John W. Westley, "Polyether Antibiotics: Naturally Occurring Aid Ionophores, Vol. 1, Chemistry", Mar-15 Cell Dekker: New York, 1983) has divided existing polyethers by class and type. Using Westley's system, the A80190 is a new member of class lb, type (1), belonging to the group of polyethers, since the compound contains a spirochetal system. Other members of this group include A28695 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).

The subject compound, A80190, has the structure shown below: OMe f '^' V'Ole i / OMe y! ± ®A Me 5 Me ile HOecX i r \ A / \ A ^,

OH

DK 159684 B

2

Characteristics of A80190

Antibiotic A80190 has been assigned the structure (1) shown above on the basis of X-ray crystallographic studies. A80190 (in its free acid form) has the following characteristics:

Condition: White crystals (from acetone-water) mp: 98 - 100 ° C or 120 - 122 ° C (more hypotensive) j probably varied with degrees of solvation; 25 pKa = 6.2 (66% aqueous dimethylformamide) O] D = 15 -26 ° (c 1, CHCl 3)

Molecular weight: 828 (mass spectrometry with field desorption) Empirical formula: C44H76 ° 14 UV: no absorbance IR: (CHCl3) Figure 1; shows absorption at the following frequencies (cm-1): 3019, 2970, 2936, 2827, 1721, 1457, 1402, 1376, 1314, 1163, 1105, 1092, 1083, 1056, 1022, 1006, 989, 980, 945, 934, 917, 982 and 859 30 35

3 DK 159684 B

EA:

Found Calculated

Carbon 63.35 63.77 5 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 hydrocarbons such as chloroform and hydrocarbons such as diethyl ether, benzene, toluene and hot hexane.

15 A80190 contains an acidic function capable of forming salts with bases. A80190 as well as the pharmaceutically acceptable salts of A80190 are useful as antibiotics, as coccidiostats, and as agents that enhance the efficiency of animal feed utilization.

A80190 and its salts (hereinafter "the A80190 compounds") are useful as antibacterial and anticoccidial agents. They increase the efficiency of feed utilization in 25 ruminants and act as growth promoters in monogastric animals. In addition, they exhibit insecticidal, herbicidal and antiviral effects. They are also useful as ionophores. 1 2 3 4 5 6

The invention further relates to synergistic supplements 2 for poultry feed consisting of A80190, or pharmaceutically acceptable salts thereof, together with a compound 4 selected from nicarbazine and 4,4'-dinitrocarbanilide. These 5 supplements are useful for controlling coccidiosis 6 in animals.

"DK 159684 B

4

The salts of A80190 are useful for separating and purifying the antibiotic. The pharmaceutically acceptable salts are particularly useful. Such salts exhibit little or no toxicity to warm-blooded animals. Examples of salts 5 of A80190 include alkali metal, alkaline earth metal and amine salts.

Representative and suitable alkali metal and alkaline earth metal salts of A80190 include sodium, potassium, lithium, cesium, rubidium, barium, calcium and magnesium salts.

Examples of suitable amine salts of A80190 may be cited as the ammonium salt as well as the primary, secondary and tertiary C1-C4 alkylammonium and hydroxy C2-C4 alkylammonium salts. Examples of illustrative amine salts include salts formed by reaction of A80190 with ammonium hydroxide, methylamine, sec-butylamine, isopropylamine, diethylamine, diisopropylamine, ethanolamine, triethylamine and 3-amino-1-propanol.

20 It is well known in the veterinary pharmaceutical field that the form of an antibiotic itself is usually not of greater importance when treating an animal with the antibiotic. In most cases, the conditions prevailing in the animal's organism will change the drug to a different form than the form in which it was administered. Therefore, the salt form in which the drug is administered is of no greater importance. However, salt form can be chosen for reasons of economy, convenience and lack of toxicity.

30

The antibiotic A80190 can be prepared by growing an A80190-producing strain of Actinomadura oligospora under sub-aerobic culture conditions in a suitable medium until substantial antibiotic activity is obtained. The antibiotic can be recovered by various isolation and purification procedures well known to those skilled in the art.

DK 159684 B

5

A new Actinomadura oligospora microorganism producing the antibiotic A80190 was isolated from a soil sample from India. Subsequently, a significantly improved variant leading to increased production of A80190 was isolated by LaVerne D. Boeck.

Cultures of these two A80190-producing organisms have been deposited in accordance with the Budapest Treaty in the Collection of Tribal Cultures at the Northern Regional Re-10 Search Center, Agricultural Research, North Central Region, 1815 North University Street, Peoria, Illinois, 61604, from which they are available to the public under the landfill numbers NRRL 15877 (parent strain) and NRRL 15878 (variant strain).

15

Taxonomic studies of the variant have been conducted by Frederick P. Mertz at the Lilly Research Laboratories. On the basis of these studies, the two new organisms have been classified as members of a new species 20 of the genus Actinomadura, for which the name Actinomadura oligospora sp. November This classification is based on direct laboratory comparisons with similar species and review of published descriptions [M. Goodfellow and G. Alderson, "Numerical Ta-25 xonomy 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," pp. 261 - 276 in FA Skinner and DW Lovelock (ed.), "Identification Methods for Microbiolo-30 gists," 2nd ed., The Society for Applied Microbiology Technical Series No. 14, Aademic Press, New York, 1979; L. H. Huang, "Actinomadura macra sp. Nov., The Producer of Antibiotics P-47, 433 and CP-47,434". Int. J. Syst. Bacteriol. 30: 565-568 (1080), and Η. A. Lechevalier and M.

35 P. Lehevalier, "A Critical Evaluation of the Genera of Aerobic Atinomycetes", pp. 393-405 in H. Prauser (ed.), "The Actinomyetales", Gustav Fischer Verlag, Jena].

DK 159684 B

6

Methods used

The methods recommended by the International Streptomyces Project (ISP) for characterizing Streptomyces species [E. B, Shirling and D. Gottlieb, "Methods for Characterization of Streptomyces Species", Int. J. Syst. Bacteriol. 16: 313-340 (1966)], have been followed with certain supplementary experiments (DJ Blazevic and GM 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 a final concentration of 1.0%. The plates were incubated at 30 ° C and read after 14 days.

The production of melanoid pigment (chromogenicity) was determined with ISP # 1 (tryptone yeast extract liquid), ISP # 6 (peptone yeast extract iron agar), ISP # 7 (tyrosine 20 agar) and modified ISP No. 7 from which the tyrosine was removed.

The starch hydrolysis was determined by testing for the presence of starch using iodine on ISP No. 4 25 (agar of inorganic salts and starch) plates (see Blazevic and Ederer, supra).

The morphology was examined using an optical light microscope. A scanning electron microscope (SEM) was used to study the trace surface ornament.

NaCl tolerance was measured by adding NaCl to ISP # 2 agar to the desired concentration. 1 ICSS-NBS Centroid Color Charts, Standard Sample 2106 (National Bureau of Standards, 1958, U.S. Department of Commerce, Washington, D.C.) and the Color Harmony Manual

DK 159684 B

7 (4th ed., Container Corporation of America Chicago, Illinois, 1958) was used to assign color names.

The isomers of diaminopimelic acid (DAP) and carbohydrates in whole cell hydrolysates were established by the chromatographic methods described by Becker et al., [B. Becker, Μ. P. Lechevalier, R. E. Gordon and Η. A. Lecheva-lier, "Rapid Differentiation between Nocardia and Strep tomyses by Paper Chromatography of Whole-Cell Hydrolysates," Appl. Microbiol. 12: 421-423 (1964)] and by Lechevalier [Μ. P. Lechevalier, "Identification of Aerobic Acti-nomycetes of Clinical Importance," J. Lab. Clin. With. 71: 934-944 (1968)].

The resistance to lysozymes 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 Mycobacterium and Nocardia as a Taxonomic Tool",

Int. J. Syst. Bacteriol. 27, 176-178 (1971)].

20

Antibiotic resistance was measured by placing slices of antibiotic sensitivity on the surfaces of grafted ISP # 2 agar plates.

Phosphatase and urease were determined by the methods described by Blazevic, supra.

The mycolic acids were determined by a method based on techniques described by Minnikin [D. E. Minnikin, L. Al-30 shamaony, and M. Goodfellow, "Differentiation of Mycobacterium, Nocardia, and Related Taxa by Thin-Layer Chromatographic Analysis of Whole-Organism Methanolysates," J.

Gen. Microbiol. 88: 200-204 (1975)]. 1

The phosphorlipid assay was performed as described by Lechevalier [Μ. P. Lechevalier, c. De Bievre and H. Lechevalier, "Chemotaxonomy of Aerobic Actinomycetes: Phospho-DK 159684 Bay Lipid Composition", Biochemical Systematics and Ecology 5, 249-260 (1977)].

Cultural characteristics 5 The growth of the organism was generally poor on chemically defined media, with better on complicated organic media. There was an absence of aerial mycelia with the exception of trace amounts on ISP # 4 and sodium butyrate agar. When 10 spores were present, these had an oyster-white color in the Tresner and Backus system [H. D. Tresner and E. J. Backhus, "System of Color Wheels for Streptomycete Taxonomy",

Appl. Microbiol. 11: 335-338 (1956)]. The color of the back was gray-yellow to brown. No soluble pigments were produced except for a very light brown soluble pigment in ISP # 2 and a dark brown soluble pigment in yeast extrusion agar. The following Table I shows these cultural characteristics; comparing the corresponding characteristics of the known strain 20 Actinomadura macra.

25 30 35

DK 159684 B

9 td Φ

H

Ό I 3 ^

5 L £ S

S ΟΛ -¾ 10 ^ s s Φ * 3 ® £ οι o> · y J? o H · cD Λ C. £ H cr> m -H ^ H 00 P tH -

1n j * ^ H Si £ H .Sj H

10 w Id 0) 03 H C -H fi OH

g g ijh.pp O.P p * ftP P ° pp Φ <2 Λ Φ6ΦΦ d · Φ Φ G Φ φ> φ φ Λ i &. “οι> ερ ρ & ε +> ρ ό · ρρ <0 • α. η oo d φ ίβ · dd 8 dd ο race ρ a <Ρί 'Φ Η 2) DlOH Η Ό in Η Η ϋ ^ ΗΗ w- (Ο Μ Ο (0 15 ε .. «i Η Φ Ο • Η Η j ri1 <- »> Η Η Η Φ Φ Φ 00 Ο) Ό 3 2 <Ο <0 'Ο' Ο Επ η · η ij 00 Ή>> [> ·. µ + J Η ^ co φ e ό σ lo ^ d G d 20 Η 0 Ja £ 'Η S' Η J Ο) Η Φ · Η Φ £ co · η d ρ> ρ £ £ o5i>. ρ d to χ! ό £ · α and ni u ^ ° (d d) o (d Φ ° w 55 So d λ η haiga &

Qj r-ι u_j · h) Q) φ D) Oi 0) Cn aw>, pp φ »Ο0. +» Φ, ·· w 0. + »φ -¾ j Ο) φ φ a> ihs φ oi ^ . e φ a> <φ ^ td ρί ό · ροι d * o «tddx> d O-tidP« * ρ p, ¾ Οί ο Η d Φ · Η 00 Οι G Η d -HOOdiEHd · η oo dd 4J 2 O VO Η 2 ttiOMflCQ ^ H κ σ> CO CO XH pi O 'Η Η

25 W

* r \ Η φ Ρ (0

H

(0 Λί Η 30 £ ιΗ ·· ·· ·· ·· ·· ····· ^ (I t · tf Οι ·· ** β Οι ·· ** s al * · ε a ι * Ο Ρί <C0 Ο Pi <C0 ϋΚιϊ C0 Ο 0 $ <C0 5 ε d Η Ό φ g 35 1 Ν 00 ^ 10 ί-ι to a * a · a * a * a coo co o coo coo <H g H g Hg Hg 10

DK 159684 B

X ′ Τ ′ fl Η • ri · φ

Dl -P

β c> 1 g ^

5 3 · .¾ R

ΰ R H <D

Λ fi -P

«I 3 -Ρ R ϋ | * 8

. C * CO

> 1 · ρ

O) -PR

. Ό Ό O

Η X · Η Η «Η β>>

Ρ Q) -Η £ Æ C

m ° <d R «Η Di, O, Φ

10 (0 R O in «I R M | H

U O O) 10> H · H

η.-PP -PP ·· -P · »-P> i ·· +» ® (d β> φ φ fi>, φ φ · R Φ CO β Φ ttRil Λ ρ>. +) + j> »4J + ) ΌΜΟ-Ρ Ό ·> -Ρ Ό · O -Ρ Λ. «Οοββ« οοββ Ο νο Di β OvOffiD Ο σνΟι β -Ρ <Η Η rjOVHH Ο Μ CO Η 0 Μ · η Η 000CGH 'χ' Ρ (0 _ C0 Τ '• Ρ 5 ~, ¾ $ -4 ^ Φ Ό ο φ ο> ® ϋ_ι rrt (0 ^ Η α Λ ® <*

HH 0> 0 R

«Η · Η (d Φ J R -Ρ Η> ω Q) IR Ο CQ> -Ρ R ___ 3 Ο β Ρ ®» g Ε-ι 3 S> Ή 3

ρη Ο) R «Η Ο Ρ R

you j η ΟΙ .Q

Ρ. R β Ο), 3 00>)> ι J) 3 Η Η * Η · IS 3 · Ρ Ρ R Ρ 3 w> η 00 «Η> Ί β - (0 β Λ 0) t3) _ · in w Ο ) 3 R <d 3 0> H · RO) id> 1 * H · -HO) 0 Η · Ρ Λ -PP • χ '· Ρ Ρ χ'> ιΡ Ρ Η · Ρ Ρ J 0) · ®Φ β > ι φ Φ g Φ φ ΟΙ Φ Φ φ Η φ DS β β σν -Ρ Μ> · Ρ Ρ Ό · Ρ Ρ Ό · ρ Ρ & · ρ ρ οί · ΗΕ ^ β> ι (Β 00 β β Ο Μ β β οοββ -Ησνββ £ Κ ΜΗ J 03 σν Η Η ο Μ Η Η 0σν · ΗΗ ΚΜΗΗ 30 ·· ·· ·· ·· »» * · ·· # · ·· ·· ·· ·· 6 Οι ·· ·· £ · Οι ·· * · β Οι ·· ·· S Qf ·· ·· 6 Οι 0 DS rtj ø 0 DS 3 ω 0 DS <0 0 DS <0 0 DS <0 1 (d

• H CO β P

Η 10 - H CO H

φ - β φ οι φ φ c x o co cd Di ε 35 i o Φ W OR Ρ Φ

L Dl R otd cdR

cd Dl · cd Φ 3 Οι ε> O) 00 N ε H CO o 3

<HZ U 0 O <HK

DK 159684 B

11 Ό <d Η m

5 P

Φ>

0 P

o> ° ro

ή P

p b>

3 ·· C X

<h P P 3 fi

- O C P -H

a 3 λ ft 10 td b> P ro p w.

u -π cq Yes> i> 1 R Η · ρ ©. p p · p p (d 0) Ό Ό) Χ fi Ό <D 0) file »®« £

S b) · <0 P> · P P> · P P C

. feoofifi fBvOCfi Φ <DJ li) CO S I ^ ISHH t-JMHH É O)

H

JJ ft

«0 JJ

οι ^ ii P 5 ©? u v ^ o ro d «Η Η © m ro

Η P

Q) H

J> 9 * ω o o 03 <0> 11 * P § P ft 20 b> P Ό ω

oo 5Λ P> S

i> ~> i c px; ^ 3 co · 3 ° {d ° <o * h

m b)> ι P ΡΛΙ P H

h -h b> Λ O) .. 0> ©

Η · P (D ·· P PP O

J © Ό © A! > i 3 © file »®® £

rK O). + J Η Ό ·> P> · P P E

S -HHfi «. OOOffifl 8 W C C P

is DJ 00 H s OOlhH OOHH m 25 3

II

© Ό

H

(0 _Λ b) 30 id Λ ·· M ·· ·· ** ·· • · ·· C3 p, ·· it β Pi ♦♦ ·· β Pi ti

O DS * 2 to CD P5 <3 CO CD 05 <5; CO

05 3 +> • h «5 0) I .Μ d) ω ep e 8 g O 3 id 3>

35 in P HP H P

p IP P> i Ό td II

(d P X P P HH

b) s © ro 3 ro ro cd <CD Ό 2Λ oe ro

DK 159684 B

12

Morphological characteristics

There was sparse production of tracer chains on ISP # 4 and sodium butyrate agar. The sporeophores contained about 5-10 spores per chain, and they were generally twisted as in Recus-flexilis (RF) configuration. However, hook-shaped sporophores could also be observed. The air hyphens tended to clump together. The spore shape was elongated, and the size ranged from 0.5 to 0.7 µm on the one joint and from 0.9 to 1.3 µm on the other joint. The average track size was 1.1 x 0.6 µm. The spore-surface ornamentation was smooth.

Physiological characteristics 15 In the following Table II, the carbohydrate utilization pattern of the strain is given in comparison with the pattern of A. macra. ISP medium # 9 was used as basal medium. By adding vitamin or by using Luedemann's medium [G. M. Luedemann and B. Brodsky, "Micromonospora carbonacea sp. N., In Everinomicin-Produce Organism," Antimicrob. Agents Chemother. 1964, 47-52] achieved slightly improved growth, but no changes in carbon utilization. Adonitol, cellobiose, glucose and ribose were utilized. Doubtful exploitation was noted with fructose and xylose. Arabinose, cellulose, dextran, galactose, i-inositol, inulin, lactose, mannitol, mannose, melizitosis, milibiosis, raffinose, rhamnose, salicin, sucrose, trehalose and xylitol were not utilized for growth.

35

DK 159684 B

13

TABLE II

a

Carbon utilization pattern for NRRL 15878 and A. macra 5 _

Carbon source NRRL 15878 A. macra control 10 adonitol + L-arabinose

Cellobiose +

cellulose - ND

dextran - ND

15 D-fructose ± D-galactose - ± D-glucose + + i-inositol - ±

inulin - ND

20 D-lactose mannitol D-mannose D-melezitose D-melibiosis 25 raffinose L-rhamnose ribose + ± salicin sucrose - + 30 trehalose - +

xylitol - ND

D-xylose ± ± a + = utilized, - = not utilized 35 ± = questionable utilization, ND = not determined.

DK 159684 B

14 Table III below shows the resistance of the strain to various antibiotics at the indicated concentrations and that is compared with the resistance of A. macra.

5

TABLE III

Antibiotic resistance of NRRL 15878 and A. macraa_ 10

Anti-Concentration NRRL 15878 A. macra biotic ion 15

Bacitracin 10 units +

Cephalothin 30 µg ++

Gentamicin 10 µg 20 Lincomycin 2 µg ++

Neomycin 30 µg

Oleandomycin 15 µg

Penicillin G 10 Units ++

Rifampin 5 µg + + 25 Streptomycin 10 µg - +

Tetracycline 30 µg

Tobramycin 10 µg

Vancomycin 30 µg 30 α + Resistance (No Inhibition Zones) - = Sensitive (Inhibition Zones) 35

DK 159684 B

15 NRRL 15878 grew at temperatures from 15 to 42 ° C and tolerated up to 2% NaCl. The organism produced catalase, phosphatase and urease.

NRRL 15878 degrades on casein, DNA, esulin and gelatin, but not on adenine, calcium malate, chitin, elastin, guanine, hippurate, hypoxanthine, keratin, starch, testosterone, tyrosine and xanthine.

Cell wall analysis

The whole cells hydrolyzed contained the meso isomer of diaminopimelic acid. The sugars present in the whole cell hydrolyzates were as follows: glucose, mannose, madurose and ribose. The type of cell wall according to Becker, supra, is type III, and the sugar pattern is of type B (Lecheevalier, 1968). A qualitative analysis of whole cell methanolysates for mycolic acids gave questionable results. Thus, it is doubtful whether the culture contains mycolic acids. A phosphor lipid pattern of type PI was found. Type PI does not contain any nitrogenous phosphorus lipids, and this type is characteristic of the genus Actinomadura (Lechevalier, 1977).

25

Identity of the strain NRRL 15878 has a type III cell wall, a sugar pattern for the whole type B cells and a phorphorlipid pattern 30 of type PI. This chemotaxonomic information as well as the general cultural characteristics are consistent with the strain attributed to the genus Actinomadura Lechevalier and Lechevalier (Lechevalier, 1970). 1

A comparison of these characteristics with those given in published descriptions of known species of Actinomadura shows that the culture is reminiscent of

DK 159684 B

16 on A. pelletieri (Laveran 1906) Lechevalier and Lecheva lier, 1070, and to A. macra (Huang, 1080).

The culture is first and foremost reminiscent of Actinomadura pelleti-tieri in the absence, or at least the rare occurrence, of aerial mycelia. When producing sporophores, these have a morphology similar to the morphology described in the literature (Goodfellow, 1979). The two cultures also possess a number of common physiological characteristics. However, the differences in cultural characteristics and the physiological differences are sufficient to distinguish the two cultures as distinct species.

According to Lechevalier (1970), A. pelletieri 15 is represented exclusively by a group of clinical isolates. Original description of A. pelletieri, given by Gordon [R. E. Gordon, "Some Criteria for the Recognition of Nocardia madurae (Vincent) Blanchard", J. Gen. Microbiol 45: 355-364 (1966)] describes the organism as being a clear red culture. NRRL 15878, on the other hand, does not produce this pigment. A. pelletieri degrades elastin, hypoxanthine, keratin and tyrosine, utilizes trehalose, reduces nitrate and grows at 45 ° C, but NRRL 15878 does not have these characteristics. NRRL 15878 utilizes adonitol and cellobiose, degrades esculin and DNA, and is resistant to lysozymes, whereas A. pelletieri does not have these characteristics. The new culture must therefore be regarded as a different species from A. pelletieri. 1 2 3 4 5 6 Due to the similarity of culture to A. macra, 2 simultaneous laboratory comparisons were made. NRRL 15878 and A.

3 macra has many common characteristics. They are both unable to break down adenine, calcium malate, chitin, ela 5 stin, guanine, keratin, starch, testosterone, tyrosine and 6 xanthine. None of the organisms produce or metalanoid pigments. Both cultures break down casein, DNA and gelatin, they produce catalase and phosphatase, and the 17

DK 159684 B

synthesizes a polyether antibiotic. They have the same tolerance to NaCl and they both grow on sodium butyrate, just as they have the same type of cell wall. A. macra and NRRL 15878 differ from each other in terms of the carbon-5 utilization pattern, degradation of esculin and hypoxanthine, resistance to antibiotics, temperature range, urease production and reduction of nitrate.

A. macra and NRRL 15878 have many cultural characteristics 10 in common. In particular, the absence of aerial mycelia. However, there are also significant differences. The back color of NRRL 15878 is gray to yellowish-brown, while in many media A. macra produces a red color. This difference is most clearly seen on glucose-asparagine agar. In this medium, A. macra produces a pink airy growth, which is not the case for NRRL 15878. These comparisons are shown in Table 1.

The morphology of NRRL 15878 corresponds to the morphology of A.

20 macra. They both have poorly developed aerial mycelia, which belong to the Rectus-flexilis (RF) section according to 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)].

The 25 surface ornamentation is smooth.

The differences and similarities between NRRL 15878 and A. macra are summarized in Table IV.

30 35

18 DK 159684 B

TABLE IV:

Comparison between NRRL 15878 and A. macra 5 _

Similarities Differences 10 Morphology Antibiotic resistance

Physiological properties Carbon utilization pattern

Polyether Synthesis Culture Characteristics 15

Deficiency of air hybrids Degradation of esculin

Nitrate reduction 20 Spore shape and size

Temperature range

Urease production 25

Table V shows these similarities and differences in detail.

30 35

DK 159684 B

5

(UP

P 6 O φ (0 Ό

jn E (d H

1 ° P 9 J β w ll + l + ll + l + lllll s

Dl 0 <^

00 H

£ H u 00 m in H $

H 0) S

15 Λ £> iJ id w

PS -P S

j tu r σ> W 2 -H Η H g e ES O 0) d

Η "I

<D, S g, ί 20 o> “3 c PH <5

• H CO CH

G IS O O w

Dl 00 tJ X

• h in ri i »

H H fi P

0 J M'S II + I + I ++ IIIIIII

1 PS

E PS

id Z

25 co

P

Q) ·· P m

co cd P G

Q) · © D) * h ε g

on> in CO -H

30 Λ O) G + J

P CO Ό (d CG

ΉΗ> i Η · Η O

DOP (d XS P

P · ΡΛ Ε O P O O

5 T3 g G G G G G CO -P G G

m o G 3 G G -ri -Η H G (d · Η H W Η · Η <d C G -Η -Η -H -H + J Η -P -Η X -P O O O Λ

Ό In GOO-P CQD (dGOld> -PO-P

03 O OHlD-rl <(dOH (dQ, P-HlOPG

to I W d (ί ®ίΖΗ (0 O d ► (OP 0)> 1®)

HG (d (dOOOOOODl D> X3 X 01 -P -P X

35 O O H

• P G £ 1 (d

CO G P -P

O (d cd cd & H Q O ϋ

DK 159684 B

ε Λ

ISLAND

κ

(S

5 s Q) r — i

d X

o) to o O) P P o o c c o -

tO <D C "H

P O 00, O O -pi

m Λ A Cd PI H

lU tj irt Qj ^^ (0 CO 00 o © · β µ hhrtin> ·

two p + I I CM + + + Z £ 1 .Q OS 0) H I 0 O

s <

P H

(0 H § 0) H = 1 15 ti o Q) p Λ 1-1 o ®

S> PI

PI <1) CT »Ξ CQ v m w °

g <D X

or> O) C O

20 P -ri o ° c ** CO C H P,

00 S- 0) Η Λ CM O

t> Η O O ^ O)

00 <D O x p β I

w> p ω C P i (0 pH p ft p to o td Hin (0 o ppfcHin m »

J P + I I CM I + + P + 0) Λ OS OlH + (0 O

DS

DS

25 Z

R

N

P O

Q) 01 • Η P> i> b oh

<D p P

30 SS S Φ P O d C

(DtM PdoicDPtD o OPfiooip ε ή C 3 β Hl φΉ P01 P Ol (1) P O) αίΛοεορ £ fi ω -η 'O x «Ό I Ή O) C Λ! > 9? ® 5 rÅ ^ (ΟΡΗΟΟΦίηεΐΟΟ ^ ,, Η, · 0 0) ΗΖΑΙΟίΡΌίΟΙ I, ί ΡΡΡΉΡΟ k <h Οΐφφπίροι-Ηωπιοιφρορερ tO Ό Ό H PP 0) CP d Ή Ή ΰ cpp ftp · © ·

HftO-HO I Φ.ί3ΦΟ®ΦΗ8> «ΐΟ · Ρ POPPiPP'HPOuixopmuffl qk ppac I ι5Ο) Ι1) [0Λ [11 · Η! (! ΦΦΦΜ0) (1)

p C0rai <0Hp {0i> i <D-rlHCQ- & PPg4 (0PP

01 ϋϋΜΗΟΡΟΗΟΙΡΟΙ OlH O O E ¢) O O

Q) fl} ffl NQMO-HdOlSddnODiDiDitDMDiDi

Bh>> S 2 2 2 CU CU DS (0 DS ffl O CQ W B * S W CQ

DK 159684 B

21

These comparisons show that the cultures NRRL 15877 and NRRL 15878 are significantly different from other species of Actinomadura, and they represent strains of a new species for which the name Actinomadura oligo-5 spora sp. November The specific nickname (0. ligo.spor.us: L. adj. "Oligo" few, L.n. "sporus" spores, "oligospora" few spores) refers to the relative lack of sporophores in the organism. The strain NRRL 15878 is the type strain of A. oligospora.

10

As with other organisms, the properties of the A80190-producing culture of the invention, Actinomadura oligospora NRRL 15878, may undergo variations. Recombinants, mutants or variants of strain 15 can be obtained by methods known per se. For example, mutants can be produced 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 inducing variants, mutants and recombinants of Actinomadura oligospora NRRL 15878, with substantially the same properties, are also useful in the preparation of A80190.

The process according to the invention for the preparation of A80190 or its salts is characterized in that according to the characterizing part of claim 2.

The culture medium used to grow Actinomadura oligospora cultures can be selected from a variety of 30 different media. However, in order to achieve economical production, optimal yield and convenient isolation of the product, certain culture media are preferred. Thus, for example, glucose is a preferred carbohydrate source for large-scale fermentation, although ribose, xylose, fructose, galactose, mannose, mannitol, potato dextrin and the like can also be used. Glycerol and lipids produce little (or no) growth or antibiotic production when used as a primary carbon source. In combination with glucose, they increase biomass, but they reduce antibiotic production.

22

DK 159684 B

A preferred nitrogen source is collagen hydrolyzate, but enzyme hydrolyzed casein, meat peptones, fish meal, liver flour and the like are also useful nitrogen sources. Among the nourishing inorganic salts which can be incorporated into the culture media are the usual soluble salts which can produce ions of zinc, sodium, magnesium and calcium as well as ammonium, chloride, carbonate, sulfate and nitrate ions.

Essential trace elements necessary for the growth and development of the organism should also be present in the culture medium. Such trace elements usually appear as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organism. Foaming is usually not a problem, but if necessary, small amounts (i.e. 0.2 ml / l) of an anti-foaming agent such as polypropylene glycol can be added in the case of large scale fermentation.

In order to produce significant amounts of antibiotic A80190, it is preferred to use submers aerobic fermentation ring in tanks. Small amounts of A80190 can be obtained by shake-bottle culture. Due to the time delay in antibiotic production, which usually occurs when grafting large tanks with the spore form of the organism, it is preferred to use a vegetative graft. This vegetative inoculum is prepared by grafting a small volume of the culture medium with the spore form or with 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 medium of the vegetative seed may be the same medium used for larger fermentations, but other mediums may be used.

23 DK 159684 B

they are also suitable.

The A80190-producing organism produces A80190 when grown at temperatures between ca. 25 and approx. 37 ° C.

5 An optimum temperature for A80190 production seems to be about 30 - 32 ° C.

As is usual in submerse aerobic culture processes, sterile air is blown into the reaction vessel 10 from the bottom while the medium is stirred with conventional turbine stirrers. The maximum absorption of oxygen in the fermentation under the conditions used up to now has not exceeded about 0.22 mmol / liter / minute. In a fully shielded 165 liter fermentation vessel containing about 115 liters of liquid, an aeration rate of 0.125 rpm is a stirring rate of 200 rpm. per minute sufficient to maintain the dissolved oxygen level of 30% saturation or greater.

20

Production of the antibiotic A80190 can be monitored during the fermentation by testing samples of the liquid for antibiotic activity against organisms known to be sensitive to the antibiotic. A test organism useful for testing A80190 is Bacillus subtilis ATCC 6633. The test is conveniently conducted using a plate test with agar wells.

After A8019Q has been formed under submerse aerobic fermentation conditions, the antibiotic can be recovered from the fermentation medium by methods known per se. The antibiotic activity produced by fermentation of the A80190-producing organism occurs both in the filtered liquid and in the myelium mass. Therefore, the maximum recovery of A80190 is achieved by first filtering the medium to separate the liquid from the mycelial mass. The filtered liquid and mycelial mass can then be purified

DK 159684 B

24 separately so that they each yield an amount of A80190.

A number of different techniques can be used for this purification. A preferred technique for purifying the filtered liquid is to set the liquid to a pH of about 9 and extract it with a suitable solvent such as ethyl acetate. The solvent used for the extraction can then be evaporated in vacuo to obtain the portion of the A80190 antibiotic derived from the fermentation liquid. A preferred method of purifying the mycelial mass consists in extracting the separated mycelial filter cake with a suitable solvent such as methanol or acetone.

The solvent used for the extraction is then evaporated under vacuum, leaving a concentrated aqueous solution. This aqueous solution is then adjusted to a pH of about 9 and extracted with a suitable solvent such as ethyl acetate. The solvent is then evaporated in vacuo to give the mycelial portion of A8019Q. The two portions of the A80190 complex resulting from the culture fluid and mycelium are further purified by conventional procedures. A preferred procedure involves chromatography over silica gel.

Alternatively, the solid constituents of the culture, including the constituents of the medium and the mycelium, may be used without extraction or separation, but preferably after water removal, as a source of A80190. For example, after the production of A80190, the whole fermentation liquid can be dried by lyophilization, by drum drying or by azeotropic distillation and drying. The dried material is then mixed directly into a feed mixture.

The cationic alkali metal and alkaline earth metal salts of A80190 are prepared by procedures usually used in the preparation of cationic salts. For example, the free acid form of A80190 is dissolved in a suitable solution

DK 159684 B

solvent such as acetone. To the solution is added a 1/3 volume of water, then the resulting solution is adjusted to a pH of about 9-10 with the base corresponding to 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 consists of dissolving A80190 (acid form) in a water-immiscible solvent such as ethyl acetate, adding a corresponding volume of water and adjusting the mixture to pH 10 with the corresponding cationic base (e.g., NaOH, KOH or the like). The separated organic phase is washed with 15 water and concentrated to dryness. The residue is lyophilized from dioxane. The salt can be crystallized from a suitable solvent such as pentane.

The salts formed with organic amines can be prepared in a similar manner. For example, the gaseous or liquid amine may be added to a solution of A80190 in a suitable solvent such as acetone. Then the solvent and the excess amine can be removed by evaporation.

25

The disclosed A80190 compounds inhibit the growth of bacteria and fungi that are pathogenic to animal and plant life. The inhibitory effect of A8Q190 is illustrated in the following Table VI. Activity was measured by the conventional disk diffusion method (6 mm slices were dipped in solutions containing 1 mg of the compound per ml of solution; the slices were placed on agar plates inoculated with the test organism).

35

26 DK 159684 B

TABLE VI

Antibacterial effect of A8Q190 5

inhibition

Test organism_ (mm diameter)

Staphylococcus aureus 17 10 Bacillus subtilis 23

Micrococcus luteus 20

Mycobacterium avium 21

Saccharomyces pastorianus 10 Neurospora crassa 14 15 Candida albicans 14

An important aspect of the antimicrobial action of the A80190 compounds relates to the effect of the compounds on anaerobic bacteria. The minimum inhibition concentrations (MICs) at which A80190 inhibits various anaerobic bacteria, as determined by standard agar dilution experiments, are summarized in the following Table VII. Endpoints were read after 24 hours of incubation.

25 30 35 27

DK 159684 B

TABLE VII

Anaerobic bacterial isolates sensitivity to A80190 5 Anaerobic bacteria MIC (µg / ml)

Clostridium difficile 2994 <0.5

Clostridium perfringens 81 <0.5 10 Clostridium septicum 1128 <0.5

Eubacterium aerofaciens 1235 <0.5

Peptococcus asaccharolyticus 1302 1

Petococcus prevoti 1281 <0.5

Peptostreptococcus anaerobius 1428 1 15 Peptostreptococcus intermedius 1624 1

Propionibacterium aenes 79 '1

Bacteroides fragilis 111> 128

Bacteroides fragilis 1877> 128

Bacteroides fragilis 1926B> 128 20 Bacteroides thetaiotamicron 1438> 128

Bacteroides melaninogenicus 1856/28> 128

Bacteroides melaninogenicus 2736> 128

Bacteroides vulgatis 1211 16

Bacteroides corrodens 1874> 128 25 Fusobacterium symbiosum 1470 8

Fusobacterium necrophorum 6054A 16

Anticoccidial action is an essential property of the 30 A80190 compounds. The following Table VIII summarizes the results of an in vitro tissue culture screening of A80190 compounds against Eimeria tenella: 35

DK 159684 B

28

TABLE III

Activity of A80190 against Eimeria tenella in vitro______ 5

Concentration (µg / ml) 10 -

Compound 5 1 0.2 0.04 0.008

A80190 Ca C A A A

15 a C = cytotoxic A = active 20 For the treatment of coccidiosis in poultry, a non-toxic anticoccidial amount of an A80190 compound is administered to infected or susceptible birds, preferably orally on a daily basis. The A80190 compound can be administered in many ways, but it is most convenient to administer it together with a pharmaceutically acceptable carrier, which is preferably the bird's feed. A variety of factors are necessary to determine an appropriate concentration of the A80190 compound, but the amounts administered generally range from about two to about 100 ppm in the feed, preferably in the range of about 15 to about 100 ppm. about 50 ppm. The invention also relates to a poultry feed supplement that can be used in the treatment of coccidiosis, which comprises an effective amount of an A80190 compound for the treatment of coccidiosis in combination with a suitable carrier.

29

DK 159634 B

The aforementioned agents for the control of coccidiosis in poultry consist of 1) an A80190 compound in combination with 5 2) a compound selected from a) nicarbazine, b) 4,4'-dinitrocarbanilide,

Nicarbazine and 4,4'-dinitrocarbanilide are described in U.S. Patent No. 2,731,382. Nicarbazine is a complex of 4,4'-dinitrocarbanilide and 2-hydroxy-4,6-dimethylpyrimidine, but the compound 4,4'- dinitrocarbanilide also exhibits anticoccidial activity alone; see Science 122, 244 (1955).

The components included in the combinations of an A80190 compound with compounds 2 (a) - (b) are used in amounts which, in combination, are synergistic with respect to at least one organism that induces coccidiosis. In general, the maximum amounts used in the combinations are the same as the maximum amounts for anticoccidial treatment with the individual components. The lower limits are generally smaller than the limits required for therapeutic treatment with the individual components. The agents are generally compositions containing (1) from about 2 to about 100 ppm of an A80190 compound and (2) (a) from about 5 to about 125 ppm nicarbazine, (b) from about 25 to about 150 ppm 4, 4'-30 dinitrocarbanilide. The A80190 compounds are particularly effective when co-administered with nicarbazine. The preferred combinations contain from about 2 to about 20 ppm of an A80190 compound having between about 5 and approx.

50 ppm nicarbazine.

Another important feature of the A80190 compounds is their ability to improve feed utilization in animals. For 35 30

DK 159684 B

For example, the A80190 compounds result in improved feed utilization in ruminants having an advanced rumen function.

The efficiency of feed utilization can be measured by observing the production and concentration of propionate compounds in the rumen using the method described by Arthur P. Raun in U.S. Patent No. 3,794,732 (see, in particular, Example 5). Table IX shows the ratio of the concentrations of volatile fatty acids (VFA) in A80190 treated flasks to the corresponding concentrations in the control flasks in this experiment.

15 20 25 30 35

DK 159684 B

<v. fo <C> fo

> H

OH _ H i-l \ 0 K IN fs OO 00

(O + JH H OO «3 W ID H

4-> c o σ \ 00 O '03 O' CJ \ o o ε ^ ».vs s ΕπΛίε o o o o o o (d 0 ni

Li (U

s ®

tr G

S Λ g> 1 +3 h, + J (0 O)> u h oo cn η σ> w

©. t> -H IT) Η Η O H CN C

µ +> co oo co in vo w ø irj d s s S S S Ό jQ O O O O O O Ή ω * y

U.S

* s S g 5> ø λ «JJ +»

+) (OVDNOVOCOVO-P

·>, + J CO θ 'IN Η σ »(N C

H c 0 σι 00 O 'O CJ> C * 0

frt O ssssssO

Hid d (OOOOi-lOO o co µ µ y m a a <Ό

E-ι 0 S

«Η Λ Λ • xd -P _ G S o” o o * * * * * cn * r | ID 00 O IS 00 Ό · * Η Η Ο, ΝίΝ'ψνΟνΟΗ 0 ο OH'sicscsooLno μ

CO

rfj QjHrHHHHHO C

«Η & * 10 Ό Ο) S g 0 fl 5> S ® x c S d u oh S 32 • h cn ^ η n 5 ►>« Η fl) S · Η (0

O M \ 00 H 0) .C

00 o dl - H S · 0 <Q * Hr-ι O H 00 H C W Λ d) η • η o ε ra m ø

H

• s 0 H

+ J + J Q) co u ε 0 & μ η Ό i ε h + j ø o C Λ μ c; 5-i 0 0 0 Ό d) «η

ri H

0 0 0 o μ ε μ B

* <0 Λ

DK 159684 B

32

It can be seen from the table that the A80190 increases propionate concentrations and thus feed utilization relative to controls when administered orally to ruminants in amounts of between approx. 0.02 mg / kg / day and approx. 1.5 mg / kg / day. The 5 preferred amounts for administration are between about 0.05 and approx. 0.5 mg / kg / day.

A preferred method of administration is to mix the compound with the animal's feed. However, the compound 10 may also be administered by other means, for example in the form of tablets, metered portions, large pills or capsules. The formulation of these various dosage forms can be done by methods well known in the veterinary pharmaceutical field. The individual dosage unit 15 should contain an amount of one of the subject compounds which is directly related to the appropriate daily dose for the animal to be treated.

The invention further relates to animal feed mixtures containing as one active ingredient one of the compounds of the present invention. These feed compositions are adapted to increase feed utilization, and these feed compositions consist of e.g. animal feed, containing about 2.5 to 25 grams of one A80190-25 compound per feed. ton.

As described above, the aforementioned A80190 compounds are active against anaerobic bacteria, especially Clostridium perfringens. The A80190 compounds are therefore useful for treating or preventing enteritis in chickens, pigs, cattle and sheep. The A80190 compounds are also useful in the treatment of enterotoxemia in ruminants.

The aforementioned A80190 compounds are also useful as 35 worms. For example, A80190 can eradicate Caenor-habditis elegance at levels as low as 20 ppm.

DK 159684 B

33 The A80190 compounds are also useful in the treatment of swine dysentery. Thus, A80190 inhibits the growth of Treponema hyodysenteriae, which is a swine dye-inducing organism, at levels as low as 0.39 µg / ml. A preferred method of administration to pigs consists of incorporating an appropriate amount of an A80190 compound into the pig's feed or drinking water. An appropriate amount will depend on whether the purpose of the treatment is to prevent or to cure the infection. Usually, the concentration of the active compound required to prevent an infection is lower than the concentration required to eliminate an infection in animals already infected. Generally, amounts of the A80190 compound are between about. 20 and approx. 100 grams per

15 tonnes of feed are effective in preventing infections. Amounts of between approx. 100 and approx. 500 g of A80190 compound per tonnes of feed are recommended for the treatment of pigs suffering from dysentery. These amounts provide concentrations of between approx. 1 and approx. 5 mg / kg body weight per day. per day (prophylactic treatment) or between approx. 5 and approx. 25 mg / kg body weight per day (treatment of infected animals). When the A80190 compound is added to the animal's drinking water, it is recommended that amounts of between approx. 0.01 and approx. 0.05 grams (prophylactic) or between approx. 0.05 and approx. 0.25 grams (therapeutic) of the compound per liters of water.

The invention further provides animal feed mixtures for the treatment of swine dysentery which comprise a swine feed and an effective amount of an A8019Q compound for this purpose. As previously stated, an effective amount of the compound will typically range from about 20 to about 500 grams of A80190 compound per day. tons of feed. The A80190 compounds can be administered orally or parenterally to the animals. They can also be administered by insufflation, ie. by blowing the antibiotic, in the form of a prepared 34

DK 159684 B

dust, into an enclosed space where the animals or poultry are. The animals or poultry will thereby inhale the prepared dust found in the air. The prepared dust can also enter the body through the eyes 5 (a process known as intraocular injection).

The most practical way of administering the A80190 compounds is to formulate them in the animal feed. For this purpose, a variety of feedstuffs may be used, including the usual dry feed type, liquid and tablet-fed feeds.

Thus, although the preferred method consists of mixing the active compound with the animal's feed, the compound may also be administered by other means, e.g. in the form of tablets, 15 measured portions, large pills or capsules. The individual dosage unit should contain an amount of the A80190 compound which is directly related to the correct daily dose for the animal to be treated.

The methods of formulating medications in animal feed are well known. A preferred method is to prepare a concentrated drug premix which is then used to prepare prepared feed. Typical premixes may contain from about 2.2 to about 440 g of the active compound per day. kg of premix. Such premixtures can be either liquid or solid preparations.

The final formulation of the animal feed or poultry will depend on the amount of drug to be administered. The usual methods of formulating, mixing, and comminuting feed can be used to prepare feed containing an A80190 compound.

The subject A80190 compounds can be formulated for parenteral administration by methods well known in the art.

DK 159684 B

35 for the veterinary pharmaceutical field. Effective injectable compositions containing the A80190 compounds may take the form of either suspensions or solutions. When the composition is in the form of a solution, the A80190 compound is dissolved in a physiologically acceptable carrier. Such carriers consist of a suitable solvent, preservatives such as benzyl alcohol (if necessary) and buffers. The solvents usable include, for example, alcohols, glycols or inert oils, such as vegetable oils or highly refined mineral oils.

Compositions in the form of injectable suspensions are prepared by using a non-solvent, i.e. a liquid which does not act as a solvent for the compound together with excipients. This non-solvent can e.g. be water or a glycol such as polyethylene glycol.

Appropriate physiologically acceptable excipients are needed to keep the compound suspended in the composition in the form of a suspension. The adjuvants may be selected from thickeners such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin and alginates. Many surfactants are also useful for suspending the active compounds. Lecithin, alkylphenol polyethylene oxide adducts, naphthalenesulfonates, alkylbenzenesulfonates and polyoxyethylene sorbitan esters are useful suspending agents in liquid non-solvents.

30

Many substances that affect the hydrophilicity, density and surface tension of the liquid non-solvent can assist in the preparation of injectable suspensions in the individual cases. For example, silicone-based anti-foaming agents, glycols, sorbitol and sugars may be useful suspending agents.

DK 159684 B

36

In the preparation of powders or powders for administration by insufflation, the compounds are typically mixed with talc, diatomaceous earth or other inert substances which act as auxiliaries.

5

The disclosed A80190 compounds are also useful as insecticides and acaricides. For example, 0180190 can control the following insects at levels as low as the levels indicated in the brackets: mosquito larvae (<50 ppm), 10 salivary larvae (35 ppm), adult house flies (35 ppm) and Mexican bean seed beetles and southern "armyworms" (<100 ppm ). A80190 is also active against mites, such as the top-spotted eider mite, when administered in amounts as low as 25 ppm.

15

The disclosed A80190 compounds also have herbicidal activity. For example, A80190 can eradicate fingeraxe, mustard and quinoa in quantities of 2.25 kg / ha, either pre-emergent or post-emergent.

20

The disclosed A80190 compounds are active against polioviruses. For example, tissue culture experiments show that A80190 is effective against polio III virus at a level of 2000 µg / ml.

25

Antibiotic A80190 exhibits ion transport properties and therefore belongs to the ionophores (ion-carrying substances) (see B. C. Pressman, Alkali metral chelators the ionophores, in "Inorganic Biochemistry", Volume 1, G.L.

30 Eichhorn, Elsevier, 1973). At a concentration of 0.5 µg / ml, A80190 exhibits ionophoric activity. The activity with Na +, K + and Rb + is better than with Cs + or Li +.

However, at a lower concentration (0.2 µg / ml), A80190 exhibited an even greater ionophoric effect with Cs than 35 with Na + as cation.

You can therefore use A80190 when you want a selective

DK 159684 B

37 removal of certain cations. Examples of such applications include the removal and recovery of silver ions from solutions in photography, the removal of toxic cations from streams of industrial wastewater before such streams are discharged into the environment, and desalination of seawater. A80190 can be used as the one component of an ion-specific electrode (see O. Kedem et al., U.S. Patent No. 3,753,887, August 21, 1973).

A80190 alters the cation permeability of both natural and artificial membranes. Therefore, A80190 can be used as a component of a membrane to be used for the selective transport of cations against a concentration gradient. A potential application of this property lies in the extraction of heavy precious metals on a commercial basis; see E. L. Cussler, D. F. Evans, and Sister Μ. A. Matesick, Science 172 ', 377 (1971).

Another aspect of the subject A80190 compounds is that they are active as inhibitors of the enzyme ATPase. This enzyme, which is an alkali metal-sensitive enzyme found in cell membranes, is involved in the energy needed for the active transport. By "active transport" is meant the energy-intensive series of operations whereby 25 intracellular and extracellular fluids maintain their compositions. Inhibitors of ATPase reduce the energy required for active transport. In vitro studies have shown that A80190 inhibits the ATPase of mitochondria with a half-effective concentration (IC, -q) of 0.5 30 µg / ml.

The A80190 compounds in question are also potential cardiotonic agents. 1

The invention is further illustrated by the following examples.

Preparation of A80190 EXAMPLE 1 38

DK 159684 B

5 A. Shake Bottle Fermentation of A80190

The culture Actinomadura oligospora NRRL 15878, either as a lyophilized tablet or as a suspension kept in liquid nitrogen, is used to inoculate a seeding medium of the following composition:

Ingredient Quantity (%)

Glucose 1.0 15 Soluble starch 2.0 Yeast extract 0.5

Enzymatic hydrolyzate of casein * 0.5

CaCOg 0.1 20 Demineralized water up to 1 liter

NaOH was added to raise the pH of the medium to about 7.2 prior to sterilization.

25 * "NZ Amine A", Sheffield Chemical Co.,

Norwich, N.Y.

Oblique cultures or plates are prepared by adding 2.5% agar to the seeding medium. The grafted slant culture is incubated at 30 ° C for a period of between ca. 10 and approx. Fourteen days.

The mature slant culture is scraped with a sterile tool to loosen the spores and remove the mycelium mat. About a quarter of the loosened spores and the culture growth thus obtained are used to seed 50 ml of a medium in the first step.

The inoculated medium in the first step is incubated in a 250 ml

DK 159684 B

39

Erlenmeyer flask at 30 ° C for about 48 hours on a shaker rotating in a circular motion (5.08 cm) at a speed of 250 rpm. minute.

This first-stage incubated medium (0.4 ml) is used to seed 50 ml of a production medium of the following composition:

Ingredient Quantity (%) 10

Glucose 3.0 "N-Z amine A" 0.4

Collagen hydrolyzate * 0.5

MgSO4 * 7H20 0.05 CaCOg 0.2

Cold tap water up to 1 liter (Prior to sterilization, pH is adjusted to 7.0) 20 * "IPC 3", Inland Industrial Molasses Co.,

Dubuque, Iowa.

The seeded production medium is incubated in a 250 ml large mouth Erlenmeyer flask at 30-32 ° C for 8 to 10 days on a shaker rotating in a circular motion (5.08 cm) at a rate of 250 rpm. minute.

B. Tank Fermentation of A80190 In order to obtain a larger volume of graft, 10 ml of a first stage incubated medium prepared as described in Section A is used to seed 400 ml of a second stage growth medium which has the same composition as the medium in the first step. This second stage vegetative medium 35 is incubated in a two-liter large-mouthed Erlenmeyer flask for about 48 hours at 30 ° C on a shaker rotating in a circular motion (5.08 cm)

DK 159684 B

40 at a speed of 250 rpm. minute.

The 5 second incubated vegetative medium thus prepared (800 ml) is used to seed 100 liters of sterile production medium prepared as described in Section A. The seeded production medium is allowed to ferment in a 165 liter fermentation tank with stirring for 8 to 10 days at a temperature of 30 to 32 ° C. A limited air flow (0.12 - 0.25 v / v / m) and a low rotational speed (150 - 200 rpm) in the container causes a dissolved oxygen level of over 30% air saturation to be maintained. .

15 C. Insulation of A80190

The entire fermentation liquid from two 100 liter tanks was combined (207 liters) and filtered through a filter press using "Hyflo supercel". The filtered mycelial cake 20 was extracted by circulating methanol (40 liters) through the filter press. Acetone can also be used as an extractant. The methanol extract, which was concentrated in vacuo to a volume of about 15 liters, was combined with the filtrate of the fermentation liquids 25 (182 liters). The mixture was adjusted to pH 9 using 1 N sodium hydroxide and the resulting solution extracted with a corresponding volume of ethyl acetate. The ethyl acetate extract was concentrated to a volume of about 700 ml. To the concentrated extract 30 was added 1 liter of water and the pH was adjusted to 9.0 with sodium hydroxide, after which the mixture was extracted twice with 1 liter of toluene, maintaining a pH of 9.0. The toluene extracts were combined and concentrated in vacuo to give an oily residue containing 35 A80190.

The residue was dissolved in toluene (100 ml) and transferred to one

DK 159684 B

41 column containing 2 liters of silica gel (Woelm, 70-150 mesh) in toluene. The column was first eluted with 10 liters of toluene and then with 10 liters of each of the two toluene-ethanol mixtures 49: 1 and 48: 2. Five fractions of 1 liter were collected. The elution was monitored by bioassay and TLC. The fractions containing A80190 were combined and concentrated. The residue was dissolved in dioxane and lyophilized to give 13.6 g of crude A80190.

10 D. Purification of A80190 20.4 g of crude A80190 obtained from four 100 liter tanks as described above were dissolved in 200 ml of acetonitrile. The solution was transferred to a column containing 2 15 liters of silica gel (Woelm, 70-150 mesh) in acetonitrile. The column was washed with 10 liters of acetonitrile and eluted sequentially with mixtures of acetonitrile and acetone (95: 5, 2 L), (9: 1, 10 L), (4: 1, 10 L) and (7: 3, 10 L). ), collecting 1 liter fractions. The elution 20 was followed by bioassay using Bacillus subtilis. The fractions containing A80190 were combined and concentrated. The residue was dissolved in dioxane and lyophilized to give 15.8 g of purified A80190.

E. Crystallization of A80190 28.2 g of purified A80190 were dissolved in 500 ml of acetone. 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 temperature for 20 hours so that crystallization could occur. The crystals were separated by filtration, washed with water and dried in a vacuum oven to give 25.9 g of crystalline A80190 (acid form). FIG. 1 shows the infrared absorption spectrum of A80190 in chloroform.

Preparation of the sodium salt of A80190 EXAMPLE 2 42

DK 159684 B

5 300 mg of A80190 (acid form) was dissolved in 30 ml of acetone. 10 ml of water was added and the pH was adjusted to 10 with NaOH. The solution was concentrated to an oily residue which was dissolved in dioxane and lyophilized to give an amorphous white powder (mp 145-150 ° C).

The molecular weight of the product as determined by field desorption mass spectrometry (FDMS) was 850. IR spectroscopy in CHCl 3, shown in FIG. 2, exhibit absorption maxima at the following frequencies (cm 2): 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 3

Alternative Preparation of the Sodium Salt of A80190 20 g of A80190 (acid form) was dissolved in 500 mg of ethyl acetate and 500 ml of water was added. The mixture was adjusted to pH 10 with 5N NaOH and stirred for 15 minutes to maintain the pH unchanged. The ethyl acetate phase 25 was separated and washed with 500 ml of water, then concentrated to dryness under vacuum. The residue was dissolved in 100 ml of dioxane and lyophilized to give 4.1 g of the sodium salt of A80190.

The water used for leaching was allowed to stand at room temperature overnight and the crystals thus formed were filtered off and dried. These crystals consisted of A80190 in acid form (mp 115-120 eC).

35

Crystallization of the sodium salt of A80190 EXAMPLE 4 43

DK 159684 B

The sodium salt of A80190 (1 g) in amorphous form was dissolved nicely (10 ml) and the solution was allowed to stand at room temperature for 3 days (thereby evaporating the solvent to a volume of about 5 ml). The crystals thus formed were filtered off and dried under vacuum to give 593 mg of the crystalline sodium salt of A80190 with a m.p. at 225 - 230 AD. The difference in melting point relative to the amorphous starting material reflects the different physical states.

EXAMPLE 5

Chromatographic identification of A80190 I. TLC on silica gel System: acetonitrile acetone (1: 1)

Rf = 0.59

Detection: Bacillus substilis

Vanillin-I ^ SO ^ Spray 25

II. HPLC

Adsorbent: "uBondapak C18" (4 x 300 mm column)

Solvent system: acetonitrile: tetrahydrofuran 30 I (O: 6: 1: 3) containing 1%

H3PO4; adjusted to pH 3.0 with NH 4 OH

Detection: refractometer

Flow rate: 3.0 ml / min 35 Retention time: 9.7 min.

EXAMPLE 6 44

DK 159684 B

A80190 is prepared by the method described in Example 1 using the culture Actinomadura oligosporus NRRL 5 15877.

Example 7 A8019O-modified chicken feed for coccidiosis control 10

A high energy balanced feed for chickens for rapid weight gain is prepared according to the following recipe: 15 Ingredient% kg

Ground yellow corn 50 454

Soybean meal solvent extracted, peeled and pre-milled, 50% protein 31.09 282.3

Animal fat (beef tallow) 6.5 59 25 Dried fish meal with soluble additives (60% protein) 5.0 45.4

Soluble residues from distillation 4.0 36.3 30

Dicalcium phosphate feed quality 1.8 16.3

Calcium Carbonate 0.8 7.3 35

Ingredient% kg 45

DK 159684 B

Vitamin premix (representing vitamins A, D, E, K and B 2 ·, choline, niacin, pantothenic acid, riboflavin, biotin and glucose as filler) 0.5 4.5 10 Trace mineral mixture (representing MnSO4, ZnO,

K1, FeSO4 and CaCOg) 0.2 1.8 2-Amino-4-hydroxybutyric acid (hydroxy analog to methionine) 0.1 0.9 A80190 (Na salt) 0.01 0.1

The substances listed above are mixed using 20 standard feed mixing techniques. Chickens fed this feed and given water ad libitum are protected from being exposed to coccidiosis. The weight gain obtained can be compared to the results obtained with coccidiosis-free chickens fed a similar feed mixture without drug content.

EXAMPLE 8 A80190 Enhanced Feed for Slaughter Cattle 30

A balanced feed with high cereal content for slaughter cattle is prepared as follows:

Ingredient% kg 46

DK 159684 B

Finely ground corn 67.8 615.6 5 Crushed corn cobs 10 90.8

Dehydrated alfalfa flour, 17% protein 5 45.4 10 Peeled ground soybeans, solvent extracted, 50% protein 9.9956 90.8 cane sugar syrup 5 45.4 15

Urea 0.6 5.5 A80190 (Na salt) 0.0044 0.04 Dicalcium phosphate, feed quality 0.5 4.54

Calcium carbonate 0.5 4.54 Sodium chloride 0.3 2.72

Trace mineral mixture 0.03 0.27 30 35

Ingredient% kg 47

DK 159684 B

Vitamin A and mixture * 0.07 0.64 Vitamin E mixture ** 0.05 0.45

Calcium propionate 0.15 1.36 * Content per kg: Approx. 4405285 I.E. vitamin A; 500440 I.E. Vitamin D2 and 850 g of soybeans added to 1% oil.

** Dried corn from spirits containing approx.

44052 I.E. d-a-tocopheryl acetate per kg.

15

The mixed feed is pressed into tablets. At an average daily administration of 6.8 kg feed per day. The animal feeds an average feed of 300 mg 20 A80190 (Na salt) per day. animals per day.

EXAMPLE 9 A80190 Enhanced Swine Feed 25

A balanced feed for pigs with piglets is prepared as follows:

Ingredient% kg / ton 48

DK 159684 B

Ground yellow corn 65,10 591,1 5 Soybean oil and flour, extracted and peeled 18,50 168 Dried beet pulp 10.00 90.8 10 Dicalcium phosphate 2.90 26.3

Calcium carbonate 1.20 10.9

Vitamin mixture ^ 1.10 10 15

Salt (NaCl) 0.55

Choline chloride, 25% 0.35 32 2 20 Trace mineral mixture 0.15 1.4

Vitamin A mixture ^ 0.10 0.9

Hydroxy Analogue to Methionine 0.05 0.4 25 Total 100.00 908

1) Each kilogram of mixture contains the following: US $ 77,161

units of vitamin 2205 I.E. vitamin E, 441 mg ri boflavin, 1620 mg pantothenic acid, 2205 mg niacin, 4.4 30 mg vitamin B ^ / 441 mg vitamin K, 19180 mg choline, 110 mg folic acid, 165 mg pyridoxine, 110 mg thiamine and 22 mg biotin.

2) Each kilogram of mixture contains the following: 50 g of manganese 35 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 at least 130 g and not more than 150 g

, 0 DK 159684 B

49 calcium as calcium carbonate.

3) Each kilo of mixture contains 6.613800 USP units of vitamin A.

5

To 90.8 kg of this feed, a premix is prepared by adding A80190 (10 g) to a small amount of solvent-extracted soybeans, grinding the mixture in a mortar and diluting the ground mixture to 0.454 kg 10 with a further amount of solvent-extracted soybeans. This premix is then added to 90.8 kg of the pig feed described above and mixed by standard technique. This drug-containing feed provides a level of 100 g A80190 per day. tons of basal feed. This feed is administered to the pregnant sow for at least one day, preferably for 7 to 10 days prior to the farrowing, and after the farrowing, the feed is administered as long as desired.

Larger or smaller amounts of the drug-containing feed containing varying levels of A80190 are prepared by varying the amount of A80190 in the premix and / or the amount of the basal feed.

A sow is administered between 2.2 and 22 g of A80190 per day. 100 kg fo-25 there. The drug-containing feed is kept available ad libitum along with water. Usually a sow consumes about 2.7 - 3.6 kg of feed per day. day.

EXAMPLE 10 A80190 formulation for piglets A80190 is dissolved in a small amount of ethanol. This ethanol solution is suspended in polyethylene glycol (MW 200).

The resulting suspension is concentrated so that each unit dose has a volume of about 0.5 to 2 ml. Such suspensions are administered to young pigs in amounts of

DK 159684 B

50 between 1.10 and 110 mg per day. kg three times daily.

EXAMPLE 11 5 Improved feed for the control of swine dysentery

A premix is prepared by standard methods using the following ingredients: 10 Ingredient g / kg A80190 Compound 150.0

Calcium Silicate 20.0 15

Calcium carbonate (Eastern peel flour) 830.0

Total weight 1000 g

This premix is added to a commercial pig feed, using standard mixing techniques so as to obtain a final content of the active compound of 100 g / t.

EXAMPLE 12 25

A demonstration of the synergistic effect of nicar-bazin in combination with A80190 was carried out as follows: One-week-old broilers were distributed in cages in groups of five and fed either a 30-drug-containing or control feed, typically for 1 day before being infected with oocysts by a coccidiosis-inducing organism. The chickens were kept on their respective feed rations for a given period of time, typically 7 days. Four repetitions 35 were performed for each treatment.

The chickens were seeded with 200000 oocysts of Eimeria

DK 159684 B

51 acervulina (Lilly strain 59), 60000 oocysts of Eimeria maxima (strain FS-177) and 40000 oocysts of Eimeria te-nella (strain FS-155).

5 The results of the experiments are shown in the tables below. It is seen that the incidence of bowel lesions is synergistically reduced relative to the reduction with increasing doses of the individual constituents, although to a lesser extent also the weight gain 10, which also exceeds that obtained by the individual components.

15 20 25 30 35

DK 159684 B

52

Intestinal lesions (points) 5 Nicarbazine j A80190 (p.p.m).

(p.p.m.) I_0_4_8_12 16 20 0 I 9.13 8.63 5.94 3.50 1.19 0.0 20 I 9.13 5.13 0.19 0.13 0.0 10 30 | 8.88 1.63 0.50 0.0 40 I 8.90 0.56 0.0 50 I 8.56 0.0 125 I 0.08 15 Colon lesions (points)

Nicarbazin | A80190 (ppm) 20 (ppm) | _0_4_8_12 16 20 0 I 3.44 2.63 1.75 1.19 0.19 0.0 20 I 3.19 2.38 0.31 0.56 0.06 30 I 3.50 0.94 0.19 0.0 25 40 I 3.17 1.00 0.58 50 I 3.19 0.54 125 I 0.33 30 35

DK 159684 B

53 Weight gain (grams per chicken)

Nicarbazin | A80190 (p.p.m.) 5 (p.p.m.) I 0_4_8_12 16 20 0 I 147 236 265 251 272 261 20 I 159 268 284 266 279 30 I 174 279 258 283 40 I 211 258 289 10 50 | 222 276 125 I 279 15 20 25 30 35

Claims (10)

A polyether antibiotic named A80190 of formula 5 OMe \ / OMe ^ Me T Me 0 Me Me T ^ V-vWY 'Me Me ϋΜ and pharmaceutically acceptable salts thereof with bases. 20 Process for preparing an A80190 compound according to claim 1, characterized in that Actinomadura oligospora NRRL 15877 or NRRL 15878 or a mutant thereof having substantially the same characteristics is grown in a culture medium containing assimilable sources of carbon , nitrogen and inorganic salts, under submerse aerobic fermentation conditions until antibiotic A80190 is formed, then separates A80190 from the culture medium and, if desired, reacting it with a base 30 to form a pharmaceutically acceptable salt. Compound A80190 and pharmaceutically acceptable salts thereof according to claim 1 for use as a veterinary product or as a health promoting product for animals. 35 Compound A80190 and pharmaceutically acceptable salts thereof according to claim 1 for use in the control of 55 DK 159684 B coccidiosis in poultry. Process for increasing the feed utilization efficiency of ruminants, characterized in that the animal is orally administered an effective propionate-increasing amount of compound A80190 or a pharmaceutically acceptable salt thereof according to claim 1. Process for promoting the growth of monogastric animals, characterized in that the animal is administered an effective amount of the compound A80190 or a pharmaceutically acceptable salt thereof according to claim 1. Animal feed mixture, characterized in that it contains as active ingredient A80190 or a pharmaceutically acceptable salt thereof according to claim 1. Poultry feed mixture according to claim 7, characterized in that it contains a first component which is A80190 or a pharmaceutically acceptable salt thereof according to claim 1, and another component selected from nicarbazine and 4,4, -dinitrocarbanilide. Poultry feed mixture according to claim 8, characterized in that the second component is nicarbazine. The Actinomadura oligosporus NRRL 15878 for use in the practice of claim 2. 30